Constructing a cost-efficient, high-throughput and high-quality single molecule localization microscope for super resolution imaging John S. H. Danial*1,2, Jeff Y. L. Lam*1,2, Yunzhao Wu*1,2, Matthew Woolley1, Eleni Dimou1,2, Matthew R. Cheetham1,2, Derya Emin1,2, and David Klenerman1,2 1 Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge, United Kingdom 2 UK Dementia Research Institute, University of Cambridge, Cambridge, United Kingdom * These authors contributed equally To whom correspondence should be addressed: John S. H. Danial (js2494@cam.ac.uk) and David Klenerman (dk10012@cam.ac.uk). Abstract Single Molecule Localization Microscopy (SMLM) leverages the power of modern optics to unleash ultra- precise structural nanoscopy of complex biological machines in their native environments as well as ultra- sensitive and high-throughput medical diagnostics with the sensitivity of one, single molecule. To achieve this remarkable speed and resolution, SMLM setups are either built by research laboratories with strong expertise in optical engineering, or commercially sold at a hefty price tag. The inaccessibility of SMLM to life scientists for technical or financial reasons is detrimental to the progress of biological and biomedical discoveries reliant on super resolution imaging. In this work, we present the NanoPro; an economic, high- throughput, high-quality, and easy-to-assemble SMLM for super resolution imaging. We show our instrument performs similarly to the most expensive, best-in-class commercial microscopes, and rivals existing open-source microscopes at a lower price and construction complexity. To facilitate its wide adoption, we compiled a step-by-step protocol, accompanied by extensive illustrations, to aid inexperienced researchers in constructing the NanoPro as well as assessing its performance by imaging ground-truth samples as small as 20 nm. The detailed visual instructions make it possible for students with little expertise in microscopy engineering to construct, validate, and use the NanoPro in less than one week, provided all components are available. Introduction Many biological molecules assemble into nanoscopic complexes to perform their biological functions1–3. Visualizing these tiny machines is of paramount importance to understanding the inner workings of the cell, and therefore the human body in health and disease. Single Molecule Localization Microscopy (SMLM) is, undoubtedly, one of the most powerful super resolution imaging tools developed over the last two decades to chart the nanoscopic organization of complex biological macromolecular assemblies in their native environments. Famous techniques, such as Stochastic Optical Reconstruction Microscopy (STORM)4,5, Photo Activated Localization Microscopy (PALM)6, Point Accumulation in Nanoscale Tomography (PAINT)7 fall within the larger umbrella of SMLM. The general principle underlying the operation of this set of technologies is that, instead of imaging the bulk of fluorophores in a sample of interest, fluorophores are allowed to be switched ‘on’ and ‘off’, one-at-a-time, so that their centroids can be localized with high, single- digit nanometer precision. By repeating this process multiple times, the large majority of all fluorophores can be precisely located. These ‘localizations’ are then used to construct a super resolved image which reveals the underlying biological structure with much higher clarity. The use of SMLM has uncovered the ultrastructure of many important biological macromolecular complexes including the nuclear pore complex1, neuronal cytoskeleton8, apoptotic pores2, amyloid aggregates9, endocytic machinery3 and many others. Although the use and optimization of these different methods has been extensively pursued in the past10–14, they all require individual fluorophores to be imaged with high precision and accuracy15. A high precision, resulting from the use of bright fluorophores, efficient detection schemes and precisely aligned illumination source(s), as well as, high accuracy, resulting from simple, mechanically stable constructions that are robust against external vibrations and thermal drift, define in a complex manner the resolving power of a system. State-of-the-art microscopy setups delivering high resolving powers are only available commercially, are expensive to acquire and maintain (costing several hundreds of thousands of US dollars), and can be limited in their capabilities. Some of these microscopy setups are even delivered with additional features that are not required to perform super resolution imaging. Amongst these features are eyepieces, which are no longer used to observe a mounted sample, objective and dichroic turrets, which are not required given the need for a single objective and dichroic mirror, and single-use control equipment (i.e. joystick) which delivers a suboptimal user experience. Furthermore, the large variety in microscopy components, as in lasers, cameras, stages and bodies, requires their integration using generic microscopy control software solutions which can be tricky to use, specifically if high-throughput measurements are aimed for. Overall, these attributes render the use of SMLM in important biomedical investigations, that require high quality results in a high-throughput manner, limited to laboratories, and imaging facilities, that have optical expertise or can afford tailored, but expensive, solutions. Open-source microscopies and components for single molecule imaging The microscopy community has, for long, advocated the development of single molecule microscopy setups that can be assembled from standard, off-the-shelf optical components, are substantially cheaper than commercially-available pre-assembled microscopy setups, and exclusively designed for SMLM experiments. To this end, four different microscopy setups were conceived in the last five years each addressing one, or more, challenges in the accessibility of single molecule imaging by the wider scientific community. The first of these is the miCube (figure 1a), a modular fluorescence microscope developed by the Holbhein lab and which costs between 23,000 USD (for the basic edition) and 127,000 USD (for the premium edition). The miCube is constructed from a combination of simple, 3D printed parts and small, Computer Numerically Control (CNC) machined aluminium parts and includes off-the-shelf medium-to-high sensitivity scientific Complementary Metal Oxide Semiconductor (sCMOS) camera, an X/Y stage with a long (100 mm) travel range, a short range piezo Z insert and a fibre-based laser combiner16. The miCube was recently used in tracking the genome editing enzyme, CRISPR-Cas9, with 40 nm precision in living, gram-positive bacteria. Despite its many features and remarkably small footprint, the miCube can be expensive if opting for the premium version, cannot be used for automated, multi-sample or long measurements given the lack of a focus stabilization system, cannot be used for Forster Resonance Energy Transfer (FRET) based measurements given the absence of choice between different emission filters, and is not suited for sensitive, intensity-based measurements due to the lack of a flat field illumination module. The K2 TIRF (https://ganzingerlab.github.io/K2TIRF/K2TIRF/index.html) is an alternative open-source microscope which was later developed by the Ganzinger lab with additional features to overcome several of the shortcomings of the miCube chassis (figure 1b) including the implementation of a focus stabilization system, flat field illumination module, and simultaneous multi-colour imaging, but at the expense of a reduced capacity to perform multi-sample measurements due to the installation of a shorter range X/Y stage, larger price tag due to the additional components, and increased assembly complexity. The second microscope setup is the liteTIRF (figure 1c) developed by the Jungmann lab for high resolution PAINT imaging entirely assembled from off-the-shelf components at a moderate cost of 24,000 USD (single edition)17. To lower its cost, the setup uses one, single-wavelength laser, economic sCMOS camera with a moderate quantum efficiency, a manual X/Y/Z stage, and a highly-compacted assembly that uses a minimum number of components. liteTIRF was shown to resolve DNA origami structures with 10 nm spaced docking strands. Although the demonstrated resolution (i.e. localization precision and accuracy) is remarkable, this was achieved through iterative alignment of the imaged origamis taking advantage of prior knowledge of their structure. Despite achieving a single digit nanometer resolution, the setup cannot be used for multi-coloured single molecule experiments due to the presence of a single laser, high-throughput, multi-sample imaging due to the absence of a motorized X/Y/Z stage, intensity-sensitive measurements due to the absence of a flat-field illumination module, and general purpose single molecule imaging experiments due to the use of a low-quality camera which is not sensitive enough for capturing the low number of photons released by single fluorophores under different imaging modalities. Unlike the miCube and the K2 TIRF, the liteTIRF is not a modular assembly owing to its highly-compacted structure. This implies that expansion of the setup to include more laser lines or a larger, higher-quality, camera might not be possible without changing the assembly architecture. The third open-source microscope setup is the WOSM (Warwick Open Source Microscope, figure 1d), an ultra-stable CNC machined assembly developed by the Cross lab for high-quality super resolution imaging (https://wosmic.org/). The WOSM chassis is a sturdy, two piece, and low-profile aluminium assembly which largely resembles the miCube. An important selling point of the microscope is the minimized distance between the mounted sample and the top side of the chassis which, according to its developers, allows for long duration, high resolution imaging without the need for drift correction. Notably too, and similar to the K2 TIRF, the WOSM comes with a comprehensive control suite. Despite its numerous advantages, particularly its remarkable stability, the WOSM is, presumably, substantially more expensive than the other microscopes given the use of highest quality components (e.g. a high-sensitivity electron multiplying Charge Coupled Detector (emCCD) camera, and commercial laser combiner), is not suited for high- throughput, multi-sample imaging given the short travel range on the X/Y/Z stage and lack of a focus stabilization system, is not capable of performing intensity sensitive measurements due to the absence of a flat field illumination module, and can be complicated to construct given the need for long milling components required to drill through a large, single block of aluminium forming the base of the chassis. Although the development of WOSM can be traced back as far as 2016, the setup is not yet presented with a complete list of components required for its assembly. These revolutionary microscopes are 1.5 to 20 times cheaper than commercially available microscopes which can range in price from 170,000 USD (for separate commercial components) to 400,000 USD (for bespoke, pre-assembled solutions). Although the reduction in price is remarkable, other developers focused their efforts on developing substantially cheaper instruments, but at the expense of reduced imaging quality. The first of these microscopes was developed by the Liu lab, an open-frame setup that costs around 3,800 USD (figure 1e)18. The setup is composed of ultra-low-cost and off-the-shelf components which would allow imaging in an epi-illumination mode and is, therefore, best-suited for STORM imaging only. Although robust quantitative resolution measurements are lacking, results based on imaging microtubules and the H3 protein demonstrate a spatial resolution between 30 nm to 100 nm. The setup cannot be used for high-speed, high-quality PAINT, or more generally TIRF, imaging due to limitations on the illumination angle (which cannot be controlled on the setup, first, given that the excitation beam cannot be translated, and, second, due to the use of an optical diffuser which homogenises the beam but creates several secondary point sources), is not suited for long duration (> 10,000 frames) imaging due to unestablished stability measurements, cannot be used for high-throughput, multi-sample imaging due to the absence of a motorized X/Y/Z stage, and requires a custom-written imaging script. The second, and final, of the cost-efficient setups is the cellSTORM (https://beniroquai.github.io/stormocheap/); a 3D printed, mobile-based super resolution microscope costing less than 1000 USD (figure 1f)19. The microscope can perform super resolution imaging with 100 nm spatial resolution. To reduce the cost of cellSTORM, the developers used a combination of 3D printed and ultra-low-cost opto-mechanical components as well as deep learning for image processing. Although this is clearly remarkable, the notable reduction in price comes at the expense of a significantly reduced spatial resolution, and inability to perform multi-sample imaging experiments. These microscopes, and others20, have pioneered the broad accessibility of SMLM, but collectively suffer from three important shortcomings that prevent them for achieving their vision. The first shortcoming is that none of the above microscopes (except for the cellSTORM and to some extent the miCube) is provided with a step-by-step guide for assembly. These microscopes were designed with the vision of being accessible to scientists with little optical expertise, who are tight in budget, and who need a super resolution microscope to answer an important biological or biomedical question. To meet this vision, it is absolutely essential that the end-user is capable of assembling and maintaining these relatively sophisticated machines with ease. The second shortcoming is that the majority of the listed setups are not designed for high-throughput, multi-sample imaging. Biological and biomedical scientists can only draw meaningful scientific conclusions by imaging several cellular and human samples under a variety of different conditions. For this reason, and since these assemblies are designed for biologists, high-throughput, multi- sample imaging counts as a fundamentally important feature to have. The third, and final, shortcoming is that none of the above microscopes is delivered with a simple user control experience. Being versatile, gaming controllers are being extensively utilized across many modern instruments, including commercial microscopes such as the Nano imager from Oxford Nanoimaging as well as the OpenStage21, to deliver an engaging user experience. The use of simple control tools is seen as important for the accessibility of these instruments by the wider community of non-experts. The above microscopes are standalone devices that have undergone extensive optimization in design to serve specific purposes. Some developers adopted a different direction which is to optimize the individual components which comprise any microscope assembly. As an example, the Ries lab has recently developed a highly economic laser combiner (https://github.com/ries-lab/LaserEngine) taking advantage of cheap, high-power diode lasers22. The downside of using such lasers is having to cope with their distorted beam profile which strongly deviates from the Gaussian shape commonly encountered with high quality, but expensive, lasers. To overcome this problem, they combine the different lasers into a single, square- core, multi-mode fibre to produce a square profile, flat-field illumination which can be used for high-quality single molecule experiments. This trick can reduce the price of a laser combiner from 45,000 USD to less than 5,000 USD. Another example is the open flexure stage (https://openflexure.org/) which attempts at democratizing high-quality microscopes through the development of high-precision, sub-micron resolution stages out of 3D printed components23. Although, to our knowledge, these stages were only tested with low-to-medium quality super resolution imaging24, they hold much promise given their versatility and the strong support provided for their production, assembly and use. A final example is the NanoJ (https://github.com/HenriquesLab/NanoJ-Fluidics/wiki) developed by the Henriques lab which relies on the use of LEGO blocks to construct an open-source fluidic exchange system that can be easily integrated with any open-source or commercial microscope system25. These, and more, components, or rather subassemblies, can be utilized in many different ways to construct robust, multi modal microscopes at a fraction of the price. Overview of NanoPro 1.0 Extending the revolutionary work described above, and mitigating its major shortcomings, as well as building on our expertise in constructing high-throughput, multi-sample SMLM setups26–28, we designed and conceived the NanoPro 1.0; an economic, ultra-high-quality custom microscope solution for high- throughput, multi-sample super resolution imaging based on the detection of single molecules (figure 2a and table 1). NanoPro 1.0 is assembled from a combination of CNC machined aluminium parts and off-the- shelf components to produce a high-line super resolution microscope under 70,000 USD (2.4 – 5.7 times cheaper than existing commercial rivals). The microscope is specifically intended at biological and biomedical scientists with no prior expertise in optical engineering, who require an autonomous machine for high-throughput, multi-sample, and multi-target super resolution imaging at a 20 nm spatial resolution, and which can be easily assembled using the detailed, IKEA style, step-by-step instructions provided in this protocol. This microscope is not intended for individuals or facilities that require ultra-low-cost microscopes, setups with ultra-high-stability and spatial resolutions much below 20 nm, setups with minimal features but high-to-modest spatial resolutions, and microscopes with an extensive set of features but increased assembly complexity. The many excellent alternative open-source microscopes described in the previous section would better serve these diverse purposes. NanoPro 1.0 achieves its unique positioning in the cost versus quality space by implementing the following features: 1. An economic, multi-laser engine powered by high-power, low-cost lasers coupled into a custom-made square-core fibre at remarkable coupling efficiencies (>80%) to deliver a square profile, flat-field illumination at the sample plane29. The square-core has a 70 µm x 70 µm core that is smaller than commercially-available fibres used in contemporary setups with low-cost laser / LED combiners22,30. Due to its small core size, our fibre system can be used to illuminate a sample of interest in EPI, HILO or TIR modes to allow high-quality STORM, PAINT or generic single molecule based experiments (figure 2b and s3). The laser engine is housed in an economic, easy-to-assemble and stable enclosure to comply with laser safety regulations. 2. A focus stabilisation system assembled from widely-available optomechanical components and controlled using the custom software provided with NanoPro 1.0 and the multipurpose controller delivered with the assembly (figure 2c). The focus stabilisation system facilitates prolonged imaging by adjusting for axial drift, and is fundamentally important for the automation of multi-sample imaging (figure s1). 3. A slip-stick X/Y/Z stage with 50 mm travel range and 1 nm movement resolution in all directions (figure 2d). The stage differentiates itself from traditional motorized X/Y stages and piezo-based Z stages in that it allows long range travel in the lateral directions which, when coupled with the focus stabilization system described above, permits high-throughput, multi-sample imaging. These features come in an economic assembly that is at least half-the-price of competing alternatives. 4. Multi-colour imaging enabled by a 4-slot, low-cost and off-the shelf filter-changer (figure 2e). Although lacking in all other open-source microscopies, this important feature would allow hybrid, FRET based techniques (e.g. FRET-PAINT31) to be easily-conceived which allows conventional PAINT experiments to be accelerated by up to 100 folds. 5. Best-in-class sCMOS camera with 95% maximum quantum efficiency, 6.5 µm pixel size, and low electron readout noise at a third of the price of conventional EMCCD cameras (figure 2f). The camera’s high quantum efficiency, homogenous noise profile and small-to-moderate pixel size allows signal oversampling. This results in high-quality single molecule imaging with median localization precisions reaching down to 2.1 nm; only 1 nm more than sophisticated and complex microscopy configurations such as the MINFLUX32. 6. A transparent enclosure that protects the microscope, minimizes disturbance to the mounted sample from air currents, and permits the controlled flow of gases to cater for the future possibility of performing live-cell super resolution imaging (not demonstrated here, figure 2g). 7. A game pad controller that controls the lasers, focus stabilization system, stage, filter-changer, camera’s live mode and acquisitions (figure 2h). The controller elevates the pressure of the Graphical User Interface (GUI) and delivers an engaging experience that is intuitive and economic. 8. A fully-illustrated, step-by-step IKEA style guide (i.e. this protocol) that explains, with ample detail, the assembly, operation, performance evaluation and troubleshooting of NanoPro 1.0 assuming no prior expertise in optical engineering or knowledge of optical, and related, components. Although this protocol focuses on the use of NanoPro 1.0 in super resolution imaging due to the lack of ground-truth, commercially-available reference standards for single molecule tracking, distancing and counting experiments, it is expected that these measurements would be readily achievable on our setup given the presence of all the required quality components. Choice of components In order for us to guarantee the high-quality of our instrument, as well as the presence of the necessary features mentioned above, we focused on acquiring components that can perform the needed functions reliably, can be easily and reproducibly integrated into the entire assembly, are economic, and can be purchased from reliable vendors with worldwide shipping services or manufactured at local facilities. There is usually a strong competition between products that occupy a unique niche in the market, such as scientific grade cameras and positioners (i.e. motorised sample stage), in which case the choice between these different products was dictated by quality-over-price. Expensive, high-quality products with additional, unrequired features were replaced with the next best competitor product on the market if it didn’t compromise the purpose of our assembly. Additionally, preference was given to products that are more expensive but can be reliably and reproducibly integrated within the entire assembly to ensure the end-user can easily replicate this protocol. As a first example of the above, the choice of excitation filters (i.e. dichroic filters used to combine the different laser lines) was based on the cost and ease of installation in widely-available optomechanical components. However, this was not the case with filters and dichroic mirrors in the emission path which were chosen of the highest quality to ensure imaging quality, particularly the localization precision, is not compromised. As a second example, the choice between expensive (> 500 USD), high-quality, low-power lasers and low- cost (100 – 500 USD), low-quality, high-power lasers, and ultra-low-cost (< 100 USD), ultra-low-quality, high-power lasers. As previously mentioned, beam shape quality does not correlate with imaging quality in our setup as all the combined laser lines are launched into a square-core fibre which produces a homogeneous illumination at the sample plane. This leaves us with the low- and ultra-low-cost lasers to choose from. Ultra-low-cost lasers, also known as laser diodes, suffer from several important shortcomings, amongst these, undocumented lifetime measurements, lack of quality control over the output intensity, imprecise engineering resulting in unexpected designs and tolerances, and need for packaging by the end- user as well as their temperatures to be controlled using external, complicated electronic circuitry. Low-cost lasers do not suffer from these shortcomings. Given that the price difference between a low-cost and an ultra-cost laser would not exceed 400 USD, which in the grandeur scheme of the entire assembly represents less than 2% of the total cost, it is sensible to choose a more expensive, but still low-cost, laser that can be easily integrated and operated. A third example is that of the cameras which are available in three categories: expensive, ultra-low-noise, and low-speed EMCCD cameras – which, for more than a decade, were the camera of choice for single molecule imaging experiments – low-cost, low-noise, and high-speed sCMOS cameras, and ultra-low-cost, medium-noise, and high-speed non-scientific CMOS camera. NanoPro 1.0 was designed to provide the highest quality single molecule imaging (i.e. lowest localization precision and highest signal-to-noise ratio). Compromising imaging quality with the use of a non-scientific CMOS camera was unquestionable, and, therefore, the choice was left between the EMCCD and sCMOS cameras. sCMOS cameras are being increasingly used for super resolution imaging given their superiority in detecting single molecules at high photon counts. Until recently, they were manufactured with peak quantum efficiencies of 82%. Currently available, equally-priced, sCMOS cameras are produced with a 95% peak quantum efficiency and intelligent noise-correction algorithms which can push the localization precision of single molecules detected in PAINT and STORM experiments down to 2 nm, as well as, allow faithful single molecule tracking, distancing and counting experiments where photon budget is low. Furthermore, currently available sCMOS cameras can be run at 5 to 10 times higher speeds than the best-in-class EMCCD cameras at full field-of-view, provide stable operation at higher temperatures, are manufactured with up to four times the number of pixels, are gaining a larger share in the global market for cameras, and are up to a third cheaper in price than EMCCDs. All these attributes were strong indicators for us to consider the use of an sCMOS in our instrument. The fourth and final example is that of the X/Y/Z stage. Conventional, commercially-available microscopes are fitted with a motorized X/Y stage and Z stage composed of a motorized part for coarse movement and a piezo positioner for fine focusing and movement. Although this configuration offers a long travel range along the X and Y axes which is suitable for imaging samples mounted on glass coverslips and multi-well plates, single-molecule, and particularly super-resolution, experiments are performed on glass coverslips where no more than 50 mm of travel range is required, and, therefore, the extra-long travel range is not required for these measurements. Additionally, this system is expensive, costing at least 23,000 USD, and is, relatively, troublesome to control due to the presence of multiple different components that use different coding libraries. There are two alternatives to this configuration, a low-cost, open-loop, piezo-electric 3-axis actuator, or a moderate-cost, closed-loop, piezo-electric 3-axis actuator both which can be readily configured to move along the X, Y and Z axes. The open-loop option does not contain a position sensor. This would prevent the end-sure from returning to a recorded or absolute position given the high error in movement reproducibility. Although an open-loop configuration can be used with random multi-field-of-view imaging, it would not be suitable for imaging at periodic or user-recorded positions. It is often the case that the end-user desires to image at set locations in which case the open-loop system would not serve their purpose. To this end, we chose the slightly more expensive closed-loop positioners to ensure multi-sample imaging is not restricted as described above. The above examples are not inclusive of all the components used in the microscope assembly. The choice of these components was dictated by the set of rules, rationally designed for constructing the NanoPro 1.0, which were described earlier. These rules ensure the microscope is well-suited for the highest precision structural measurements. At times, this comes at a cost. Users who can sacrifice precision for a substantially lower cost, may consider replacing some of the components listed in the procedure below with those listed in table 3. Key performance indicators Establishing the performance of a super resolution microscope is an important task that helps the end-user answer a basic, but important, question: what is the smallest distance between any two biological objects / structures that a microscope can resolve. Answering this question requires the use of a ground-truth sample; one where the dimensions of the underlying structure(s) are known a priori. There are numerous ground-truth samples which can be used to measure the spatial resolution of a microscope. In order to choose the best-in-class and ensure our results are easily reproducible by the end-user, our sample had to satisfy the below requirements: 1. It has to be purchased ready for mounting. This excludes a large number of excellent samples including: immuno-stained nuclear pore complexes, actin filaments, microtubules, and Clathrin-coated pits, which have to, either, be obtained from collaborators with strong and established expertise in producing these samples to ensure high-quality control standards on their production, or be prepared in-house in which case quality cannot be appropriately controlled. 2. It can be ordered in a range of sizes, below and above the predicted spatial resolution of the system. Many of the biological samples listed above are larger than the predicted spatial resolution of NanoPro 1.0 (i.e. 20 nm). As an example, the average diameter of the nucleoporins forming nuclear pore complexes lies between 70 nm and 120 nm33 and of microtubules around 100 nm34. Although image based algorithms, such as the Fourier Ring Correlation (FRC)35, can be used to quantify the spatial resolution of a system from images acquired of these samples, seeing is believing. The end-user has the right to, not just quantify the spatial resolution of the system but, be able to confirm that they can observe biological objects at that limit. 3. It can be imaged with different dyes. As previously mentioned, the spatial resolution of a system is dependent on the localization precision which in turn is dependent on the quality of detection at the emission wavelength of the fluorophore used in an experiment. This might be a deterrent to using some of the cellular samples listed above in some imaging modalities, such as DNA-PAINT, where some dyes can be seen to bind to cellular structures in a nonspecific manner. 4. It can be ordered with fiducial markers. Nearly every microscope requires correction against thermal and mechanical drift either through the application of drift correction algorithms, based on cross correlation36, or, using fiducial markers, such as fluorescent beads or nanoparticles. The spatial resolution of a microscope system is clearly dependent on which, or if both, of these methods is used for drift correction. As such, the sample to be used for establishing the performance of NanoPro 1.0 has to contain fiducial markers to allow the end-user quantify the lower limit of the spatial resolution. DNA origami structures are the only ground-truth samples which satisfy the above requirements. A DNA origami is a precisely engineered array of different, single-stranded DNA molecules held together by a DNA scaffold37,38. These tiny structures were originally developed for a range of applications in nanotechnology but found their way in developing DNA-PAINT7 and their subsequent use in establishing the resolving power of PAINT based microscopes. The preparation of DNA origami structures is facilitated by Picasso11; a software tool that can be readily used to design and simulate origami structures where docking strands can be placed along a 2D periodic array at distances as short as 2.5 nm and up to 50 nm. The complication in using origami structures is that their assembly is a meticulous process requiring the careful addition of over 150 different DNA strands at fixed proportions. Imperfections in the synthesized strands, or a single error during the assembly process, can result in faulty structures which are not suitable for establishing the performance of a microscope. To ameliorate this problem, we reverted to commercially-available origami structures, known as ‘nanorulers’, which are composed of three, equidistant docking strands placed in a line at set dimensions of 20 nm and 40 nm. These nanorulers are pre-assembled and pre-mounted on sealed coverslips that are delivered ready-to-use. To this end, we used the NanoPro 1.0 in imaging the nanorulers as described in the protocol (see procedure). Using a simple cross-correlation drift correction algorithm, we could readily uncover the ultra-structure of the 40 nm and 20 nm nanorulers (figure 3a and b). Our drift correction curves (figure 3c) demonstrate that the NanoPro 1.0 is exceptionally stable, capable of excluding frame-to-frame perturbations, and allowing image recovery without the use of fiducial markers, or sophisticated drift correction algorithms, for structures as small as 20 nm. Furthermore, our median localization precision (2.1 nm, figure 3d) measurements indicate that our instrument can rival the best-in-class, commercially- available systems and can provide localization precisions that are only 1.1 nm higher than those provided with alternative, non-single molecule based methods, such as MINFLUX. The stability and precision of the NanoPro 1.0 are strong indicators to its suitability for high-quality super-resolution imaging. Usage and limitations As with any microscope setup, NanoPro 1.0 comes with a number of limitations which inform its usage. In order to aid the end-user in choosing whether to construct the instrument described here, or else how to best use it, we have decided to divide these limitations into five mutually-exclusive categories: sample-, readout-, accuracy-, design- and investment-based limitations. With regards to sample-based limitations, the NanoPro 1.0 was designed for high-throughput, multi-sample super resolution imaging. As such, the sample holder designed for our instrument can accommodate the following: 26 x 76 mm, #1.5 thickness glass coverslips (VWR, cat. no. MENZBC026076AC40), alone or coupled with CultureWell™ gaskets composed of 50, 3 mm wells (Grace Bio-Labs, cat. no. GBL103250), as well as ibidi ® µ-Slide 8 or 18 Well glass bottom chambers. These hosts can accommodate up to 50 different recombinant, cellular or human-derived samples, at volumes ranging from 200 µL to 3 µL per sample, to be imaged in one, single run. Furthermore, and given that these silicon gaskets can be readily mounted, the coverslips can be functionalized in various ways to enable, not only super resolution imaging, but also surface-based, single-molecule based assaying39. As for readout-based limitations, the microscope is not capable of performing measurements of single molecule FRET due to the absence of wavelength-splitting equipment in the emission path, 3D SMLM imaging due to the absence of a cylindrical lens5 or other optical elements40 for engineering the Point Spread Function (PSF), and spectrally-resolved SMLM due to the absence of a diffraction grating41 or dispersive prism42 in the emission path. Although some of these methods are already well-established, the decision not to integrate them was based on the rationale of simplifying both, the assembly and readout, to what is commonly required. Nevertheless, given the modular nature of the instrument, some or all of these features may be included in future upgrades. With regards to accuracy-based limitations, the NanoPro 1.0 achieves a 20 nm spatial resolution using simple algorithms based on cross correlation for drift correction. A higher resolution might be achieved using iterative alignment of origami-based fiducial markers. However, deep axial imaging might prevent the user from appropriately observing these markers and some surface preparation protocols could be incompatible with their incorporation. Other drift correlation algorithms, such as those based on residual entropy43 were reported to achieve a single digit nanometre resolution, however, these were not tested here and, therefore, their performance cannot be guaranteed. As for the design-based limitations, although the NanoPro 1.0 is more compact than many of the commercially-available setups, it is assembled on a 1.5 m x 0.9 m optical table and, therefore, requires at least 2.5 m x 1.5 m of room space. The instrument is also not suitable for placement inside an enclosed biosafety cabinet. Finally, even though the NanoPro 1.0 is an economic, state-of-the-art setup providing many of the advanced features delivered with commercial microscopes at a fraction of the price, it still requires a substantial financial investment as well as partially-dedicated research staff to build and maintain the instrument as described here (table 2). These five important factors, when considered with the set of biological or biomedical questions that need to be answered, should allow the end-user to inform a balanced decision on whether it is appropriate to establish our instrument in their own lab, or facility, and how to best make use of it. Overview of the procedures The protocol starts by planning for the procurement, fabrication and installation of all components making up the NanoPro 1.0 as well as obtaining access, and negotiating for, a suitable room to accommodate the microscope’s assembly. We first describe how to assemble the setup starting with the laser engine, microscope box, focus stabilization system, detection module, sample stage and holder, and, finally, all enclosures. We then describe how to set up the electrical connections and install the NanoPro 1.0 control software to align the lasers and focus stabilization system prior to assessing the performance of the system and operating it thereafter. Due to the sheer number of components comprising, and steps involved in building, the assembly, we have introduced the following: 1. The assembly, alignment and operation procedures are accompanied by detailed, illustrated guides (see supplementary) that assume no expertise in mechanical or optical engineering. 2. The reference number of each item (see materials) is listed in each section, once, to ensure similar items are not confused. Materials Fibre • [item 1] 2x 70 µm x 70 µm, NA=0.22, L=2m, FC/PC connector, square-core fibre cable (CeramOptec GmbH, cat. no. 05806-1 Rev. A) • [item 2] 40 mm focal length, FC/PC connector, achromatic fibre collimator (Thorlabs, cat. no. C40FC-A) Light • [item 3] 1x 561 nm, 100 mW laser (Roithner LaserTechnik, cat. no. RLTMLL-561-100-3) • [item 4] [optional] 1x 10 KHz, analog modulation unit (Roithner LaserTechnik, cat. no. RLTMXL ANALOG 10KHZ) • [item 5] 1x 350 mW, 405nm diode laser with cooling option (Lasertack GmbH, cat. no. PD-01254-E) • [item 6] 1x 55 mW, 488nm diode laser with cooling option (Lasertack GmbH, cat. no. PD-01339-E) • [item 7] 1x 700 mW, 635nm diode laser with cooling option (Lasertack GmbH, cat. no. PD-01229-E) • [item 8] 1x 20 mW, 850 nm compact diode laser module with shutter (Thorlabs, cat. no. LDM850) • [item 9] 1x 740 mW, 4900 K, 1225 mA mounted LED (Thorlabs, cat. no. MNWHL4) Light analysis • [item 10] 1x 320-1100 nm, 2D lateral effect position sensor (Thorlabs, cat. no. PDP90A) • [item 11] 1x 700-1400 nm, IR detector card (Thorlabs, cat. no. VRC5) • [item 12] 1x Digital LCD, compact power and energy meter console (Thorlabs, cat. no. PM100D) • [item 13] 1x 400-1100 nm, 500 mW, Standard Photodiode Power Sensor (Thorlabs, cat. no. S121C) • [item 14] 1x Prime BSI Express, sCMOS camera (Teledyne Photometrics, cat. no. O1_PRIME_BSI_EXP) Motion control • [item 15] 3x Enhanced blocking force and integrated sensor, 51 mm travel, positioner (Smaract, cat. no. SLC-1780-D-S) • [item 16] 1x MSC2 sensor module (Smaract, cat. no. MCS2-S-0001) • [item 17] 1x MSC2 control system (Smaract, cat. no. MCS2-C-0002) • [item 18] 1x K-Cube PSD auto aligner (Thorlabs, cat. no. KPA101) Optics • [item 19] 1x Apochromatic, 100x magnification, 1.49 numerical aperture, TIRF objective (Nikon, cat. no. MRD01991) • [item 20] 1x 405/488/561/635/800-1050 nm, BrightLine® multiphoton super resolution dichroic splitter (Semrock, cat. no. DI01-R405/488/561/635/800-T1-25X36) • [item 21] 1x 390/482/564/640 nm, BrightLine® quad-band bandpass filter (Semrock, cat. no. FF01- 390/482/563/640-25) • [item 22] 1x 446/523/600/677 nm, BrightLine® quad-band bandpass filter (Semrock, cat. no. FF01- 446/523/600/677-25) • [item 23] 1x 525/30 nm, BrightLine® single-band bandpass filter (Semrock, cat. no. FF01-525/30- 25) • [item 24] 1x 600/37 nm, BrightLine® single-band bandpass filter (Semrock, cat. no. FF01-600/37- 25) • [item 25] 1x 676/29 nm, BrightLine® single-band bandpass filter (Semrock, cat. no. FF01-676/29- 25) • [item 26] 1x 700 nm, BrightLine® multiphoton short pass dichroic beam splitter (Semrock, cat. no. FF700-SDI01-25X36) • [item 27] 1x 425 nm, long pass dichroic mirror (Thorlabs, cat. no. DMLP425) • [item 28] 1x 505 nm, long pass dichroic mirror (Thorlabs, cat. no. DMLP505) • [item 29] 1x 605 nm, long pass dichroic mirror (Thorlabs, cat. no. DMLP605) • [item 30] 1x 805 nm, long pass dichroic mirror (Thorlabs, cat. no. DMLP805) • [item 31] 1x 700 nm, premium long pass filter (Thorlabs, cat. no. FELH0700) • [item 32] 10x 400-750 nm, broadband dielectric mirror (Thorlabs, cat. no. BB1-E02) • [item 33] 1x 750-1100 nm, broadband dielectric mirror (Thorlabs, cat. no. BB1-E03) • [item 34] 1x 125 mm focal length, achromatic doublet (Thorlabs, cat. no. AC254-125-A-ML) • [item 35] 1x 200 mm focal length, tube lens (Thorlabs, cat. no. TTL200-A) • [item 36] 1x 11 mm focal length, 0.25 numerical aperture, aspheric lens (Thorlabs, cat. no. C220TMD-A) • [item 37] 1x 1.0 optical density, neutral density filter (Thorlabs, cat. no. NE10B-A) • [item 38] 1x 700-1100 nm, 30 mm, non-polarising beam splitter (Thorlabs, cat. no. CCM1-BS014/M) • [item 39] 1x Variable line grating test target (Thorlabs, cat. no. R1L3S6P) • [item 40] 1x Pack of 1000, 26x76 mm, #1.5 Menzel Glaser glass coverslips (VWR, cat. no. MENZBC026076AC40) Optoelectronics • [item 41] 1x Intel® Core™ i9 processor, NVIDIA® GeForce® GTX 1650 Ti 4GB, 32 GB RAM, 1 TB SSD, laptop (Dell, XPS 15 (9500)) • [item 42] 1x 7-Port USB Desktop Hub (Currys, cat. no. ACH115EU) • [item 43] 1x Sony PlayStation DualShock 4 controller (Onecall, cat. no. CS30605) • [item 44] 1x 4-output, digital analog converter device (Active Robots Ltd., cat. no. 1002_0B) • [item 45] 1x 6-output, control hub (Active Robots Ltd., cat. no. HUB0000_0) • [item 46] 1x 5 V, constant DC power source device (Active Robots Ltd., cat. no. PSU2000_0) • [item 47] 1x 1.5 V-5 V, variable DC power source device (Active Robots Ltd., cat. no. PSU2001_0) • [item 48] 2x 60 cm, mini-USB cable (Active Robots Ltd., cat. no. 3036_0) • [item 49] 2x 60 cm, converter device cable (Active Robots Ltd., cat. no. 3002_0) • [item 50] 1x 12 outlet, surge-protected power strip (Thorlabs, cat. nos. HDPS12-UK [for UK], HDPS12-US [for US], HDPS12-EU [for EU]) • [item 51] 1x 1 m, black cable trunking (Thorlabs, cat. no. CMS002) • [item 52] 1x 15 V, 2.4 A power supply unit for one K- or T-cube (Thorlabs, cat. no. KPS101) • [item 53] 1x 15 V/5 V, power supply unit for K- or T-cubes (Thorlabs, cat. no. TPS002) • [item 54] 1x T-cube LED driver (Thorlabs, cat. no. LEDD1B) • [item 55] 2x 914 mm, male to male, BNC coaxial cable (Thorlabs, cat. no. 2249-C-36) • [item 56] 2x BNC to test clips (Thorlabs, cat. no. T3788) • [item 57] 3x 12 V, 80 W, 100-240 VAC, power supply for laser modules (Lasertack GmbH, cat. no. PD-01341-E) • [item 58] 1x 30 A, 250 V, terminal strip (RS Components, cat. no. 782-2857) • [item 59] 1x 10 m, unscreened flat ribbon cable (RS Components, cat. no. 214-0683) • [item 60] 3x 1m, IEC power cable (RS Components, cat. nos. 731-6185 [for UK], 731-6163 [for US], 626-6751 [for EU]) • [item 61] 5x 5 mm, vibration motor (Precision Microdrives Ltd., cat. no. 304-111) • [item 62] 1x 50 cm, Micro B to USB A 2.0 cable (XMA Ltd., cat. no. CDL-160-05M) Optomechanics • [item 63] 1x 700 mm x 900 mm x 1500 mm, heavy-duty passive frame (Thorlabs, cat. no. PFH90150-8) • [item 64] 1x 110 mm x 900 mm x 1500 mm, breadboard (Thorlabs, cat. no. B90150B) • [item 65] 1x Passive isolation foot pump (Thorlabs, cat. no. PTA127) • [item 66] 1x Fibre launch system (Thorlabs, cat. no. KT110/M) • [item 67] 1x Four-position slider bundle (Thorlabs, cat. no. ELL9K) • [item 68] 4x 225 mm, square construction rail (Thorlabs, cat. no. XE25L225/M) • [item 69] 12x 50 mm length, 25 mm diameter, pedestal pillar post (Thorlabs, cat. no. RS2P/M) • [item 70] 4x 38 mm length, 25 mm diameter, pedestal pillar post (Thorlabs, cat. no. RS1.5P/M) • [item 71] 1x 2 in length, 1 in diameter, pedestal pillar post (Thorlabs, cat. no. RS2P) • [item 72] 2x Pack of 5, 50 mm length, 12.7 mm diameter, optical post (Thorlabs, cat. no. TR50/M- P5) • [item 73] 1x Pack of 5, 30 mm length, 12.7 mm diameter, optical post (Thorlabs, cat. no. TR30/M- P5) • [item 74] 3x Pack of 5, 50 mm length, 12.7 mm diameter, post holder (Thorlabs, cat. no. PH50/M- P5) • [item 75] 1x 1.24 in slot length, clamping fork (Thorlabs, cat. no. CF125) • [item 76] 9x Pack of 4, 0.5 in length, 6 mm diameter, cage assembly rod (Thorlabs, cat. no. ER05- P4) • [item 77] 8x 0.25 in length, 6 mm diameter, cage assembly rod (Thorlabs, cat. no. ER025) • [item 78] 2x Pack of 4, 6 mm diameter, rod adaptor (Thorlabs, cat. no. ERSCB-P4) • [item 79] 4x Kinematic mirror mount (Thorlabs, cat. no. KM100CP/M) • [item 80] 3x 30 mm, right-angle kinematic mirror mount (Thorlabs, cat. no. KCB1C/M) • [item 81] 3x 30 mm, cage cube with dichroic filter mount (Thorlabs, cat. no. CM1-DCH/M) • [item 82] 3x 30 mm, 0.5 in thickness, cage plate (Thorlabs, cat. no. CP33T/M) • [item 83] 8x Compact kinematic mirror mount (Thorlabs, cat. no. KMS/M) • [item 84] 8x 1 in diameter, 2.5-6.1 mm thickness, mirror holder (Thorlabs, cat. no. MH25) • [item 85] 3x Rotation mount for 1 in optics (Thorlabs, cat. no. LRM1) • [item 86] 1x 2 in travel, SM1 zoom housing (Thorlabs, cat. no. SM1NR1) • [item 87] 3x SM1 end cap (Thorlabs, cat. no. SM1CP2) • [item 88] 2x SM1 coupler (Thorlabs, cat. no. SM1T2) • [item 89] 2x Adaptor with external SM1 threads and internal SM2 threads (Thorlabs, cat. no. SM1A2) • [item 90] 1x Adaptor with external SM1 threads and internal M25 x 0.75 threads (Thorlabs, cat. no. SM1A12) • [item 91] 1x Adaptor with external SM05 threads and internal SM1 threads (Thorlabs, cat. no. SM1A1) • [item 92] 1x Adaptor with external C-mount threads and external SM1 threads (Thorlabs, cat. no. SM1A39) • [item 93] 1x 2 in, SM1 lens tube (Thorlabs, cat. no. SM1L40) • [item 94] 1x Pack of 5, 0.3 in, SM1 lens tube (Thorlabs, cat. no. SM1L03-P5) • [item 95] 1x 0.5 in, SM1 lens tube without external threads (Thorlabs, cat. no. SM1M05) • [item 96] 1x 1 in, SM1 lens tube spacer (Thorlabs, cat. no. SM1S10) • [item 97] 1x FC/PC fibre adaptor cap with internal SM1 threads (Thorlabs, cat. no. S120-FC) • [item 98] 1x SM1 thread spanner wrench (Thorlabs, cat. no. SPW606) • [item 99] 1x 9-piece hex key set (Thorlabs, cat. no. CCHK/M) • [item 100] 1x 4-40 cap screw and hardware kit (Thorlabs, cat. no. HW-KIT5) • [item 101] 1x M6 cap screw and hardware kit (Thorlabs, cat. no. HW-KIT2/M) • [item 102] 1x Pack of 25, 0.25"-20 in thread, 0.5 in length, stainless steel setscrew (Thorlabs, cat. no. SS25S050) • [item 103] 1x Pack of 2, microscope slide spring clips (Thorlabs, cat. no. SLH1/M) • [item 104] 1x Bullseye level (Thorlabs, cat. no. LVL01) • [item 105] 1x Pack of 2, handles (Thorlabs, cat. no. BBH1) • [item 106] 2x Hinge for rail enclosures (Thorlabs, cat. no. XE25H) • [item 107] 1x Lid stop for rail enclosures (Thorlabs, cat. no. XE25LS) • [item 108] [consult workshop] 2x 20 mm x 235 mm x 275 mm, 5080 aluminium tool plate (Aalco, cat. no. 277120) • [item 109] [consult workshop] 2x 20 mm x 160 mm x 235 mm, 5080 aluminium tool plate (Aalco, cat. no. 277121) • [item 110] [consult workshop] 2x 20 mm x 200 mm x 275 mm, 5080 aluminium tool plate (Aalco, cat. no. 277122) • [item 111] [consult workshop] 3x 20 mm x 105 mm x 130 mm, 5080 aluminium tool plate (Aalco, cat. no. 277124) • [item 112] [consult workshop] 1x 20 mm x 105 mm x 230 mm, 5080 aluminium tool plate (Aalco, cat. no. 277125) • [item 113] [consult workshop] 1x 20 mm x 80 mm x 90 mm, 5080 aluminium tool plate (Aalco, cat. no. 277129) • [item 114] [consult workshop] 1x 20 mm x 45 mm x 105 mm, 5080 aluminium tool plate (Aalco, cat. no. 277131) • [item 115] 1x Wire stripper (RS Components, cat. no. 663-617) • [item 116] 1x Pack of 15, 32 mm length, 6 mm diameter plain steel dowel pin (RS Components, cat. no. 270-653) • [item 117] 1x Pack of 20, 24 mm length, 4 mm diameter plain steel dowel pin (RS Components, cat. no. 270-596) • [item 118] 1x Pack of 100, 20 mm length, M1.6, TORX®, stainless steel screw (RS Components, cat. no. 179-5712) • [item 119] 1x Pack of 10, 15 mm x 15 mm, steel angle bracket (RS Components, cat. no. 427-991) • [item 120] 1x Pack of 250, 0.3 mm thickness, M1.6, stainless steel plain washer (RS Components, cat. no. 179-5724) • [item 121] 1x Pack of 10, M6, clamping knob (RS Components, cat. no. 830-4158) • [item 122] 1x Planar connecting element (Smaract, cat. no. SCE-CN) • [item 123] 1x Rectangular connecting element (Smaract, cat. no. SCE-RN) • [item 124] 1x TORX® T5, screwdriver (Farnell, cat. no. 4432850) • [item 125] 1x 2.5 mm x 50 mm, slotted, screwdriver (Farnell, cat. no. 3378478) • [item 126] 1x 525 pieces, M3 to M6, socket cap screw kit (Onecall, cat. no. TRFAKIT0005) • [item 127] [consult workshop] 1x 2440 mm x 1220 mm x 5 mm, black foam PVC (Vision Plastics Ltd.) • [item 128] [consult workshop] 1x 1000 mm x 1000 mm x 8mm, clear Perspex (Engineering & Design Plastics Ltd.) • [item 129] 1x Laser plates (Custom fabrication) • [item 130] 1x Laser combiner enclosure (Custom fabrication) • [item 131] 1x Positioner base (Custom fabrication) • [item 132] 1x Microscope box (Custom fabrication) • [item 133] 1x Sample enclosure (Custom fabrication) • [item 134] 1x Sample holder (Custom fabrication) Sample • [item 135] 1x 40 nm, Cy3B, immobilized high-resolution DNA-PAINT nanorulers (GATTAquant GmbH, cat. no. 3030) • [item 136] 1x 20 nm, Cy3B, immobilized high-resolution DNA-PAINT nanorulers (GATTAquant GmbH, cat. no. 3031) Safety • [item 137] 1x 12% visible light transmission, universal style, laser safety glasses (Thorlabs, cat. no. LG4) Software • [item 138] Supplementary software • [item 139] ImageJ software (https://imagej.nih.gov/ij/) Other • [item 140] 1x Low autofluorescence immersion oil (Thorlabs, cat. no. MOIL-30) • [item 141] 1x Fibre and vibration motor mount (Custom fabrication) • [item 142] 1x Lens cleaning tissue (Merck, cat. no. WHA2105862) • [item 143] 1x Park of 25, thick Viton O Ring (Hooper Ltd., cat. no. OR26X2V175) • [item 144] 1x Pack of 50, thin Viton O Ring (Hooper Ltd., cat. no. OR26X1V175) • [item 145] 1x 25.1 mm focal length, Plano convex lens (Thorlabs, cat. no. LA1951-ML) • [item 146] 1x 1 in, SM1 lens tube (Thorlabs, cat. no. SM1L10) Procedure Step 1: procurement and space preparation (timing: up to 6 months) 1. To guarantee the high performance (i.e. minimized localization accuracy and precision) of NanoPro 1.0, obtain access to a suitable room that satisfies the following requirements: a. Is on the ground, or preferably basement, floor of the building, and does not face, or is close to, a street. Having the setup on the ground, or basement floors, avoids low frequency vibrations which cannot be dampened using the air isolation table (item 63) and that can affect the resolving power of the microscope. The host institution can be consulted on whether the building can accommodate such an optical table on a basement or ground floor room. b. Is laminated with a vibration isolation material and, if possible, be mechanically isolated from the rest of the building. c. Is fitted with card access, or keypad lock as well as a signal device to indicate when the microscope is under operation (i.e. lasers are on). d. Is fitted with an air conditioner for controlling the temperature and humidity. e. Is at least 4.5 m x 3.5 m in dimensions. The door of the room should be at least 85 cm wide and the hallway along which the room is should be at least 1.5 m wide. All hallways and doors leading to the room should be at least 1.35 m wide. f. Does not contain any large equipment that generates heat or noise (e.g. freezers, pumps, robots …). g. Does not contain any windows or other uncontrollable light sources. This room could contain windows in case they are blacked out. 2. It is advisable that all components are procured (i.e. ordered) one after the other in the following order: positioners and all auxiliary components (items 15, 16, 17, 122, 123), objective lens (item 19), lasers (items 3, 4, 5, 6, 7, 57), fibre cable (item 1), filters (items 20 – 26) and samples (items 135, 136) followed by all other items. The following should be considered when items are procured: a. For the positioners and all their auxiliary components, the supplier should be asked for all the necessary screws; this is a collection of M2 and M1.6 screws that are only produced by the supplier and that are required for assembly. b. Critical step: for the lasers, the supplier should be requested not to fix the adjustable lenses using glue, or any other material, and to ensure that the rotating threads are extended out of the laser heads for fine adjustment of collimation. c. For the optical table (items 63, 64), the supplier should be, first, advised with a suitable date for delivery. These are large and heavy items, they need to be dismantled (i.e. unpackaged, see unpacking and installation) and delivered inside the building as fast as possible to prevent road blockages. Second, the supplier should be requested to place the two items comprising the optical table far from one another to ensure easy unpacking. Third, the supplier should be requested to provide on-site unpackaging and installation of the optical table. If agreed, points 9 to 17 can be skipped. d. All suppliers should be asked for delivery notes to satisfy grant requirements (if applicable). e. Some of the components are listed with UK-based suppliers. The catalogue number of these components is shared between worldwide suppliers and can be ordered using these numbers from local suppliers. 3. It is highly-recommended that components to be fabricated (items 129 – 134) are manufactured in a mechanical workshop where quality can be easily monitored and controlled (e.g. mechanical workshop facility within institute or department). Due to the precision required in fabricating these components, it is advised that these components are not procured from companies that perform accelerated, computerized manufacturing and prototyping due to the high cost incurred for the accelerated service and the lack of control on quality. Although complete instructions on assembly are provided below, it is advised that the mechanical workshop assembles these components in their ready-to-use form to ensure reproducibility during assembly (see fabrication for further information). Step 2: fabrication (timing: up to 3 months) 4. Follow these instructions for manufacturing the laser plates (item 129): a. If applicable, provide the mechanical workshop with the aluminium tool plates (items 111, 112). b. Provide the mechanical workshop with the supplementary drawing files [Drawing 1.pdf and Drawing 2.pdf] and CAD files [CAD_File 1.f3d and CAD_File 2.f3d]. 5. Follow these instructions for manufacturing the microscope box, positioner base and sample holder (items 131 – 133): a. If applicable, provide the mechanical workshop with the aluminium tool plates (items 108, 109, 110, 113, 114). b. Provide the mechanical workshop with the supplementary drawing files [Drawing 3.pdf – Sheets 1 to 9] and CAD files [CAD_File 3 to 10.f3d]. 6. Follow these instructions for manufacturing the sample enclosure (item 134): a. If applicable, provide the mechanical workshop with the clear Perspex (item 128). b. Provide the mechanical workshop with the supplementary drawing files [Drawing 3.pdf – Sheets 1 and 10 to 17] and CAD files [CAD_File 11 to 17.f3d]. 7. Follow these instructions for manufacturing the laser combiner enclosure and fibre and vibration motor mount (items 130 and 141): a. If applicable, provide the mechanical workshop with the black foam PVC (item 127). b. Provide the mechanical workshop with the supplementary drawing files [Drawing 4.pdf] and CAD files [CAD_File 20 to 26.f3d] for the laser combiner enclosure. c. Provide the mechanical workshop with the supplementary CAD file [CAD_File 18.f3d] for 3D printing the fibre and vibration motor mount. Step 3: unpacking and installation (timing: 2 days) 8. Clear the microscope room of all equipment and packages which were delivered beforehand to ensure there is enough space for installing the optical table (items 63, 64). Critical step: at least eight individuals need to carry out points 9 to 17. These individuals need to be contacted well ahead to ensure their presence on the day of delivering the optical table. Furthermore, ensure that none of the contacted individuals are medically-unfit for lifting heavy material (25 kg). Critical step: points 9 to 17 involve the unpackaging and lifting of heavy material. Lifting heavy material is a serious risk. To avoid this risk, the supplier can be contacted well ahead to arrange an on-site unpacking and installation service, as advised in point 2c. In case agreed, points 9 to 17 can be skipped. In case not agreed, a risk assessment form (see supplementary risk assessment form and guidance [Form 1.pdf]) needs to be filled out to ensure all activities are compliant with health and safety regulations. Critical step: request from the goods-in, or mechanical workshop, facilities a set of large hex keys to unpackage the optical table. It is recommended to obtain a cordless screwdriver set where available. Critical step: request from the goods-in, or mechanical workshop, facilities a forklift (also known as hand pallet truck) to be used for lifting one of the two items comprising the optical table. 9. The optical table will be delivered in two large wooden pallets secured by long, Philipps-head screws. For the larger pallet (i.e. the frame), Loosen all the screws on the top and front / back wooden panels using the screwdriver set borrowed from your local facility. Carefully remove the panels and stow in a safe area. Caution: a small number of the screws will be hard to remove. Note the location of these screws and avoid them to prevent injury. Caution: wear thick cotton gloves and enclosed shoes before dismantling. 10. Pierce the plastic film wrapping the frame and lift, with the aid of all participating individuals, the frame a few centimetres off the bottom of the wooden pallet, carefully slide it out of the wooden pallet, and, finally, rest it on the ground. 11. Lift the frame off the ground. two individuals should lift the left, shorter side of the frame, and another two should lift the right, shorter side of the frame. Two of the four individuals should walk backward facing and the other two forward facing. 12. Deliver the frame to the microscope room and orient the frame so that the back, longer side is 30 cm away from a wall with at least one AC power socket protected by a circuit breaker to avoid any short circuits. 13. For the smaller pallet (i.e. the breadboard), Loosen all the screws on the top, front, back, left and right wooden panels using the screwdriver set borrowed from your local facility. Carefully remove the panels and stow in a safe area. 14. Observe the gaps between the bottom of the breadboard and the wooden pallet. Insert the fork lift (i.e. hand pallet truck) in the gaps. Push the handle of the forklift downwards to lift the forks, and the breadboard, upwards. The longer side of the breadboard will be perpendicular to the fork. To ensure better stability during delivery, and allow easier access through narrow doors and hallways, carefully rotate the breadboard on the fork. One of the eight individuals has to hold the fork lift in place to ensure the bread board does not slip off the fork. Drive the fork lift to the entrance of the microscope room ensuring someone holds the doors open. 15. Push the breadboard to slip it off the fork and allow its longer edge to touch the ground. Lift the breadboard from the other longer edge upwards until the breadboard sits perpendicular to the ground. 16. Slide the breadboard (item 64) into the microscope room parallel to the installed frame (item 63) but at least 2 m away. Two of the eight individuals should tilt the breadboard such that it is almost level with the ground, but ensuring a small gap remains along the long edge. At this point, two other individuals should lift the breadboard from each corner of the other long edge touching the ground. 17. Lift the breadboard and walk sideways to bring it on top of the installed frame. Carefully bring the level breadboard down and closer to the top of the frame taking care not to trap hands and fingers between the breadboard and the frame. Rest the breadboard on the frame. 18. The 4 passive isolators (https://www.thorlabs.com/thorproduct.cfm?partnumber=PWA075) pre-installed on the frame, and on which the breadboard rests, have to be inflated. To inflate the isolators, bring the foot pump (item 65) close to one of the isolators. Loosen the black cap on the needle of the intended isolator. Press the clip on the hose of the foot pump and insert the hose into the needle (as shown in figure 1.3 of the following manual. https://www.thorlabs.com/drawings/1879b4d9f8b482d1-011CC6B6- 00C0-0936-C626E65B55115564/PWA075-Manual.pdf). Inflate the isolator by pressing on the foot pump until it is correctly inflated (as shown in figure 1.4 of the following manual. https://www.thorlabs.com/drawings/1879b4d9f8b482d1-011CC6B6-00C0-0936- C626E65B55115564/PWA075-Manual.pdf). Press the clip and quickly release the hose off the needle. Screw the black cap on the needle. 19. Repeat point 18 with the remaining three passive isolators. 20. All panels comprising the wooden pallets in which the optical table were delivered, should be stowed, or discarded, as advised by the building administrator. Caution: unremoved screws or sharp objects protruding off the wooden pallet should be removed or bent inwards to prevent serious injury. 21. Unpack the remaining components delivered beforehand and place them in a large space (e.g. ground of the microscope room) in numerical order (see Materials). Step 4: assembling the laser combiner (timing: 3 hours) Critical step: the individual performing the assembly process is requested to stand facing the longer edge of the breadboard (item 64). Wherever there is reference to the front side, this will be the long side closest to the individual performing the assembly process; wherever there is reference to the back side, this will be the long side furthest from the individual performing the assembly process. Critical step: for all items that need to be screwed to the breadboard, the screwing position is indicated by a two-coordinate label (X,Y) – where X refers to the position along the longer side of the breadboard and Y refers to the position along the shorter side of the breadboard. The position (1,1) is the thread to the front / right hand side. Moving to the left increases the X position. Moving to the back increase the Y position. 22. Screw an M6 x 20 mm set screw (item 101) to the lower side of the 50 mm long, 25 mm wide pedestal pillar post (item 69) (Figure 1, supplementary visual assembly guide). Repeat the same for the remaining 11 pedestal pillar posts. 23. Screw the (12) assemblies formed in the previous point to the breadboard at positions (7,3), (7,6), (11,3), (11,6), (7,13), (7,16), (11,13), (11,16), (7,18), (7,21), (11,18) and (11, 21) (Figure 2, supplementary visual assembly guide). 24. Repeat point 22 with the four 38 mm long, 25 mm wide pedestal pillar posts (item 70) and screw them to the breadboard at positions (3,8), (3,11), (11,8) and (11,11) (Figure 3, supplementary visual assembly guide). 25. Take one of the three small laser plates (item 129) and place it over the closest set of four 50 mm long, 25 mm wide, breadboard-screwed pedestal pillar posts from the front side. Screw the laser plate to the posts using four M6 x 12 mm cap screws (item 101). Tighten the screws using the 5 mm L-shaped hex key with green-coloured band (item 99) (Figure 4, supplementary visual assembly guide). Repeat the same for the remaining two small laser plates screwing them to the second and third closest sets of four 50 mm long, 25 mm wide, breadboard-screwed pedestal pillar posts from the front side (Figure 5, supplementary visual assembly guide). 26. Take the large laser plate (item 129) and place it over the closest set of four 38 mm long, 25 mm wide, breadboard-screwed pedestal pillar posts from the front side. Screw the laser plate to the posts using four M6 x 16 mm cap screws (item 101). Tighten the screws using the 5 mm L-shaped hex key with green-coloured band. 27. The ultra violet (405 nm), blue (488 nm) and red (638 nm) lasers (items 5, 6 and 7 respectively) are packaged into two separate components; a laser head and an electronic circuit board. Connect the male green terminal wired to one of the laser heads to the female green terminal, labelled LD+, LD-, NTC, NTC, TEC+ and TEC-, on the accompanying electronic circuit board (Figure 6, supplementary visual assembly guide). Repeat the same for the remaining 2 lasers. Critical step: do not randomly connect any laser head with any electronic circuit board. The circuit boards are configured to work with the accompanying lasers. Only connect laser heads to electronic circuit boards from the same package. 28. Slot the board-connected ultra violet (405 nm) laser head into the central groove of the furthest small laser plate from the front side. Screw the laser head to the laser plate using two M5 x 12 mm cap screws (item 126). Tighten the screws using the 4 mm L-shaped hex key with cyan-coloured band (item 99) (Figure 7, supplementary visual assembly guide). Place the connected electronic circuit board on top of the four 3 mm threads on the laser plate next to the recently fixed laser head. Secure the electronic circuit board using four M3 x 12 mm cap screws (item 126). Fix the screws using the 2 mm L- shaped hex key with orange-coloured band (item 99) (Figure 8, supplementary visual assembly guide). Repeat the same with the blue (488 nm) and red (638 nm) lasers, placing them on the second closest and closest small laser plates from the front side, respectively. 29. The green (561 nm) laser (items 3, 4) is packaged into two separate components; a large laser head and a large control box. Slot the laser head into the central groove of the large laser plate. Secure the laser head using four M4 x 25 mm cap screws (item 126). Tighten the screws using the 3 mm L-shaped hex key with blue-coloured band (item 99) (Figure 9, supplementary visual assembly guide). Note that the laser head will be connected to the control box in point 172. 30. Slot an M6 x 16 mm cap screw into a 50 mm post holder (item 74) (Figure 10, supplementary visual assembly guide). Screw, and tighten, the cap screw into the thread on the post holder using the 5 mm L-shaped hex key with green-coloured band (Figure 11, supplementary visual assembly guide). Repeat the same for another seven 50 mm post holders. Screw the eight post holders onto the breadboard at positions (16,4), (16,9), (16,14), (16,19), (14,7), (14,12), (14,17) and (14,22) (Figure 12, supplementary visual assembly guide). 31. Screw a 50 mm long, 12.7 mm wide optical post (item 72) to a compact kinematic mirror mount (item 83) (Figure 13, supplementary visual assembly guide). Repeat the same for the remaining seven 50 mm long, 12.7 mm optical posts and compact kinematic mirror mounts. 32. Dismantle the mirror holder (item 84) by holding the protruding screw and loosening the outer ring (Figure 14, supplementary visual assembly guide). Insert a 400-750 nm mirror (item 32) into the groove and screw the outer ring to secure in place (Figure 15, supplementary visual assembly guide). Repeat the same for the remaining seven 400-750 nm mirror holders and mirrors. Caution: handle all optical components carefully to avoid fingerprints or scratches and make sure no lasers are connected, or turned on, for the following steps. 33. Screw one of the assemblies formed in point 32 to one of the assemblies formed in point 31 (Figure 16, supplementary visual assembly guide). Repeat the same for the remaining 14 assemblies formed in points 31 and 32. 34. Slot one of the assemblies formed in point 33 to one of the post holders added to the microscope in point 30. Repeat the same for the remaining seven assemblies and seven post holders (Figure 17, supplementary visual assembly guide). 35. Slot an M6 x 16 mm cap screw into a 50 mm post holder. Screw, and tighten, the cap screw into the thread on the post holder using the 5 mm L-shaped hex key with green-coloured band. Repeat the same for another three 50 mm post holders. Screw the four post holders onto the breadboard at positions (18,6), (18,11), (18,16) and (18,21). 36. Screw a 30 mm long, 12.7 mm wide optical post (item 73) to a kinematic mirror mount (item 79) (Figure 18, supplementary visual assembly guide). Repeat the same for the remaining three optical posts and kinematic mirror mounts. 37. Loosen the set screw on the groove of a kinematic mirror mount assembly formed in point 36 using the 2 mm L-shaped hex key with yellow-coloured band (item 99). Insert the 805 nm long pass dichroic mirror (item 30) in the groove of the kinematic mirror mount and screw the set screw to fix the mirror in place (Figure 19, supplementary visual assembly guide). Ensure that the non-reflective surface of the dichroic mirror is facing in the direction of the two black capped knobs on the kinematic mirror mount. The reflective surface can be identified by looking on both sides of the dichroic mirror at an angle (check the following website for more information: https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3313). Repeat the same for the 425 nm, 505 nm and 605 nm dichroic mirrors (items 27, 28, 29, respectively) and the remaining three assemblies formed in point 36. Note which of the assemblies holds the 425 nm, 505 nm, 605 nm and 805 nm dichroic mirrors. 38. Slot the 425 nm, 505 nm, 605 nm and 825 nm dichroic mirror holding assemblies formed in point 37, in order starting from the furthest and working towards the front, into the breadboard-screwed post holders added in point 35 (Figure 20, supplementary visual assembly guide). 39. Loosen the four set screws on the cage plate of the fibre launch system (item 66) using the 2 mm L- shaped hex key with yellow-coloured band to remove it (Figure 21, supplementary visual assembly guide). Loosen and remove the four rods screwed to the XY translation mount of the fibre launch system (Figure 22, supplementary visual assembly guide). 40. Loosen the four set screws on the sides of the Z translation mount fixed to the fibre launch system using the 1.3 mm L-shaped hex key with orange-coloured band (item 99) to remove it (Figure 23, supplementary visual assembly guide). This results in two components, an XY translation mount and a Z translation mount. 41. Loosen and remove the SM1 ring inside the XY translation mount using the SM1 spanner wrench (item 98) (Figure 24 and 25, supplementary visual assembly guide). 42. Screw the aspheric lens (item 36) to the extended RMS adaptor delivered with the fibre launch system (Figure 26, supplementary visual assembly guide). 43. Screw the RMS adaptor delivered with the fibre launch system to the assembly formed in point 42 (Figure 27, supplementary visual assembly guide). 44. Screw the assembly formed in point 43 to the XY translation mount. Ensure the protruding part of the assembly formed in point 43 is pointing towards the rods fixed on the XY translation mount. Screw the SM1 ring on top of the assembly formed in point 43 by hand, followed by the SM1 spanner wrench, to secure it in place (Figure 28, supplementary visual assembly guide). 45. Loosen, and remove, the SM1 ring inside the Z translation mount using the SM1 spanner wrench (Figure 29, supplementary visual assembly guide). 46. Screw the FC/PC fibre adaptor plate delivered with the fibre launch system to the Z translation mount using the adjustable spanner wrench delivered with the fibre launch system (Figure 30, supplementary visual assembly guide). Ensure the protruding part of the FC/PC fibre adaptor plate is pointing in the same direction as the knob of the Z translation mount. Screw the SM1 ring removed in point 45 to the Z translation mount using the SM1 spanner wrench (Figure 31, supplementary visual assembly guide). 47. Slide the Z translation mount onto the rods fixed to the XY translation mount. Ensure the knob on the Z translation mount is pointing in the opposite direction to the XY translation mount (Figure 32, supplementary visual assembly guide). 48. Screw the assembly formed in point 47 to the breadboard using four M6 x 20 mm cap screws at positions (17,24), (18,23), (18,25) and (19,24). Tighten the screws using the 5 mm L-shaped hex key with green-coloured band (Figure 33, supplementary visual assembly guide). 49. Remove the black cap on one end of the fibre cable (item 1). Insert the bare end of the fibre end to the protruding part on the Z translation mount formed in point 47. Ensure the fibre is properly inserted into the protruding part by aligning the notch on the bare end of the fibre with the groove on the protruding part of the Z translation mount (Figure 34, supplementary visual assembly guide). Screw and hand- tighten the female metallic barrel on the bare end of the fibre cable with the male thread on the protruding part of the Z translation mount. Critical step: avoid scratching or touching the bare end of the fibre as this may damage the core. Avoid bending the fibre at small angles during operation. Do not screw the metallic barrel before aligning the notch on the bare end of the fibre with the groove on the protruding part of the Z translation mount to avoid damage to the fibre. Step 5: first partial assembly of the microscope box (timing: 1 hour) Critical step: it is critically important that all screws used in points 50 to 200 are strongly tightened (unless otherwise stated) to guarantee high stability of the instrument during imaging. 50. Take the bottom plate of the microscope box on which the number ‘4’ is engraved (item 132) and place it somewhere empty on the breadboard (item 64) with the engraved number facing upwards and towards the front. Insert a 6 mm dowel in one of the side holes on the top surface of the plate (Figure 35, supplementary visual assembly guide). Repeat with the three other side holes on the top surface of the plate (Figure 36, supplementary visual assembly guide). 51. Take the side plate of the microscope box on which the number ‘3’ is engraved (item 132) and seat it upright over the two dowels on the right-hand side of the bottom plate (Figure 37, supplementary visual assembly guide). Ensure the engraved number is at the bottom / front corner facing left. 52. Take the side plate of the microscope box on which the number ‘2’ is engraved (item 132) and seat it over the two dowels on the left-hand side of the bottom plate (Figure 38, supplementary visual assembly guide). Ensure the engraved number is at the bottom / front corner facing right. 53. Flip the assembly formed in point 52 backwards at 90 degrees so the underside of the bottom plate now faces the front. 54. Screw an M6 x 20 mm cap screw in one of the side counter bores on the now front-facing surface of the bottom plate. Tighten the screw using the 5 mm L-shaped hex key with green-coloured band (item 99) (Figure 39, supplementary visual assembly guide). Repeat the same for all the five remaining side counter bores on the front-facing surface of the bottom plate (Figure 40, supplementary visual assembly guide). 55. Flip the assembly formed in point 54 forwards at 90 degrees so the bottom plate now faces downwards again. Ensure the bottom plate is correctly positioned on the breadboard so that the four central counter bores on the top surface of the plate are perfectly aligned with the breadboard at positions (34,4), (34,8), (41,4) and (41,8). 56. Screw an M6 x 20 mm cap screw in one of the central counter bores at the top surface of the bottom plate (Figure 41, supplementary visual assembly guide). Tighten the screw using the 5 mm L- shaped hex key with green-coloured band. Repeat the same for the remaining three central counter bores at the top surface of the bottom plate. Step 6: assembling the excitation module and focus stabilization system (timing: 2 hours) 57. Remove the plastic stickers wrapped around four of the rod adaptors (item 78). Screw the four rod adaptors to the four threads on the right-facing surface on the right-hand side plate. 58. Strongly tighten the plate-fixed rod adaptors by inserting the 1.5 mm L-shaped hex key with purple- coloured band (item 99) through the two holes of one adaptor and firmly turning (Figure 42, supplementary visual assembly guide). Repeat the same for the remaining three rod adaptors. 59. Loosen the small screw on one side of a cage cube with dichroic filter mount (item 81) using the 1.5 mm L-shaped hex key with purple-coloured band (Figure 43, supplementary visual assembly guide). Repeat the same with the other small screw on the adjacent side of the cage cube with dichroic filter mount. Place the two screws on a flat surface with their heads facing downwards. 60. Pull apart the mounting base and cage cube. Caution: point 61 requires two individuals. Wear gloves before handling optical components. 61. Press the two clips on the mounting base and gently insert the 700 nm short pass dichroic beam splitter (item 26) with the engraved ‘Semrock’ logo facing the ‘THORLABS’ logo printed on the mounting base (Figure 44, supplementary visual assembly guide). Gently remove your fingers off the clips, and off the dichroic beam splitter, to secure it in place. 62. Place the mounting base into the cage cube. Ensure the two white dots on the mounting base are aligned with the two white dots on the cage cube to ensure correct insertion (Figure 45, supplementary visual assembly guide). Hold the inserted mounting base and cage cube tight. 63. Screw the two small screws removed in point 59 to the cage cube. Tighten the screws using the 1.5 mm L-shaped hex key with purple-coloured band. 64. Take four 0.25” rods (item 77). The rods are delivered with a set screw mounted on each end. For one of the four rods, loosen only one set screw using the 1.3 mm L-shaped hex key with orange-coloured band (item 99) (Figure 46, supplementary visual assembly guide). Given the small size of the rod, you might need to wear a latex glove, if available, to grip whilst loosening the set screw. Repeat the same for the other three rods. 65. Screw one of the rods to one of the corner threads on a specific side of the cage cube with dichroic filter mount. To find this correct side, hold the cage cube with dichroic filter mount with your left hand, ensure the protruding part is pointing downwards and the dichroic beam splitter inside the assembly is pointing towards the left and front sides (Figure 47, supplementary visual assembly guide). Repeat the same with the other three rods, screwing them to the other three corner threads on the same side of the cage cube with dichroic filter mount. 66. Insert the four rods now attached to the cage cube with dichroic filter mount into the four rod adaptors on the right-hand side plate (Figure 48, supplementary visual assembly guide). Ensure proper insertion by first loosening all the set screws on all four rod adaptors using the 2 mm L-shaped hex key with yellow-coloured band (item 99), second inserting the cage cube with dichroic filter mount, and third tightening all the eight set screws on all the four rod adaptors using the 2 mm L-shaped hex key with yellow-coloured band to firmly secure the assembly in place. 67. Take four 0.5” rods (item 76). Repeat point 64 with these four rods. 68. Screw the four rods to the four corner threads on the right-hand side of newly mounted cage cube with dichroic filter mount (Figure 49, supplementary visual assembly guide). 69. Take a right-angle kinematic mirror mount (item 80) and loosen the set screw on the side of the diagonal face using the 2 mm L-shaped hex key with yellow-coloured band. Insert a 400-750 nm mirror (item 32) into the groove on the diagonal face of the right-angle kinematic mirror mount. Ensure the frosted side of the mirror (i.e. the one with the ‘THORLABS’ logo) is facing in the same direction as the two silver knobs on the right-angle kinematic mirror mount. Carefully tighten the set screw using the 2 mm L-shaped hex key with yellow-coloured band (Figure 50, supplementary visual assembly guide). 70. Loosen all eight set screws on the two triangular sides of the right-angle kinematic mirror mount using the 2 mm L-shaped hex key with yellow-coloured band (Figure 51, supplementary visual assembly guide). 71. Slide the right-angle kinematic mirror mount into the four 0.5” rods on the right-hand side of the cage cube with dichroic filter mount (Figure 52, supplementary visual assembly guide). Ensure the frosted side of the mirror on the right-angle kinematic mirror mount is facing downwards. Tighten all the set screws on the mirror mount assembly using the 2 mm L-shaped hex key with yellow-coloured band to secure it in place. 72. Screw the rotation mount (item 85) into the SM1 thread in the centre of the top side of the right-angle kinematic mirror mount (Figure 53, supplementary visual assembly guide). 73. Loosen and remove the SM1 ring threaded inside the rotation mount using the SM1 spanner wrench (item 98). Insert the 390/482/564/640 nm band pass filter (item 21) into the groove wherefrom the SM1 ring was removed. Screw and tighten the removed SM1 ring into the SM1 thread inside the rotation mount first by hand, then using the SM1 spanner wrench (Figure 54, supplementary visual assembly guide). 74. Screw the fibre collimator (item 2) into the SM1 thread on the rotation mount (Figure 55, supplementary visual assembly guide). 75. Remove the black cap on the other end of the fibre cable (item 1). Insert the bare end to the protruding part on the fibre collimator. Ensure the fibre cable is properly inserted into the protruding part by aligning the notch on the bare end of the fibre cable with the groove on the protruding part of the fibre collimator (Figure 56, supplementary visual assembly guide). Screw and hand-tighten the female metallic barrel on the bare end of the fibre cable with the male screw on the protruding part of the fibre collimator. 76. Take four 0.5” rods. Repeat point 64 with these four rods. 77. Screw the four rods to the four corner threads on the back side of the cage cube with dichroic filter mount. 78. Take a right-angle kinematic mirror mount and loosen the set screw on the side of the diagonal face using the 2 mm L-shaped hex key with yellow-coloured band. Insert a 750-1100 nm mirror (item 33) into the groove on the diagonal face of the right-angle kinematic mirror mount. Ensure the frosted side of the mirror is facing in the same direction as the two silver knobs on the right-angle kinematic mirror mount. Finger-tighten the set screw using the 2 mm L-shaped hex key with yellow-coloured band. 79. Loosen all eight set screws on the two triangular sides of the right-angle kinematic mirror mount using the 2 mm L-shaped hex key with yellow-coloured band. 80. Slide the right-angle kinematic mirror mount into the four 0.5” rods on the back side of the cage cube with dichroic filter mount (Figure 57, supplementary visual assembly guide). Ensure the frosted side of the mirror on the right-angle kinematic mirror mount is facing backwards. Finger-tighten the set screws on the top and bottom sides of the right-angle kinematic mirror mount closest to the front side using the 2 mm L-shaped hex key with yellow-coloured band to secure in place. 81. Take four 0.5 in rods. Repeat point 64 with these four rods. 82. Hold the non-polarising beam splitter (item 38) so that the two arrows printed on the splitter are pointing towards the left and front sides. Screw the four rods to the four corner threads on the left-hand side of the non-polarising beam splitter. 83. Insert the four rods attached to the non-polarising beam splitter into the four counter bores on the right- hand side of the right-angle kinematic mirror mount (Figure 58, supplementary visual assembly guide). Tighten the four set screws on the top and bottom sides of the right-angle kinematic mirror mount closest to the right-hand side using the 2 mm L-shaped hex key with yellow-coloured band to secure in place. 84. Loosen and remove one of the two rings screwed to the SM1 coupler (item 88) (Figure 59, supplementary visual assembly guide). 85. Screw the SM1 coupler to the SM1 thread on the back side of the non-polarising beam splitter (Figure 60, supplementary visual assembly guide). Tighten the ring on the SM1 coupler to secure the coupler to the non-polarising beam splitter. 86. Take a rotation mount. Loosen and remove the SM1 ring threaded in the rotation mount using the SM1 spanner wrench. Insert the neutral density filter (item 37) into the groove wherefrom the SM1 ring was removed (Figure 61, supplementary visual assembly guide). Screw and tighten the removed SM1 ring in the SM1 thread inside the rotation mount first by hand, then using the SM1 spanner wrench. 87. Screw and tighten the rotation mount to the SM1 coupler. 88. Screw the compact diode laser (item 8) to the rotation mount (Figure 62, supplementary visual assembly guide). Rotate the laser so that the engraved word ‘CLOSED’ faces upwards. Tighten the set screw on the rotation mount using the 2 mm L-shaped hex key with yellow-coloured band to secure in place. 89. Remove the circular transparent plastic film on the active region of the lateral effect position sensor (item 10). 90. Screw the SM05-to-SM1 adaptor (item 91) to the lateral effect position sensor (Figure 63, supplementary visual assembly guide). 91. Loosen and remove the SM1 ring threaded inside a 0.3” SM1 lens tube (item 94) using the SM1 spanner wrench. Insert the 700 nm long pass filter (item 31) into the groove of the SM1 lens tube with the arrow on the outer edge of the filter pointing away from the groove (Figure 64, supplementary visual assembly guide). Screw and tighten the removed SM1 ring to the SM1 lens tube first by hand then using the SM1 spanner wrench. 92. Screw the SM1 lens tube to the SM1 thread on the right-hand side of the non-polarising beam splitter (Figure 65, supplementary visual assembly guide). Screw the 25.4 mm focal length Plano convex lens (item 145) to the SM1 lens tube. Loosen and remove the SM1 ring threaded inside a 1” SM1 lens tube (item 146) using the SM1 spanner wrench. Screw the 1” SM1 lens tube to the lens tube mounting the Plano convex lens. 93. Screw an SM1 coupler to the SM1 lens tube. Tighten the ring on the left-hand side of the SM1 coupler to secure it in place (Figure 66, supplementary visual assembly guide). 94. Screw the lateral effect position sensor assembly to the SM1 coupler. Rotate the lateral effect position sensor through two full turns until the protruding wire is pointing upwards (Figure 67, supplementary visual assembly guide). Rotate and firmly tighten the ring on the right-hand side of the SM1 coupler to secure the lateral effect position sensor in place. Step 7: second partial assembly of the microscope box (timing: 1 hour) 95. Take the top plate of the microscope box on which the number ‘1’ is engraved (item 132) and place it somewhere empty on the breadboard (item 64) with the engraved number facing upwards and towards the front. 96. Take four 0.5” rods (item 76). Repeat point 64 with four these rods. 97. Screw a rod to one of the threads at the centre of the plate (Figure 68, supplementary visual assembly guide). Repeat the same for the remaining three threads. 98. Loosen the small screw on one side of a cage cube with dichroic filter mount (item 81) using the 1.5 mm L-shaped hex key with purple-coloured band (Figure 43, supplementary visual assembly guide). Repeat the same with the other small screw on the adjacent side of the cage cube with dichroic filter mount. Place the two screws on a flat surface with their heads facing downwards. 99. Pull apart the mounting base and cage cube. Caution: point 100 requires two individuals. 100. Press the two clips on the mounting base and gently insert the 405/488/561/635/800-1050 nm super resolution dichroic beam splitter (item 20) with the engraved dot mark facing the ‘THORLABS’ logo printed on the mounting base (Figure 69, supplementary visual assembly guide). Gently remove your fingers off the clips, and off the dichroic beam splitter, to secure the dichroic beam splitter in place. 101. Place the mounting base into the cage cube. Ensure the two white dots on the mounting base are aligned with the two white dots on the cage cube to ensure correct insertion. Hold the inserted mounting base and cage cube tight. 102. Screw the two small screws removed in point 98 to the cage cube. Tighten the screws using the 1.5 mm L-shaped hex key with purple-coloured band. 103. Hold the cage cube with dichroic filter mount with your left hand with the dichroic beam splitter contained within facing upwards and to the left-hand side. 104. Remove the plastic stickers wrapped around four of the rod adaptors (item 78). Screw the four rod adaptors to the four corner threads on the top side of the cage cube with dichroic filter mount. Strongly tighten the rod adaptors by inserting the 1.5 mm L-shaped hex key with purple-coloured band through the two holes of one of the four adaptor and firmly turning. Repeat the same for the remaining three rod adaptors. 105. Loosen all the set screws on the rod adaptors using the 2 mm L-shaped hex key with yellow- coloured band (item 99) (Figure 70, supplementary visual assembly guide). Slide the four rod adaptors on the cage cube with dichroic filter mount onto the four rods attached to the top plate (Figure 71, supplementary visual assembly guide). Ensure full insertion of the rods into the rod adaptors. Tighten the set screws on the rod adaptors using the 2 mm L-shaped hex key with yellow-coloured band. 106. Screw the achromatic lens (item 34) to the SM1 thread on the left side of the cage cube with dichroic filter mount (Figure 72, supplementary visual assembly guide). 107. Screw an SM1 end cap (item 87) to the SM1 thread on the right side of the cage cube with dichroic filter mount (Figure 73, supplementary visual assembly guide). 108. Take four 0.5” rods. Repeat point 64 with these four rods. 109. Screw the four rods to the four corner threads on the top side of the cage cube with dichroic filter mount (Figure 74, supplementary visual assembly guide). 110. Take a right-angle kinematic mirror mount (item 80). Loosen all the set screws on the right-angle using the 2 mm L-shaped hex key with yellow-coloured band. Slide the right-angle kinematic mirror mount onto the four rods on the top side of the cage cube with dichroic filter mount (Figure 75, supplementary visual assembly guide). Ensure the two silver knobs on the right-angle kinematic mirror mount are pointing upwards and towards the front. Tighten the four set screws on the two triangular faces at the bottom using the 2 mm L-shaped hex key with yellow-coloured band to secure it in place. 111. Insert a 400-750 nm mirror (item 32) into the groove on the diagonal face of the right-angle kinematic mirror mount. Ensure the frosted side of the mirror (i.e. the one with the ‘THORLABS’ logo) is facing in the same direction as the two silver knobs on the right-angle kinematic mirror mount (Figure 76, supplementary visual assembly guide). Tighten the set screw on the diagonal face of the right- angle kinematic mirror mount using the 2 mm L-shaped hex key with yellow-coloured band. 112. Loosen and remove the SM1 ring threaded into a 0.3” SM1 lens tube using the SM1 spanner wrench (item 98). Insert the 446/523/600/677 nm band pass filter (item 22) into the groove of the SM1 lens tube. Screw and tighten the removed SM1 ring in the SM1 lens tube first by hand, then using the SM1 spanner wrench. 113. Screw the SM1 lens tube into the SM1 thread on the back side of the right-angle kinematic mirror mount. 114. Insert four 6 mm dowels (item 116) in the holes on the top edges of the left and right side microscope box plates (Figure 77, supplementary visual assembly guide). 115. Carefully flip the top microscope box plate upside down and place on top of the four dowels (Figure 78, supplementary visual assembly guide). Gently tap, using your hands, on the plate until fully secure. 116. Insert six M6 x 20 mm cap screws (item 101) in the counter bores on the top face of the top microscope box plate. Tighten the screws using the 5 mm L-shaped hex key with green-coloured band (item 99) (Figure 79, supplementary visual assembly guide). 117. Take the back plate of the microscope box on which the number ‘5’ is engraved (item 132) and place it somewhere empty on the breadboard with the engraved number facing upwards and towards the front. 118. Take four 0.25” rods. Repeat point 64 with these four rods. 119. Screw the four rods to the four threads on the top side of the back plate (Figure 80, supplementary visual assembly guide). 120. Take the four-position slider (item 67) and orient it so that the black slider is facing upwards and the connector on the red electronic board is facing the front. Roughly align the four silver threads on the red electronic board on top of the four rods screwed to the back plate. Slide the black slider to the left so that the two rightmost silver threads on the red electronic board are accessible. Screw two 4-40 x 3/16” cap screws (item 100) to the two rightmost silver threads. Finger-tighten the screws using the Hex 3/32” L-shaped hex key (item 100) (Figure 81, supplementary visual assembly guide). Slide the black slider to the right so that the two leftmost silver threads on the red electronic board are accessible. Screw two 4-40 x 3/16” cap screws to the two leftmost silver threads. Tighten the screws using the Hex 3/32” L-shaped hex key. 121. Loosen and remove the SM1 rings threaded in three 0.3” SM1 lens tubes using the SM1 spanner wrench. Insert the 525/30 nm, 600/37 nm and 676/29 nm band bass filters (items 23, 24 and 25, respectively) into the grooves of the three SM1 lens tubes. Screw and tighten the removed SM1 rings inside the three SM1 lens tubes first by hand, then using the SM1 spanner wrench. 122. Screw the SM1 lens tubes containing the 525/30 nm, 600/37 nm and 676/29 nm band pass filters to the second, third and fourth SM1 threads (counting from the left-hand side) on the four-position slider (Figure 82, supplementary visual assembly guide). 123. Plug the ribbon cable into the off-white connector on the red electronic board (Figure 83, supplementary visual assembly guide). 124. Insert four 6 mm dowels in the holes on the back edges of the top and bottom plates of the microscope box. 125. Slide the back plate onto the four dowels with the four-position slider facing the front. Ensure the ribbon protruding from the four-position slider is inserted into the thin groove formed between the bottom and right plates of the microscope box (Figure 84, supplementary visual assembly guide). Ensure the holes on the back plate are aligned with the dowels. Gently tap, using your hands, on the back plate until fully secure. 126. Insert ten M6 x 20 mm cap screws in the counter bores on the back side of the back plate. Tighten the screws using the 5 mm L-shaped hex key with green-coloured band. Step 8: assembling the emission module (timing: 1 hour) 127. Take four 0.5” rods (item 76). Repeat point 64 with these four rods. 128. Screw the four rods to the four threads on the back side of back plate (Figure 85, supplementary visual assembly guide). 129. Loosen all the set screws on the sides of a cage plate (item 82) using the 2 mm L-shaped hex key with yellow-coloured band (item 99). Slide the cage plate onto the four rods on the back plate (Figure 86, supplementary visual assembly guide). Tighten the screws using the 2 mm L-shaped hex key with yellow-coloured band. 130. Loosen and remove the two SM1 rings threaded to the cage plate using the SM1 spanner wrench. Screw an SM1-to-SM2 adaptor (item 89) to the cage plate (Figure 87, supplementary visual assembly guide). 131. Screw the tube lens (item 35) to the SM1-to-SM2 adaptor. Ensure the arrow printed on the tube lens is pointing towards the front (i.e. towards the microscope box) (Figure 88, supplementary visual assembly guide). 132. Screw an SM1-to-SM2 adaptor to the tube lens (Figure 89, supplementary visual assembly guide). 133. Loosen and remove the two SM1 rings screwed to the SM1 lens tube without external threads (item 95) using the SM1 spanner wrench. 134. Screw the SM1 lens tube without external threads to the SM1-to-SM2 adaptor (Figure 90, supplementary visual assembly guide). 135. Screw the SM1 zoom housing (item 86) to the SM1 lens tube without external threads (Figure 91, supplementary visual assembly guide). 136. Loosen and remove the SM1 ring threaded to a 2” SM1 lens tube (item 93) using the SM1 spanner wrench. 137. Screw the SM1 lens tube to the SM1 zoom housing (Figure 92, supplementary visual assembly guide). 138. Loosen and remove the SM1 ring threaded to a rotation mount (item 85) using the SM1 spanner wrench. 139. Screw the rotation mount to the SM1 lens tube (Figure 93, supplementary visual assembly guide). 140. Screw the SM1-to-C-mount adaptor to the rotation mount (Figure 94, supplementary visual assembly guide). Note that the two threads on the SM1-to-C-mount adaptor are visually similar and care should be taken to ensure that the correct (i.e. SM1) thread is fitted to avoid damage. 141. Remove the yellow plastic film on the active region of the sCMOS camera (item 14). 142. Screw the sCMOS camera to the SM1-to-C-mount adaptor (Figure 95, supplementary visual assembly guide). 143. Rotate the barrel on the SM1 zoom housing to adjust the distance between the active region of the sCMOS camera and the middle of the tube lens to 15 cm as measured by a ruler (Figure 96, supplementary visual assembly guide). 144. Tighten the set screw on the barrel of the SM1 zoom housing using the 1.3 mm L-shaped hex key with orange-coloured band (item 99) (Figure 97, supplementary visual assembly guide). 145. Loosely screw a stainless-steel screw (item 102) half-way through the thread on the smaller base of the 2” pedestal pillar post (item 71). 146. Rotate the sCMOS camera so that the power switch is on the top left corner. Screw the pedestal pillar post on the right side of the sCMOS camera (Figure 98, supplementary visual assembly guide). 147. Rotate the sCMOS camera so that the attached pedestal pillar post sits flat on the breadboard (item 64) (and the power switch is on the bottom left corner). 148. Slide the clamping fork (item 75) on the base of the pedestal pillar post. Insert an M6 x 20 mm cap screw into the groove of the clamping fork. Tighten the cap screw using the 5 mm L-shaped hex key with green-coloured band (item 99) (Figure 99, supplementary visual assembly guide). Step 9: assembling the sample stage (timing: 2 hours) 149. Insert two M4 dowels (item 117) in the holes on the top plate of the microscope box (Figure 100, supplementary visual assembly guide). 150. Take the positioner base (item 131) in one hand and hold it so that the counter bores are facing upwards. Insert an M1.6 x 20 cap screw (item 118) into one of the six counter bores on the back left corner of the top side of the positioner base (Figure 101, supplementary visual assembly guide). Take one of the three positioners (item 15) with the other hand and hold it so that the face of the sliding rail of the positioner is pointing downwards. Bring the positioner underneath the positioner base aligning the inserted screw with the thread on the back left corner of the top surface of the positioner. Tighten the cap screw into the thread using the T5 screwdriver (item 124) (Figure 102, supplementary visual assembly guide). Repeat the same with the remaining five counter bores on the back middle, back right, front left, front middle and front right corners of the positioner base (Figure 103, supplementary visual assembly guide). 151. Flip the positioner base upside down and slide it onto the two M4 dowels inserted in the top plate of the microscope box. Shuffle the positioner base whilst pushing down, to ensure its fully secure. Ensure the wire protruding from the positioner is pointing towards the left. Insert four M4 x 25 mm cap screws (item 126) into the counter bores on the top side of the positioner base (Figure 104, supplementary visual assembly guide). Tighten the cap screws to the top plate of the microscope box using the 3 mm L-shaped hex key with light blue-coloured band (item 99). 152. Take one of the two V-shaped elements (item 122) and orient it so that the protruding teeth are pointing to the right-hand side and downwards. Fix the V-shaped element to the fifth thread (counting from the left-hand side) on the sliding rail of the positioner using the supplied M2 cap screw (item 122). Take care not to over tighten the screw. Take the second of the three positioners and orient it so that the face of the sliding rail is pointing upwards and perpendicular to the first positioner, with its protruding wire pointing to the back. Insert the teeth protruding from the fixed V-shaped element into the third and fifth grooves on the left side of the base of the second positioner (Figure 105, supplementary visual assembly guide). Take the other V-shaped element and orient it so that it opposes the existing one. Fix the second V-shaped element to the fifth thread (counting from the right-hand side) on the sliding rail of the first positioner using the supplied M2 screw. Insert the teeth protruding from the second V- shaped element into the third and fifth grooves on the right side of the base of the second positioner (Figure 106, supplementary visual assembly guide). Tighten the two M2 screws using the 1.5 mm L- shaped hex key with purple-coloured band (item 99). 153. Take the planar connecting element (item 122) and orient it so that its longer base is upright and facing to the front and the shorter base is facing downwards, towards the two fixed positioners. Align the two holes on the shorter base of the planar connecting element with the second and third threads (counting from the front edge) on the sliding rail of the second positioner (Figure 107, supplementary visual assembly guide). Note that the odd-numbered holes are threaded and the even-numbered holes are dowel holes. Screw and tighten two of the supplied M2 cap screws through the shorter base of the planar connecting element and into the referred-to threads on the second positioner using the 1.5 mm L-shaped hex key with purple-coloured band (Figure 108, supplementary visual assembly guide). 154. Take the third positioner and orient it so that the face of sliding rail is pointing downwards. Take the rectangular connecting element (item 123) and orient it so that the wider base faces the top side of the positioner. Orient and place the rectangular connecting element so that it is flush with the top side of the positioner (Figure 109, supplementary visual assembly guide). 155. Insert one of the supplied M2 cap screws through the back right groove on the rectangular connecting element and into the thread on the back right corner of the third positioner. Tighten the cap screw using the 1.5 mm L-shaped hex key with purple-coloured band (Figure 110, supplementary visual assembly guide). Repeat the same with the remaining five cap screws, positioning them in the back middle, back left, front right, front middle and front left threads on the positioner (Figure 111, supplementary visual assembly guide). 156. Rotate and align the top of the rectangular connecting element with the longer base of the planar connecting element so that the third positioner now faces the front. Ensure the wire protruding from the positioner is pointing upwards. Align the first hole (counting from the top) of the longer base of the planar connecting element with the fourth thread (counting from the top) of the rectangular connecting element. 157. Insert one of the supplied M2 cap screws through the first hole (counting from the top) of the longer base of the planar connecting element. Tighten the screw into the fourth thread (counting from the top) rectangular connecting element using the 1.5 mm L-shaped hex key with purple-coloured band (Figure 112, supplementary visual assembly guide). Repeat the same with the remaining six M2 cap screws, inserting them through the six holes below the first hole (counting from the top) of the longer base of the planar connecting element (Figure 113, supplementary visual assembly guide). 158. Take four 0.5” rods (item 76). Repeat point 64 with these four rods. 159. Screw the four rods to the four threads on the top plate of the microscope box (Figure 114, supplementary visual assembly guide). 160. Loosen all the set screws on the sides of a cage plate (item 82) using the 2 mm L-shaped hex key with yellow-coloured band (item 99). Slide the cage plate onto the four rods. Tighten the set screws on the sides of the cage plate using the 2 mm L-shaped hex key with yellow-coloured band (Figure 115, supplementary visual assembly guide). 161. Loosen and remove the two SM1 rings threaded to the cage plate by hand or using the SM1 spanner wrench (item 98). 162. Screw the SM1-to-M25 adaptor (item 90) to the SM1 thread on the top side of the cage plate (Figure 116, supplementary visual assembly guide). 163. Screw the objective lens (item 19) to the M25 thread on the SM1-to-M25 adaptor (Figure 117, supplementary visual assembly guide). Step 10: setting up electrical connections (timing: 2 hours) Critical step: before carrying out points 164 to 185 ensure that all equipment is electrically tested as set by institutional or departmental regulations. Critical step: the following points will result in the installation of many cables. Ensure these cables are managed properly to keep the equipment tidy. You may want to use the black cable trunk (item 51) for this purpose. Critical step: ensure all components are turned off before carrying out points 164 to 185. 164. Place the power strip (item 50) at the back left corner of the breadboard (item 64) (Figure 118, supplementary visual assembly guide). Fix both ends of the power strip to the bread board using two M6 x 20 mm cap screws (item 101). Tighten the screws using the 5 mm L-shaped hex key with green- coloured band (item 99). 165. Loosen the six screws on the green terminal of the electronic circuit board attached to the ultra violet (405 nm) laser head (item 5) using the slotted screwdriver (item 125) (Figure 119, supplementary visual assembly guide). Repeat the same for the blue (488 nm) and red (638 nm) laser heads (items 6 and 7, respectively). 166. Insert the bare wires with red- and black-coloured ribbons (item 57) into the green terminals labelled ‘Vin+’ and ‘Vin-’ on the green terminal of the electronic circuit board attached to the ultra violet (405 nm) laser head (Figure 120, supplementary visual assembly guide). Tighten the two screws adjacent to the inserted wires using the slotted screwdriver to secure the wires in place. Connect the other end of the wires (i.e. female socket) to the male plug wired to the DC adaptor (item 57) (Figure 121, supplementary visual assembly guide). Connect the female socket of a power cable (item 60) to the male plug on the DC adaptor (Figure 122, supplementary visual assembly guide). Repeat the same for the blue (488 nm) and red (638 nm) laser heads. Connect the male plugs of the three power cables to the first, second and third (counting from the right-hand side) female sockets on the power strip. 167. Cut 2 m from the ribbon cable wheel (item 59) using a pair of scissors. Separate a group of six wires from the cut ribbon cable (Figure 123, supplementary visual assembly guide). 168. Separate 2 cm of each of the six wires from both ends (Figure 124, supplementary visual assembly guide). From one end of the cable, separate 1 m into three pairs of wires (Figure 125 and 126, supplementary visual assembly guide). 169. Remove 0.5 cm of the insulating material at the end of one wire using the wire stripper (item 115) (Figure 127, supplementary visual assembly guide). Repeat the same for the remaining lanes (Figure 128, supplementary visual assembly guide). 170. Insert the bare wires of a pair into the green terminals labelled ‘MOD+’ and ‘MOD-’ of the electronic circuit board attached to the ultra violet (405 nm) laser head (Figure 129, supplementary visual assembly guide). Tighten the two screws adjacent to the inserted wires using the slotted screwdriver to secure the wires in place. Repeat the same for the blue (488 nm) and red (638 nm) laser heads. Make note of the colour of each wire, the corresponding label ‘MOD+ / MOD-’ on each green terminal and corresponding laser head. This will be referred to as the connections table. 171. Cut 5 cm from the ribbon cable wheel using a pair of scissors. Separate three wires individually from the entire length of the cut ribbon cable. Remove 0.5 cm of the insulating material from both ends of each wire using the wire stripper. Insert the two bare wires of one of the three wires in the two green terminals labelled ‘INT’ of the electronic circuit board attached to the ultra violet (405 nm) laser head (Figure 130, supplementary visual assembly guide). Tighten the two screws adjacent to the inserted wires using the slotted screwdriver to secure the wires in place. Repeat the same for the blue (488 nm) and red (638 nm) laser heads. 172. Connect the female socket attached to the cable protruding from the green (561 nm) laser head (item 3) to the male plug labelled ‘LD&TEC’ on the laser control box (item 4) (Figure 131, supplementary visual assembly guide). Place the control box on the back right corner of the breadboard with the key pointing to the right side. Connect the female socket of a power cable to the male plug on the control box. Connect the male plug of the power cable to the fourth (counting from the right-hand side) female socket on the power strip. 173. Connect the male plug attached to the cable protruding from the lateral position sensor (item 10) to the female socket labelled ‘DETECTOR IN’ on the auto aligner (item 18) (Figure 132, supplementary visual assembly guide). Connect the non-USB male plug of the connection cable (item 18) to the female socket labelled ‘USB’ on the auto aligner (Figure 133, supplementary visual assembly guide). Tighten the two screws on the male plug to secure in place. Connect the male plug on the DC adaptor (item 18) with the female socket on the cord extension (item 18) (Figure 134, supplementary visual assembly guide). Connect the male plug on the cord extension to the female socket labelled ‘POWER’ on the auto aligner. Ensure the notch on the male plug is aligned with the groove on the female socket (Figure 135, supplementary visual assembly guide). Connect the female socket of a power cable to the male plug on the DC adaptor. Connect the male plug of the power cable to the fifth (counting from the right-hand side) female socket on the power strip. 174. Insert the key (item 8) in the lock on the back side of the compact laser diode (item 8) (Figure 136, supplementary visual assembly guide). Connect the male plug of the DC adaptor (item 18) to the female socket labelled ‘9V, 0.6A’ on the back side of the compact laser diode (Figure 137, supplementary visual assembly guide). Insert the appropriate continental plug into the power adaptor (Figure 138, supplementary visual assembly guide) and connect it to the sixth (counting from the right-hand side) female socket on the power strip. 175. Connect the female socket of the grey ribbon cable protruding from the right side of the microscope box to the male plug on the red control board (item 67) (Figure 139, supplementary visual assembly guide). Ensure the socket and plug are properly connected by firmly pushing them together. Connect the male plug of the DC adaptor (item 67) to the black female socket on the red control board (Figure 140, supplementary visual assembly guide). Connect the power plug of the DC adaptor to the seventh (counting from the right-hand side) female socket on the power strip. Connect the male micro- USB plug (item 67) into its corresponding female socket on the red control board (Figure 141, supplementary visual assembly guide). 176. Connect the male plug of the DC adaptor (item 14) to the female socket labelled ‘12V DC SA’ on the back side of the sCMOS camera (Figure 142, supplementary visual assembly guide). Connect the power plug of the DC adaptor to the eighth (counting from the right-hand side) female socket on the power strip. Connect one of the two male ends of the USB-C cable (item 67) to the female socket labelled ‘USB 3.2 GEN 2 / 10 Gbps’ on the back side of the sCMOS camera (Figure 143, supplementary visual assembly guide). 177. Connect the three male multi-pin plugs protruding from the first, second and third positioners (item 15) to the three female sockets on the MSC2 sensor module (item 16) labelled ‘CH1’, ‘CH2’ and ‘CH3’, respectively (Figure 144, supplementary visual assembly guide). Tighten the screws on the male multi-pin plugs to secure them in place. Connect the male multi-pin plug of the MSC2 sensor module to the female socket labelled with the hazard symbol on the MSC2 control system (item 17) (Figure 145, supplementary visual assembly guide). Connect the D-type male plug (item 17) into the female socket labelled ‘USB’ on the MSC2 control system (Figure 146, supplementary visual assembly guide). Connect the male plug of the DC adaptor (item 17) to the female socket on the MSC2 control system (Figure 147, supplementary visual assembly guide). Connect the power plug of the DC adaptor to the ninth (counting from the right-hand side) female socket on the power strip. 178. Cut and discard the test clips on the two BNC-to-test clip connectors (item 56) using a pair of scissors (Figure 148, supplementary visual assembly guide). Remove 0.5 cm of the insulating material at the end of each of the four cut wires using the wire stripper (Figure 149, supplementary visual assembly guide). Connect one female BNC socket to the BNC male plug of a coaxial cable (item 55) (Figure 150, supplementary visual assembly guide). Repeat the same for the remaining BNC female socket and coaxial cable. Connect the free BNC male plug on one coaxial cable to the BNC female socket on the control box of the green (561 nm) laser head (item 4) (Figure 151, supplementary visual assembly guide) and the free BNC male plug of the other remaining coaxial cable to the BNC female socket on the LED driver (item 54). 179. Connect the green male plug from the LED (item 9) to the female socket labelled ‘LED’ on the LED driver (item 54) (Figure 152, supplementary visual assembly guide). Connect the male plug of the DC adaptor (item 54) to the female socket labelled ‘DC 15V 1A’ on the LED driver (Figure 153, supplementary visual assembly guide). Connect the power plug of the DC adaptor to the tenth (counting from the right-hand side) female socket on the power strip. 180. Take the analog digital convertor device (item 44) and orient it so that the attached mini-USB female socket is pointing to the left and the board components are facing upwards. Loosen all the screws on the green terminals on the right-side of the device using the slotted screwdriver (Figure 154, supplementary visual assembly guide). 181. Refer to the connections table. Insert the other end of the wires connected to the terminals labelled ‘MOD-’ and ‘MOD+’ on the electronic circuit board of the ultra violet (405 nm) laser head to the first and second (counting from the back) green terminals on the analog digital converter device, respectively. Finger-tighten the screws on the green terminals adjacent to the inserted wires using the slotted screwdriver. Repeat the same for the other ends of the wires connected to the terminals labelled MOD- and MOD+ on the electronic circuit boards of the blue (488 nm) and red (638 nm) laser heads connecting them to the third, fourth, seventh and eighth (counting from the back) green terminals. Insert the ends of the red and black wires of the male BNC cable connected to the control box of the green (561 nm) laser head to the fifth and sixth (counting from the top) green terminals. Tighten the screws on the green terminals adjacent to the inserted wires using the slotted screwdriver (Figure 155, supplementary visual assembly guide). Connect the mini-USB male plug of one of the two USB connectors (item 48) to the mini-USB female plug on the analog digital converter device (Figure 156, supplementary visual assembly guide). 182. Connect the control hub (item 45) as follows: a. Connect one end of each of the two converter device cables (item 49) to the ports labelled ‘0’ and ‘1’ on the control hub (Figure 157, supplementary visual assembly guide). b. Connect the mini-USB male plug of the other USB connector (item 48) to the mini-USB female socket on the control hub (Figure 158, supplementary visual assembly guide). c. Using the converter device cables, connect the ports labelled ‘1’ and ‘0’ on the control hub to the constant DC power source device (item 46) and the variable DC power source device (item 47), respectively (Figure 159, supplementary visual assembly guide). d. Loosen all the screws on the green terminals on the constant and variable DC power source devices using the slotted screwdriver. e. Insert the ends of the red and black wires of the BNC-to-test clip connectors attached to the LED driver to the green terminals labelled ‘5V’ and ‘GND’ on the constant DC power source device, respectively (Figure 160, supplementary visual assembly guide). Tighten the adjacent screws using the slotted screwdriver to secure the wires in place. f. Cut 1 m from the ribbon cable wheel using a pair of scissors. Separate two wires from the entire length of the cut ribbon cable. Further separate 2 cm from the individual wires at both ends. Remove 0.5 cm of the insulating material at both ends of each wire using the wire stripper. Insert one end of the pair of wires to the green terminals, labelled ‘Vout’ and ‘GND’, on the variable DC power source device and tighten the screws adjacent to the inserted wires using the slotted screw driver to secure them in place. Loosen the screws on the white terminal strip (item 58). Insert the free end of the pair of wires to the two sockets on one side of the terminal strip (Figure 161, supplementary visual assembly guide). Tighten the screws adjacent to the inserted wires secure them in place. Insert the red and blue wires of the vibration motor (item 61) to the other side of the terminal strip (Figure 162, supplementary visual assembly guide). Tighten the screws adjacent to the inserted wires to secure them in place. Ensure, under this configuration that the red wire of the vibration motor is connected to the wire connected to the green terminal labelled ‘Vout’ on the variable DC power source device and the blue wire of the vibration motor is connected to the wire connected to the green terminal labelled ‘GND’. g. Mount the vibration motor on the fibre using the fibre and vibration motor mount (item 141). The mount comes in two pieces. Each piece contains three grooves; one in the centre and two on the sides. Take one piece and insert the vibration motor in the central groove and loop the fibre to insert it in the two side grooves. Secure this arrangement with your fingers. Take the remaining piece of the mount and place it on top of the vibration motor and fibre. Insert two M4 x 20 mm cap screws (item 126) into the side holes of the mount and secure it in place by finger- tightening two M4 washers on each cap screw. 183. Connect the male micro-USB plug of the micro-USB-to-USB-A cable (item 62) to the female socket on the control joystick (item 43) (Figure 163, supplementary visual assembly guide). 184. Connecting the USB hub (item 42): a. Connect the male plug of the DC adaptor (item 42) to the USB hub. b. Connect the power plug of the DC adaptor to the eleventh (counting from the right-hand side) female socket on the power strip. c. Connect the D-type male plug of the USB cable (item 42) to the D-type female socket on the USB hub (Figure 164, supplementary visual assembly guide). d. Connect the male USB plug of the USB cable to the USB C-to-USB-A adaptor (item 41) (Figure 165, supplementary visual assembly guide). e. Connect the male USB-C plug of the adaptor to one of the USB-C sockets on the laptop (item 41). f. Connect the other male USB-C plug connected to the sCMOS camera (item 14) to one of the free USB-C sockets on the laptop (item 41). g. Connect the male USB-C plug connected of the DC adaptor (item 14) to the free remaining USB-C socket on the laptop. h. Connect all the six USB-A plugs from the control joystick, control hub, analog digital converter device, MSC2 control system, lateral position sensor and red electronic board of the position slider to the USB hub (Figure 166, supplementary visual assembly guide). i. Connect the power plug of the DC adaptor to the twelfth (counting from the right-hand side) female socket on the power strip. 185. Connect the power plug of the power strip to the nearest wall socket. Step 11: assembling the laser combiner enclosure (timing: 30 minutes) 186. Screw an M6 x 20 mm set screw (item 101) to one end of a rail (item 130) (Figure 167, supplementary visual assembly guide). Repeat the same with the remaining three rails. Screw the four rails to the bread board (item 64) at positions (1,1), (1,29), (22,1) and (22,29) (Figure 168, supplementary visual assembly guide). Ensure without over-tightening that the rails are square with the breadboard (item 64). 187. Insert the four smallest PVC panels (item 130) into the grooves of the rails to form the sides of the enclosure. Ensure that the panel with the cut-out slot is inserted between the two rails at the back and that all the wires connected to the lasers are fitted through the slot. Place the largest PVC panel on top of the rails so that the holes on the panel are aligned with the threads on the rails. Fix the large top panel to the rails using four M6 clamping knobs (item 121) (Figure 169, supplementary visual assembly guide). Step 12: assembling the sample enclosure (timing: 2 hours) 188. Take the largest rectangular Perspex panel (item 133) with the semi-circular slot and place it along its longer edge on a flat surface. Take one of the two pentagon-shaped Perspex panels (item 133) and place it adjacent to the rectangular panel with the edge containing threads facing the counter bores on one side of the rectangular panel. Insert an M4 x 12 mm cap screw (item 126) into one of the three counter bores and tighten using the 2.5 mm L-shaped hex key with burgundy-coloured band (item 99) (Figure 170, supplementary visual assembly guide). Repeat the same with the two remaining threads along the same edge. Take the remaining pentagon-shaped Perspex panel and place it adjacent to the other edge with counter bores. Insert an M4 x 12 mm cap screw into one of the three counter bores and hand-tighten using the 2.5 mm L-shaped hex key with burgundy-coloured band. Repeat the same with the two remaining threads along the same edge (Figure 171, supplementary visual assembly guide). 189. Rotate the assembly formed in point 188 so that the rectangular panel is at the back. Place the narrow rectangular Perspex panel (item 133) containing only four counter bores at the front (Figure 172, supplementary visual assembly guide). Insert four M4 x 12 cap screws into the four counter bores and tighten them using the 2.5 mm L-shaped hex key with burgundy-coloured band. 190. Place the yet-unused narrow rectangular Perspex panel (item 133) containing several counter bores on top of the assembly (Figure 173, supplementary visual assembly guide). Insert five M4 x 12 cap screws into the five counter bores on the top face of the narrow rectangular panel and tighten using the 2.5 mm L-shaped hex key with burgundy-coloured band. Take one of the two hinges (item 106) and place it on top of the narrow rectangular panel so that two of the counter bores on the hinge are aligned with two of the four threads on the top face of the narrow rectangular panel. Screw a low-profile screw (item 106) into one of the counter bores of the hinge and tighten using the 2.5 mm L-shaped hex key with burgundy-coloured band (Figure 174, supplementary visual assembly guide). Repeat the same with the other counter bore. Repeat the same with the remaining hinge. 191. Take the large rectangular Perspex panel (item 133) without a hole and place it so that it lies flat on the surface with the counter bores facing down. Take the large rectangular Perspex panel with hole (item 133) and place it so that one of its longer edges is lying on the flat surface, the hole is in its lower- most position and the longer side with the three counter bores are aligned with the three edge threads on the other panel without a hole. Insert three M4 x 12 mm cap screws into the three counter bores on the longer side of the large rectangular panel with the hole and tighten using the 2.5 mm L-shaped hex key with burgundy-coloured band (Figure 175, supplementary visual assembly guide). Take the triangular Perspex panels (item 133) and place them so that their edge threads are aligned side counter bores on the new assembly. Insert six M4 x 12 mm cap screws into counter bores on the sides of one of the two rectangular panels and tighten using the 2.5 mm L-shaped hex key with burgundy-coloured band. Flip the assembly so that the remaining six free counter bores are accessible. Insert six M4 x 12 mm cap screws into the counter bores on the sides of the rectangle panel and tighten using the 2.5 mm L-shaped hex key with burgundy-coloured band. (Figure 176, supplementary visual assembly guide). 192. Flip the assembly formed in point 191 so that the large rectangular panel with a hole is lying on the flat surface. Take a handle (item 105) and place it against the threads on the large rectangular panel. Insert an M5 x 16 mm cap screw (item 126) into an M6 washer (item 101) and then into one of the counter bores on the handle and tighten using the 4 mm L-shaped hex key with cyan-coloured band (item 99) (Figure 177, supplementary visual assembly guide). Repeat the same with the other counter bore. 193. Align the assembly formed in point 192 with that formed in point 190 (Figure 178, supplementary visual assembly guide). Align the free four side threads on the assembly formed in point 192 with the 4 counter bores on the hinges of the assembly formed in point 190. Screw a low-profile screw into one of the two counter bores of one of the two hinges and tighten using the 2.5 mm L-shaped hex key with burgundy-coloured band (Figure 179, supplementary visual assembly guide). Repeat the same with the 3 remaining counter bores. Gently place the entire assembly (i.e. sample enclosure) on a flat surface 194. Take a steel angle bracket (item 119) and orient it so the side with the smaller hole is lying on the flat surface and the elongated hole is aligned with one of the four threads on the two opposite faces of the sample enclosure. Insert a M4 x 8 mm cap screw (item 126) into the elongated hole of the bracket and screw it into the adjacent thread (Figure 180, supplementary visual assembly guide). Tighten the screw using the 2.5 mm L-shaped hex key with brick rid-coloured band (item 99). Repeat the same with three more steel angle brackets. 195. Take the sample enclosure and place it over the microscope box (Figure 181, supplementary visual assembly guide). Align the holes in the four steel angle brackets on the enclosure with the threads on the top plate of the microscope box. Place an M4 x 8 mm cap screw into one of the four steel angle brackets and screw it into the adjacent thread on the top plate (Figure 182, supplementary visual assembly guide). Tighten the screw using the 2.5 mm L-shaped hex key with brick red-coloured band. Repeat the same with the other three threads on the top plate. 196. Take four 0.5” rods (item 76). Repeat point 64 with these four rods. 197. Screw the four rods to the four threads on the top panel of the sample enclosure (Figure 183, supplementary visual assembly guide). 198. Screw one end of the SM1 lens tube spacer (item 96) to the mounted LED (Figure 184, supplementary visual assembly guide). Screw the other end to a cage plate (Figure 185, supplementary visual assembly guide). Loosen all the set screws on the sides of the cage plate using the 2 mm L-shaped hex key with yellow-coloured band (item 99). Slide the cage plate onto the four screwed rods. Tighten the set screws using the 2 mm L-shaped hex key with yellow-coloured band (Figure 186, supplementary visual assembly guide). Step 13: assembling and aligning the sample holder (timing: 30 minutes) 199. Take the sample holder (item 134) and place it so that its longer edge sits flat against the third positioner. Insert a supplied M2 cap screw (item 14) in one of the two counter bores on the sample holder. Ensure that the screw is aligned with the sixth thread (eleventh hole, counting from the top) on the positioner. Note that only the odd-numbered holes are threaded. Loosely screw the cap screw using the 1.5 mm L-shaped hex key with purple-coloured band (item 99) (Figure 187, supplementary visual assembly guide). Repeat the same with the other counter bore. 200. Place the bullseye level (item 104) on the top plate of the microscope box. Note the position of the air bubble on the bullseye level as accurately as possible. Place a coverslip (item 40) inside the groove of the sample holder (Figure 188, supplementary visual assembly guide). Secure the coverslip using the installed clips. Place the bullseye level on the secured coverslip. Adjust the rotation of the sample holder until the position of the air bubble on the bullseye level on the coverslip is identical to the position noted previously. When the positions are matched, tighten the two M2 screws inserted in the counter bores of the sample holder using the 1.5 mm L-shaped hex key with purple-coloured band checking afterwards that the position on the bullseye level has not changed (Figure 189, supplementary visual assembly guide). Step 14: switching on the microscope (timing: 5 minutes) ?Troubleshooting Caution: wear the protective goggles (item 137) from points 201 to 239. 201. To switch on the device for the first time: a. Switch on the main power outlet to which the power strip (item 50) is connected. b. Switch on the power button on the power strip. c. Switch on the power button on the MSC2 control system (item 17). d. Switch on the power button on the sCMOS camera (item 14). e. Switch on the power button on the LED driver (item 54). f. Switch on the power button on the position aligner (item 18). g. Switch on the power button on the green laser control box (item 4). h. Turn on the key on the green laser control box to the position labelled ‘ON’. i. Turn on the key at the back side of the compact diode laser (item 8) (Figure 1, supplementary visual operation guide). j. Press the brown button labelled ‘ENABLE’ at the back side of the compact diode laser (Figure 2, supplementary visual operation guide). k. Flip the handle labelled ‘LASER APERTURE SHUTTER’ on the compact diode laser to the position labelled ‘OPEN’ (Figure 3, supplementary visual operation guide). l. Open and switch on the computer. 202. To switch on the device for subsequent times, follow points 201a, j and l. Step 15: setting up the computer and installing the NanoPro 1.0 control software (timing: 2 hours) ?Troubleshooting 203. Download and install the National Instruments LabVIEW 2020 SP1 64-bit runtime engine from the following link (https://www.ni.com/en-gb/support/downloads/software- products/download.labview.html#369642). 204. Download and install the National Instruments VISA 20.0 from the following link (https://www.ni.com/en-gb/support/downloads/drivers/download.ni-visa.html#346210). 205. Unzip the supplementary software folder and copy its contents to a new folder on the desktop renamed to ‘NanoPro 1.0’. Ensure the contents of the unzipped folder are as follows: two folders named [data] and [Dependencies], five files named [Configuration.csv], [Sequence.csv], [NanoPro_v1.3.aliases], [NanoPro_v1.3.exe] and [NanoPro_v1.3.ini]. 206. Download and install the Kinesis 64-bit software for 64-bit windows from the following link (https://www.thorlabs.com/Software/Motion%20Control/KINESIS/Application/v1.14.28/KINESIS%20Inst all%20x64/kinesis_18247_setup_x64.exe). Copy the contents from the folder path [C:\Program Files\Thorlabs\Kinesis] and paste them into the desktop folder path […\NanoPro 1.0\Dependencies\Thorlabs]. 207. Install the [PVCamSDK_Setup.exe] and [PMQI-LabViewSamples_Setup_1.2.2.1.exe] software files provided with the sCMOS camera (item 14). Copy the files [pvcam_helpher_-coloured_v1.dll] and [PVCamNET.dll] from the folder path [C:\Program Files\Photometrics\PMQI- LabViewSamples\Examples\2018\Dependencies] and paste them into the desktop folder path […\NanoPro 1.0\Dependencies\Photometrics]. 208. Request the drivers’ file from SmarAct (supplier of item 15). Unzip the contents of the drivers’ file and install the file [CDM21226_Setup.exe]. Install the [MCS2_Installer_2.1.3.exe] software file provided with the MSC2 control system (item 17). During installation, ensure that the check boxes for ‘MSC2 Tools and Programs’ and ‘Support for MSC2 with USB Interface’ are checked. 209. Download and install the 64-bit version of the Phidget control software from the following link (https://www.phidgets.com/downloads/phidget22/libraries/windows/Phidget22-x64.exe). Copy the contents from the folder path [C:\Program Files\Phidgets\Phidget22] and paste them into the desktop folder path […\NanoPro 1.0\Dependencies\Phidgets]. 210. Download and install ImageJ (item 139) from the following link (https://wsr.imagej.net/distros/win/ij153-win-java8.zip). Download and install ThunderSTORM from the following link (https://zitmen.github.io/thunderstorm/). Follow the installation instructions provided. 211. Open the [Configuration.csv] file in the folder unzipped in point 205. Edit the file using the following information: a. Enter the serial number of the analog digital convertor device (item 44) in the field opposite to ‘Phidget 1 Serial Number’. The six-digit serial number is printed on a white sticker fixed to the analog digital convertor device. b. Enter the serial number of the control hub (item 45) in the field opposite to ‘Phidget 2 Serial Number’. The six-digit serial number is printed on a white sticker fixed to the control hub. c. Enter the serial number of the position aligner (item 18) in the field opposite to ‘Photo Sensitive Detector Serial Number’. The eight-digit serial number is printed on a black sticker fixed to the position aligner. d. Enter the COM port number of the four-position slider (item 67) in the field opposite to the ‘Filter Slider COM port’. To find the COM port number of the slider, type ‘device manager’ in the search box of Windows 10 located at the bottom left corner of the screen. Left-click the {Device Manager} tab that appears under the Best Match list. A long list of components will be shown. Browse to the component named ‘Ports (COM & LPT)’ and left-click the grey arrow on the left side of the component. The COM port number will be dropped-down. 212. Run the NanoPro 1.0 control software by double-clicking the [NanoPro_v1.3.exe] software file in the ‘NanoPro 1.0’ folder. The NanoPro 1.0 control software window should appear (Figure 4, supplementary visual operation guide) as well as another blank camera view window (Figure 5, supplementary visual operation guide). The [message box] on the NanoPro 1.0 control software window should display ‘Ready’. See Troubleshooting section if this message is not displayed. Step 16: aligning the laser combiner (timing: 4 hours) ?Troubleshooting 213. Familiarise yourself with the control joystick (item 43) (Figures 6 and 7, supplementary visual operation guide). 214. Switch on the red laser (item 7) using the control joystick (Figure 6, supplementary visual operation guide). Increase the power of the red laser to 0.5 as indicated on the [Power level box] on the NanoPro 1.0 control software window (Figure 7, supplementary visual operation guide). Press the ‘Live’ button on the control joystick to activate laser emission (Figure 7, supplementary visual operation guide). 215. Connect the power sensor (item 13) to the power meter (item 12). Switch on the power meter and if needed connect to an appropriate power plug. Change the settings on the power meter so that the wavelength is set to 638 nm. Check the manual for operating the power meter. Caution: red laser will be emitting at 70 mW. Wear safety glasses (item 137) and do not look directly into the beam. Ensure room is secured against uncontrolled entry. Any reflective items (e.g. jewellery, watches, etc.) must be removed for alignment. Caution: do not increase the power of the red laser more than 3.5 to prevent overheating of the laser head under ambient conditions. 216. Loosen the four clamping knobs (item 121) securing the top panel of the laser enclosure assembled in point 187. Lift the panel and stow in an appropriate place. 217. Adjust the power meter settings according to the vendor’s instructions. Place the active area of the power sensor in front of the red laser and record the reading on the power meter, hereafter referred to as the first recorded reading (Movie S1). 218. Loosen the black capped screw on the post holder (item 74) on the breadboard (item 64) at position (16,4) to liberate the post assembly (hereafter referred to as the first post assembly). Rotate the first post assembly so that the laser beam reflected off the mounted mirror is steered towards the centre of the mirror on the post assembly on the breadboard at position (14,7) (hereafter referred to as the second post assembly) (Figure 1, supplementary visual alignment guide). Tighten the black capped screw using the 5 mm L-shaped hex key with green-coloured band (item 99) to secure the first post assembly in place (Movie S2). Loosen the black capped screw on the post holder on the breadboard at position (14,7) to liberate the second post assembly. Rotate the second post assembly so that the laser beam reflected off the mounted mirror is steered 2 mm off the front edge of the 805 nm long pass dichroic mirror (item 30) which is part of the post assembly on the breadboard at position (18,6) (hereafter referred to as the third post assembly) (Figure 2, supplementary visual alignment guide). Tighten the black capped screw using the 5 mm L-shaped hex key with green-coloured band to secure the second post assembly in place and (Movie S3). Take an A4 piece of paper, fold in half and rest it in front of the fibre launch system (item 66). Loosen the black capped screw on the post holder on the breadboard at position (18,6) to liberate the third post assembly. Rotate the third post assembly so that the laser beam reflected off the mounted 805 nm long pass dichroic mirror is steered through all the mounted long pass dichroic mirrors (items 27, 28 and 29) and towards the folded piece of paper (Figure 3, supplementary visual alignment guide). Remove the piece of paper. Tighten the black capped screw using the 5 mm L-shaped hex key with green-coloured band to secure the third post assembly in place (Figure 4, supplementary visual alignment guide and Movie S4). 219. Rotate the two knobs on the kinematic mirror mount (item 79) on the third assembly, one after the other, so that the beam is steered towards the centre of the fibre launch system (Movie S5). 220. Screw the FC/PC fibre adaptor (item 97) to the thread on the power sensor (item 13) (Figure 5, supplementary visual alignment guide). Attach the fibre end (item 1) screwed to the collimator (item 2) to the FC/PC connector on the fibre adaptor (Figure 6, supplementary visual alignment guide and Movie S6). 221. Rotate the two knobs on the kinematic mirror mount on the third assembly, one after the other, to gradually increase the power reading on the power meter (Figure 7, supplementary visual alignment guide). Repeat until the reading on the power meter is maximised. Record the reading on the power meter and proceed to next point (Movie S7). 222. Rotate the lowermost silver knob on the first assembly in one direction a small turn and observe the decrease in the reading on the power meter (Figure 8, supplementary visual alignment guide). Remember the direction of rotation. Rotate the lowermost silver knob on the second assembly in one direction a small turn and observe the increase in the reading on the power meter. If the reading on the power meter does not increase rotate the knob in the other direction a small turn and observe the increase in the reading on the power meter. Repeat until the reading on the power meter is maximised. Record the reading on the power meter and proceed to next point. 223. Rotate the uppermost silver knob on the first assembly in one direction a small turn and observe the decrease in the reading on the power meter. Remember the direction of rotation. Rotate the uppermost silver knob on the second assembly in one direction a small turn and observe the increase in the reading on the power meter. If the reading on the power meter does not increase rotate the knob in the other direction a small turn and observe the increase in the reading on the power meter. Repeat until the reading on the power meter is maximised. Record the reading on the power meter and proceed to next point (Movie S8). 224. Rotate the black knob pointing up on the fibre launch system to gradually increase the power reading on the power meter (Figure 9, supplementary visual alignment guide). Repeat until the reading on the power meter is maximised. Rotate the black knob pointing left on the fibre launch system to gradually increase the power reading on the power meter (Figure 10, supplementary visual alignment guide). Repeat until the reading on the power meter is maximised. Record the reading on the power meter and proceed to next point (Movie S9). 225. Loosen all four set screws on the side of the Z translation mount fixed to the fibre launch system using the 1.3 mm L-shaped hex key with orange-coloured band (Figure 11, supplementary visual alignment guide and Movie S10). Move the Z translation mount in one direction to gradually increase the power reading on the power meter. If the reading on the power meter does not increase move the Z translation mount in the other direction and observe the increase in the reading on the power meter. Repeat until the reading on the power meter is maximised (Movie S11). Tighten all the four set screws on the side of the Z translation mount fixed to the fibre launch system using the 1.3 mm L-shaped hex key with orange-coloured band whilst holding the Z translation mount in place (Movie S12). Rotate the black knob pointing back on the fibre launch system to gradually increase the power reading on the power meter. Repeat until the reading on the power meter is maximised (Movie S13). Record the reading on the power meter and proceed to next point. 226. Repeat points 221 to 225 and observe the increase in the reading on the power meter. Repeat until the reading on the power meter is maximised. Record the reading on the power meter, hereafter referred to as the last recorded reading. 227. If the last recorded reading is greater than 75% of the first recorded reading proceed to point 229, otherwise proceed to next point. 228. Take an A4 piece of paper, fold in half and rest it in front of the fibre launch system. Insert the 0.7 mm L-shaped hex key with red-coloured band (item 99) through the two grooves of the external thread on the head of the red laser (Figure 12, supplementary visual alignment guide). Rotate the L- shaped hex key in one direction and observe the reduction in the beam size on the folded piece of paper. If the beam is increased in size, rotate the L-shaped hex key in the other direction and observe the reduction in the beam size. Repeat until beam size is reduced to minimum. Repeat points 221 to 227. 229. Repeat points 214 to 228 (excluding points 216, 223, 224, 225) to align the green, blue and ultra violet lasers (in this order), one at a time, noting the following (Movies S14 to S18): a. In point 214, switch on the green, blue or ultra violet lasers. b. In point 215, set the wavelength to 561 nm (for the green laser), 488 nm (for the blue laser) and 405 nm (for the ultra violet laser). c. In point 217, place the active area of the power sensor in front of the switched-on laser. d. In points 218 to 223, note the following: i. For the green laser, the first assembly is inserted into the post holder on the breadboard at position (16,9), the second assembly is inserted into the post holder on the breadboard at position (14,12) and the third assembly is inserted into the post holder on the breadboard at position (18,11). ii. For the blue laser, the first assembly is inserted into the post holder on the breadboard at position (16,14), the second assembly is inserted into the post holder on the breadboard at position (14,17) and the third assembly is inserted into the post holder screwed to the breadboard at position (18,16). iii. For the ultra violet laser, the first assembly is inserted to the post holder screwed on the breadboard at position (16,19), the second assembly is inserted to the post holder on the breadboard at position (14,22) and the third assembly is inserted to the post holder on the breadboard at position (18,21). e. Point 228 is excluded for the green laser. 230. Place the stowed top panel of the laser enclosure on top of the rails so that the holes on the panel are aligned with the threads on the rails. Fix the large top panel to the rails using all four clamping knobs. Critical step: press the ‘Live’ button on the control joystick to stop the emission from the ultra violet laser. Ensure the ‘Ready’ message is displayed in the [Message box] on the NanoPro 1.0 control software window. 231. Loosen the fibre end from the FC/PC fibre adaptor and fix it back to the collimator. 232. Switch on the red laser using the control joystick. Increase the power of the red laser to 2.0 as indicated on the [Power level box] on the NanoPro 1.0 control software window. Press the ‘Live’ button on the control joystick to activate laser emission. Switch off the lights of the room. 233. Rotate the silver knobs on the right-angled kinematic mirror mount (item 80, added in point 71) so that the red beam comes straight out of the objective (item 19) and onto the ceiling (Figure 13, supplementary visual alignment guide). Rotate the red barrel on the collimator (Figure 14, supplementary visual alignment guide) so that the beam is reduced in size and eventually squared at the ceiling (Figure 15, supplementary visual alignment guide). Rotate the rotation mount (item 85, added in point 72) (Figure 16, supplementary visual alignment guide) so that the beam is square with the microscope box. Tighten the set screw on the collimator using the 1.3 mm L-shaped hex key with orange-coloured band (Figure 17, supplementary visual alignment guide). Tighten the set screw on the rotation mount using the 2 mm L-shaped hex key (item 99) with yellow-coloured band to secure in place (Figure 18, supplementary visual alignment guide). Step 17: aligning the excitation and emission paths (timing: 20 mins) ?Troubleshooting 234. Squirt two drops of the immersion oil (item 140) on top of the objective (item 19). 235. Mount the 40 nm nanorulers sample (item 135) on the sample holder (item 134) and secure from both sides using the clips with the smaller coverslip facing downwards. Ensure that the sample is properly-mounted according to the delivered instructions. 236. Switch on the green laser (item 3) using the control joystick (item 43) (Figure 6, supplementary visual operation guide). Increase the power of the green laser to 4.0 as indicated on the [Power level box] on the NanoPro 1.0 control software window (Figure 7, supplementary visual operation guide). Press the ‘Live’ button on the control joystick to activate laser emission (Figure 7, supplementary visual operation guide). 237. Rotate the lowermost silver knob on the kinematic mirror mount (item 80, added in step 71) so that the squared-beam emerging out of the objective is inclined towards the left hand side and until the beam undergoes Total Internal Reflection (TIR). TIR is roughly reached when the beam is seen to form three dots at the glass interface of the objective; two on the side and one in the middle. Ensure the beam is inclined straight to the left and not at an angle by rotating the uppermost silver knob on the kinematic mirror mount. 238. Move the positioners (item 15) downwards (coarse) using the control joystick (Figure 6, supplementary visual operation guide) until the immersion oil on top of the objective touches the bottom of the coverslip. Move the positioners downwards (coarse) step-wise using the control joystick until you observe the following pattern on the camera view window (Figure 20, supplementary visual alignment guide). Move the positioners downwards or upwards (fine) step-wise using the control joystick until you observe the following pattern on the camera view window (Figure 20, supplementary visual alignment guide). The pattern might not be centred on the camera (Figure 21, supplementary visual alignment guide). If this is the case, rotate the silver knobs on the right-angle kinematic mirror mount inside the microscope box (item 80, added in point 110) to centre the pattern on the camera (Figure 22, supplementary visual alignment guide). Further adjust the angle of the beam to ensure proper TIR excitation as described in point 237. The angle where the image shows highest contrast is the angle at which TIR occurs. 239. Press the ‘Live’ button on the control joystick to de-activate laser emission. Step 18: aligning the focus stabilization system (timing: 30 minutes) 240. Insert the detector card (item 11) into the microscope box and place the orange active area in front of the achromatic doublet lens (item 34). Observe the bright spot on the detector card resulting from the detection of the infrared beam produced by the compact diode laser (item 8). Rotate the silver knobs on the kinematic mirror mount (item 80, added in point 80) to roughly centre the beam on the achromatic doublet lens. 241. Place the detector card 2 cm away from the objective lens with the orange active area facing downwards. Observe the bright spot on the detector card (Figure 23, supplementary visual alignment guide). 242. Rotate the uppermost silver knob on the kinematic mirror mount whilst tracking the bright spot on the detector card as it inclines to the back (Figure 24, supplementary visual alignment guide) and until the spot disappears. If it inclines at an angle, rotate the lowermost silver knob to centre the bright spot. Ensure ‘Focused’ is displayed in the [Focus status box] (Figure 4, supplementary visual operation guide). Observe the auto aligner (item 18). Rotate the uppermost silver knob on the kinematic mirror mount until the white circle on the square screen on the auto aligner is displaced to the centre of the y-axis. Move the positioners (item 15) upwards then downwards (fine) using the control joystick (Figure 6, supplementary visual operation guide). Ensure the white circle moves in a straight line along the y-axis as the positioners are moved. 243. Take the front plate of the microscope box on which the number ‘6’ is engraved (item 132) and use it to cover the front opening of the microscope box (Figure 190, supplementary visual assembly guide). 244. Insert an M6 x 20 mm cap screw (item 101) into one of the eight counter bores on the front side of the front plate. Screw and tighten the cap screw using the 5 mm L-shaped hex key with green-coloured band (item 99) (Figure 191, supplementary visual assembly guide). Repeat the same with the remaining seven counter bores. This marks the end point of the assembly and alignment of the microscope (Figure 192, supplementary visual assembly guide). 245. Move the positioners upwards 1 cm above the objective lens using the control joystick. Unmount the sample. Step 19: measuring the camera pixel size (timing: 30 minutes) ?Troubleshooting 246. Mount the variable line grating (item 39) on the sample holder (item 134) and secure from both ends using the clips. Ensure the variable line grating is mounted with the black inscribing facing downwards. 247. Switch on the LED (item 9) using the control joystick (item 43) (Figure 6, supplementary visual operation guide). Increase the power of the LED by rotating the knob on the LED driver (item 54) to half-way between the labels ‘0’ and ‘LIMIT’. Ensure the switch on the LED driver is flipped to the position labelled ‘TRIG’. 248. Press the ‘Live’ button on the control joystick to activate laser emission (Figure 7, supplementary visual operation guide). Ensure ‘Live’ is displayed in the [Message box] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide). 249. Move the positioners (item 15) downwards (coarse) using the control joystick (Figure 6, supplementary visual operation guide) until the immersion oil on top of the objective touches the bottom of the coverslip. Move the positioners to the left / right and back / front to position the objective lens underneath the furthest inscribed band (counting from the side with the band with four thick lines). Move the positioners (item 15) downwards (coarse or fine) using the control joystick (Figure 6, supplementary visual operation guide) to focus the sample as described in point 238. Observe the following pattern on the camera view window (Figure 25, supplementary visual alignment guide). Press the ‘Live’ button on the control joystick to de-activate LED emission. Ensure ‘Ready’ is displayed in the [Message box] on the NanoPro 1.0 control software window. 250. Set the [Exposure time] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide) to 50. Set the [Number of frames] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide) to 1. 251. Create a new folder on the desktop and rename to ‘Pixel’. 252. Select the [Acquisition path] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide). 253. Press the ‘Acquire’ button on the control joystick to start acquisition. Ensure ‘Acquire’ is displayed in the [Message box] on the NanoPro 1.0 control software window. Gently rest the control joystick on the breadboard (item 64) to reduce perturbations to the microscope. 254. Wait until acquisition is complete. Acquisition is complete when LED emission is automatically de- activated and ‘Ready’ is displayed in the [Message box] on the NanoPro 1.0 control software window. 255. Move the positioners upwards 1 cm above the objective lens using the control joystick. Unmount the sample. 256. The acquisition of the variable line grating will be found in the folder named ‘1’ in the folder created in point 252. Open the ‘.tif’ file contained within using the installed ImageJ software (item 139). Press the *straight* line tool on the ImageJ toolbar and draw a horizontal line from the middle of the darkest mark on the right of the image to the middle of the eleventh darkest mark (counting from the right of the image) by left-clicking the mouse button on the image and dragging across. Before releasing the mouse button, record the length of the drawn line, in pixels, as displayed in the message bar of ImageJ. 257. To calculate the pixel size (𝑝𝑠), use the following formula: 𝑝𝑠 ( 𝑛𝑚 𝑝𝑥 ) = 40000 #𝑝𝑖𝑥𝑒𝑙𝑠 . Step 20: imaging ground-truth samples to establish performance (timing: 1 hour) ?Troubleshooting 258. Take the 40 nm nanorulers (item 135) from the fridge and leave it in the microscope room for at least 30 minutes for its temperature to equilibrate to prevent excessive drift during acquisition. Mount the 40 nm nanorulers sample on the sample holder (item 134) and secure, from both ends, using the clips. 259. Switch on the (561 nm) green laser (item 3) using the control joystick (item 43) (Figure 6, supplementary visual operation guide). 260. Press the ‘Live’ button on the control joystick to activate laser emission (Figure 7, supplementary visual operation guide). Ensure ‘Live’ is displayed in the [Message box] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide). 261. Move the positioners (item 15) downwards (coarse or fine) using the control joystick (Figure 6, supplementary visual operation guide) to focus the sample as described in point 238. A small portion of the image detected by the camera will be shown. To see the full image, zoom out by right-clicking on the camera view window (Figure 5, supplementary visual operation guide) and left-clicking ‘Zoom -’ from the drop-down menu several times. 262. Activate the autofocus system using the control joystick (Figure 6, supplementary visual operation guide) to lock the focus. Ensure ‘Locked’ is displayed in the [Focus status box] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide). 263. Press the ‘Live’ button on the control joystick to de-activate laser emission. Ensure ‘Ready’ is displayed in the [Message box] on the NanoPro 1.0 control software window. 264. Set the [Exposure time] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide) to 150. Set the [Number of frames] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide) to 20000. 265. Create a new folder on the desktop and rename to ‘Test’. 266. Select the [Acquisition path] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide) and choose the created folder. 267. Crop the area of the field of view by right-clicking on the camera view window then left-clicking ‘Add region‘ from the drop-down menu. A small green box will appear. Expand the green box by dragging its corners and move it by dragging it from the centre so that it only covers the region of interest. 268. Press the ‘Acquire’ button on the control joystick to start acquisition. Ensure ‘Acquire’ is displayed in the [Message box] on the NanoPro 1.0 control software window. Gently rest the control joystick on the breadboard (item 64) to reduce perturbations to the microscope. 269. Wait until acquisition is complete. Acquisition is complete when laser emission is automatically de- activated and ‘Ready’ is displayed in the [Message box] on the NanoPro 1.0 control software window. Remove the crop on the field of view by right-clicking on the camera view window then left-clicking ‘Delete region‘ from the drop-down menu. 270. Move the positioners upwards 1 cm above the objective lens using the control joystick. Unmount the 40 nm nanorulers sample and store in the fridge. 271. Squirt one drop of the immersion oil (item 140) on top of the objective (item 19). 272. Take the 20 nm nanorulers (item 136) from the fridge and leave in the microscope room for at least 30 minutes for its temperature to equilibrate to prevent excessive drift during acquisition Mount and secure the 20 nm nanorulers sample as described in point 235. 273. Repeat points 260 to 271 (excluding points 266 to 268). The two acquisitions of the 40 nm nanorulers sample and the 20 nm nanorulers sample will be found in the folders named ‘1’ and ‘2’, respectively, in the folder created in point 266. Step 21: data processing (timing: 1 hour) 274. Open the installed ImageJ software (item 139). 275. Drag the folder labelled ‘1’ inside the folder created in point 266 and drop it on the message bar of ImageJ. A new dialog window will appear. Check the box labelled ‘Use Virtual Stack’ and click the button labelled ‘Yes’. Wait for the video to load as indicated in the message bar of ImageJ. 276. Press ‘Plugins’ on the menu bar, hoover over ‘ThunderSTORM’ then hoover over ‘Run analysis’. A new dialog window will appear. In the container labelled ‘Camera’, press the button labelled ‘Camera setup’. Enter the pixel size calculated in point 258 in the field labelled ‘Pixel size [nm]’, enter 0.25 in the field labelled ‘Photoelectrons per A/D count’ (consult vendor for exact figure quoting 100 MHz/12 bit as Readout Speed / Data Bits), enter 100 in the field labelled ‘Base level [A/D counts]’ and uncheck the box labelled ‘EM gain’. In the container labelled ‘Approximate localization of molecules’, enter std(Wave.F1) in the field labelled ‘Approximate localization of molecules’. In the container labelled ‘Visualisation of the results’, enter 26 in the field labelled ‘Magnification’ and enter 1000 in the field labelled ‘Update frequency [frames]’. Press the button labelled ‘Ok’. Wait for the video to be processed as indicated in the message bar of ImageJ. During processing, a window will appear containing the super-resolved image. The super-resolved image is not drift-corrected and, therefore, the nanorulers will not be seen. 277. Once processing is complete, a new table will appear. The table contains information on each detected burst as well as tools to manipulate each burst. Press the tab labelled ‘Drift correction’ and press the radio button labelled ‘Cross correlation’. Press the double arrow button in front of the radio button and enter 10 in the field labelled ‘Number of bins’ and enter 26 in the field labelled ‘Magnification’. Wait for the super-resolved image to be drift-corrected as indicated in the message bar of ImageJ. Once completed, a super-resolved image (Figure 3a) of the 40 nm nanorulers should be seen. 278. Repeat point 276 with the folder labelled ‘2’ inside the folder created in point 266. 279. Once processing is complete, a new table will appear. The table contains information on each detected burst as well as tools to manipulate each burst. Press the tab labelled ‘Drift correction’ and press the radio button labelled ‘Cross correlation. Press the double arrow button in front of the radio button and enter 10 in the field labelled ‘Number of bins’ and enter 26 in the field labelled ‘Magnification’. Wait for the super-resolved image to be drift-corrected as indicated in the message bar of ImageJ. Once completed, a super-resolved image (Figure 3d) of the 20 nm nanorulers will be displayed. Step 22: multi-sample acquisitions (timing: dependent on the sequence of events) ?Troubleshooting Critical step: ensure a spreadsheet software, such as Microsoft Excel, is installed on the computer before proceeding with points 281 to 292. Critical step: if a large number of samples is to be imaged, add an extra drop of oil on top of the objective. 280. Open the [Sequence.csv] file in the folder unzipped in point 205. 281. Edit the file using the following information: a. Enter the number of times (i.e. repeats) the mounted sample is to be imaged in the field opposite to ‘Number of repeats’. As an example, if you are imaging each well in an ibidi ® µ- Slide 8 Well chambers twice, enter 2 in this field. b. Enter the number of wells to be imaged in the direction parallel to the longer side of the optical table (item 63) in the field opposite to ‘Number of X wells’. As an example, if you are imaging all wells in an ibidi ® µ-Slide 8 Well chambers, enter 4 in this field. Beware that if you would like to image at non-periodic locations, the entry in this field will be ignored. Use the control joystick to select the positions to acquire at (Figure 6, supplementary visual operation guide). c. Enter the number of wells to be imaged in the direction parallel to the shorter side of the optical table in the field opposite to ‘Number of Y wells’. As an example, if you are imaging all wells in an ibidi ® µ-Slide 8 Well chambers, enter 2 in this field. Beware that if you would like to image at non-periodic locations, the entry in this field will be ignored. d. Enter the number of Field Of Views (FOVs) to be imaged in the direction parallel to the longer side of the optical table in the field opposite to ‘Number of X FOVs’. As an example, if you are imaging 4 regions of each well, enter 4 in this field. Beware that the product of this field and the field opposite to ‘Number of Y FOVs’ has to equal the number of regions to be imaged in each well. e. Enter the number of Field Of Views (FOVs) to be imaged in the direction parallel to the shorter side of the optical table in the field opposite to ‘Number of Y FOVs’. As an example, if you are imaging four regions of each well, enter 4 in this field. Beware that the product of this field and the field opposite to ‘Number of X FOVs’ has to equal the number of regions to be imaged in each well. f. Enter the laser(s) to be used for imaging each region in the fields opposite to ‘Lasers’. Lasers will be switched on according to the sequence in which they are entered. As an example, if you would like to image each region using the (638 nm) red, then (561 nm) green, lasers (items 3 and 7), enter ‘Red’ in the field opposite to ‘Lasers’ and ‘Green’ in the field opposite to ‘Red’. Beware the first letter of each enter laser line has to be upper case. g. Enter the distance between each well in the direction parallel to the longer side of the optical table in the field opposite to ‘X wells distance (um)’ in micrometres. As an example, if the distance between each well in the direction parallel to the longer side of the optical table is 4.5 mm, enter 4500 in this field. Beware that if you would like to image at non-periodic locations, the entry in this field will be ignored. h. Enter the distance between each well in the direction parallel to the shorter side of the optical table in the field opposite to ‘Y wells distance (um)’ in micrometres. As an example, if the distance between each well in the direction parallel to the shorter side of the optical table is 4.5 mm, enter 4500 in this field. Beware that if you would like to image at non-periodic locations, the entry in this field will be ignored. i. Enter the distance between each field of view in the direction parallel to the longer side of the optical table in the field opposite to ‘X FOVs distance (um)’ in micrometres. As an example, if the distance between each well in the direction parallel to the longer side of the optical table is 0.1 mm, enter 100 in this field. j. Enter the distance between each field of view in the direction parallel to the shorter side of the optical table in the field opposite to ‘Y FOVs distance (um)’ in micrometres. As an example, if the distance between each well in the direction parallel to the shorter side of the optical table is 0.1 mm, enter 100 in this field. k. Enter the number of frames to be imaged for each entered laser in the fields opposite to ‘Frames’. Each laser will be switched on for the number of frames entered. As an example, if you would like to acquire 10 frames with the red laser, then 10000 frames with green laser, enter 10 in the field opposite to ‘Frames’ and 10000 in the field opposite to 10. l. [Optional, only for STORM imaging]. Enter the increase in the power of the (405 nm) ultra violet laser (item 5) after a set duration (see point 282m) in the field opposite to ‘UV activation step intensity (V)’ in volts. As an example, if you would like to increase the power of the ultra violet laser in steps of 0.1 V, enter 0.1 in the field opposite to ‘UV activation step intensity (V)’. Beware, if you are not performing STORM imaging, or would not like to use an activation laser, enter 0 in the field opposite to ‘UV activation step intensity (V)’. m. [Optional, only for STORM imaging]. Enter the time duration after which the power of the ultra violet laser is increased (see point 282m) in the field opposite to ‘UV activation step duration (min)’ in minutes. As an example, if you would like to increase the power of the ultra violet laser after every 10 minutes, enter 10 in the field opposite to ‘UV activation step duration (min)’. Beware, if you are not performing STORM imaging, or would not like to use an activation laser, enter 1000 in the field opposite to ‘UV activation step duration (min)’. 282. If periodic imaging is to be performed (i.e. no positions were selected using the control joystick), move the positioners to the centre of the well on back / right corner of the sample using the control joystick (Figure 6, supplementary visual operation guide). 283. Switch on each laser entered in point 281f using the control joystick (Figure 6, supplementary visual operation guide). Increase, or decrease, the power of each laser. The power level is indicated on the [Power level box] on the NanoPro 1.0 control software window (Figure 7, supplementary visual operation guide). 284. Press the ‘Live’ button on the control joystick to activate laser emission (Figure 7, supplementary visual operation guide). Ensure ‘Live’ is displayed in the [Message box] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide). 285. Move the positioners (item 15) downwards (coarse or fine) using the control joystick (Figure 6, supplementary visual operation guide) to focus the sample as described in point 238. 286. Activate the autofocus system using the control joystick (Figure 6, supplementary visual operation guide) to lock the focus. Ensure ‘Locked’ is displayed in the [Focus status box] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide). 287. Press the ‘Live’ button on the control joystick to de-activate laser emission. Ensure ‘Ready’ is displayed in the [Message box] on the NanoPro 1.0 control software window. 288. Set the [Exposure time] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide) to 200. 289. Create a new folder on the desktop and rename it as wished. 290. Select the [Acquisition path] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide) and choose the created folder. 291. Toggle the [Sequence switch] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide). 292. Select the [Sequence path] on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide) and choose the [Sequence.csv] file in the folder unzipped in point 205. 293. Perform points 268 to 271. Step 23: switching off the microscope (timing: 5 minutes) 294. Press the [Stop] button on the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide). Close the NanoPro 1.0 control software window. 295. Close and switch off the computer (item 41). 296. Switch off the power button (item 50) on the power strip. 297. Switch off the main power outlet to which the power strip is connected. 298. Clean the objective from excess oil using lens cleaning tissue (item 142) and acetone / ethanol (see https://microscopy.duke.edu/guides/clean-objective). Timing Step 1, procurement and space preparation: up to 6 m Step 2, fabrication: up to 3 m Step 3, unpackaging and installation: 2 d Step 4, assembling the laser combiner: 3 h Step 5, first partial assembly of the microscope box: 1 h Step 6, assembling the excitation module and focus stabilization system: 2 h Step 7, second partial assembly of the microscope box: 1 h Step 8, assembling the emission module: 1 h Step 9, assembling the sample stage: 2 h Step 10, setting up electrical connections: 2 h Step 11, assembling the laser combiner enclosure: 30 min Step 12, assembling the sample enclosure: 2 h Step 13, assembling and aligning the sample holder: 30 min Step 14, switching on the microscope: 5 min Step 15, setting up the computer and installing the NanoPro 1.0 control software: 2 h Step 16, aligning the laser combiner: 4 h Step 17, aligning the excitation and emission paths: 20 min Step 18, aligning the focus stabilization system: 30 min Step 19, measuring the camera pixel size: 30 min Step 20, imaging ground-truth samples to establish performance: 1 h Step 21, data processing: 1 h Step 22, multi-sample acquisitions: dependent on the sequence of events Step 23, Switching off the microscope: 5 min Anticipated results The assembly of NanoPro 1.0 is a relatively long process which although is extensively documented and illustrated, it is expected that the microscope would either turn to be misaligned (figure 4a – d), dysfunctional, or incapable of producing high-quality images (figure 4e – f). All these problems, their causes, and solutions are summarized in the Troubleshooting section. When well aligned and operational, the NanoPro 1.0 will be capable of acquiring high, 20 nanometre resolution images of ground-truth nanorulers (figure 3), single- and multi-target cellular structures using dSTORM and DNA-PAINT with different fluorophores (figure 5a – c, supplementary note 1), as well as multiple samples (figure 5d, supplementary note 1) without user intervention and from 8 and up to 50 samples (dependent on the type of multi-well chamber used). Imaging cellular or recombinant structures using the different SMLM techniques requires extensive expertise in sample preparation and data processing. Here, we performed imaging of these structures without significant optimization or the use of advanced image processing algorithms which could improve the representation of the underlying biological structures and push the resolution below 10 nanometres. Users of the NanoPro 1.0 must optimize these factors depending on their imaging requirements. Troubleshooting Step Problem Cause(s) Solution(s) Steps 14 and 23 Power trips when power strip (item 50) is switched on or off. Power strip is faulty. Current overload. Replace power strip. Switch off power from the wall socket, first, rather than the power strip. If problem persists, consult the electronic / electrical workshop of your department / institution to advice on the root cause of the problem and devise an appropriate solution. Step 14 Some, or all of the, microscope’s components are not switched on when main power outlet is switched on. Current is not flowing through the main power outlet. Power strip is faulty. Switch on one or more of the microscope’s components is not switched on. Plug the power strip to another main power outlet. If problem persists, consult the electronic / electrical workshop of your department / institution to advice on the root cause of the problem and devise an appropriate solution. Replace power strip. Switch on all of the microscope’s components as described in point 201. One or more of the microscope’s components are damaged. Contact the supplier of the damaged components for advice on the root cause of the problem and to arrange for maintenance or replacement. Step 15 Supplementary NanoPro 1.0 software (item 138) cannot be opened giving an error. One, or more, libraries are not installed. Follow points 203 to 212, carefully, to ensure all libraries are installed and the folder named dependencies contains all the files required by NanoPro 1.0 to operate. Step 15 The message ‘(Component) is not connected’ is displayed in the [message box] of the NanoPro 1.0 control software window (Figure 4, supplementary visual operation guide). Component is not connected. Component is not switched on. Component is damaged Component is not properly registered in the [Configuration.csv] file (see step 211). Component is not setup, yet. USB hub is not working (item 42) Connect the component as described in points 164 to 184. Switch on component as described in point 201. Contact the supplier of the damaged component for advice on the root cause of the problem and to arrange for maintenance or replacement. Register the component as described in point 211. Wait a few minutes after switching on the computer before trying to open the NanoPro 1.0 software. If problem persists, switch off the power strip, wait a few seconds, switch on the power strip, restart the computer and open the NanoPro 1.0 software. Connect USB hub to power strip as described in point 184. Connect USB to computer as described in point 184. USB hub is faulty and needs replacement. Replace with a new hub, or any other electrically-powered, USB 2.0 hub with, at least, 6 USB ports. Steps 16, 17, 20, and 22 Laser light is not emitted when the ‘Live’ or ‘Acquire’ buttons are pressed on the control joystick (item 43) (Figure 6, supplementary visual operation guide). One, or more, of the lasers is not connected. Laser power is low. One, or more, of the lasers is over- heated. One, or more, of the lasers is not aligned into the fibre. Connect the lasers (items 4 – 8) to the digital analog converter device (item 44) as described in points 165 – 172, 180 and 181. Ensure three LEDs on the electronic circuits connected to the ultra violet (405 nm), blue (488 nm) and red (638 nm) are emitting. If less than three LEDs are emitting this would indicate that connections are not properly secured. If more than three LEDs are emitting this would indicate that laser(s) is overheating (see below for solution). Make sure that the key for the green (561 nm) laser driver is at the on position. Increase the laser power using the control joystick. Laser head is faulty. Contact the supplier of the damaged laser for advice on the root cause of the problem and to arrange for maintenance or replacement. Allow the laser to cool down for at least 1 hour. Do not operate the laser at a power level greater than 4.0 as indicated on the [Power level box] on the NanoPro 1.0 control software window (Figure 7, supplementary visual operation guide). Align the lasers as described in points 213 to 229. Steps 19, 20, and 22 White light is not emitted from the LED (item 9) when the ‘Live’ or ‘Acquire’ buttons are pressed on the control joystick. LED is not connected to LED driver (item 54). LED driver is not connected to computer. LED power is low. Connect the LED to the LED driver as described in step 179. Connect the LED driver to the computer as described in points 182 and 184. Rotate the knob on the LED driver clockwise to the position Switch on LED driver is not in a correct position. labelled ‘LIMIT’. Flick the switch on the LED driver to the position labelled ‘TRIG’. Steps 16, 17, 19, 20, and 22 Four-position slider (item 67) not moving when different lasers are switched on using the control joystick. Four-position slider is damaged. Four-position slider is stuck in position. Four-position slider is not connected to red electronic board. Red electronic board is not connected to computer. Contact the supplier of the four- position slider for advice on the root cause of the problem and to arrange for maintenance or replacement. Refer to the instructions’ manual provided by the supplier on moving the four-position slider from the three black buttons on the connected red electronic board before attempting to control the movement of the slider using the NanoPro 1.0 software. Connect the four-position slider to the red electronic board as described in points 123, and 175. Connect the red electronic board to the computer as described in point 184. Steps 17, 19, 20, and 22 Image is moving / vibrating when lightly tapping on the breadboard (item 64). Screws are not strongly tightened. It is critical to strongly tighten (unless otherwise stated) all screws used in points 50 to 200. Step 17 Field of view is brighter on one side (figure 4b) Excitation path misaligned (not illuminating in TIRF) Align the emission path as described in point 237. Step 17 Field of view is cropped (figure 4c) Emission path misaligned Align the emission path as described in point 238. Steps 17, 19, 20, and 22 Bursts do not appear as symmetric concentric circles out of focus (figure 4d). Sample holder (item 134) is not aligned parallel to the top plate of the microscope box (item 132). Align the sample holder, properly, as described in points 199 and 200. Pay close attention to match, as perfectly as possible, the position of the air bubble in the spirit level on the top plate of the microscope box and the sample holder. Steps 17, 19, 20, and 22 Bursts appear elongated in and out of focus. Vibration motor (item 61) is rotating at a high speed causing vibrations to the sample and microscope. Ensure the O Rings (items 143 and 144) are appropriately slotted as described in point 73. Steps 17, 19, 20, and 22 Image appears patchy (i.e. with speckles or dark regions, figure 4f). Vibration motor (item 61) is rotating at a low speed causing ineffective elimination of the speckle pattern resulting from the propagation of light in the multimode fibre (item 1). Vibration motor not working Increase the rotation speed of the vibration motor by rotating the screw on the variable DC power source (item 47) anti- clockwise using the slotted screwdriver (item 125) until maximum limit. Connect the vibration motor to the variable DC power source, as described in points 182 f and g. Vibration motor is broken. Replace vibration motor. Vibration motor loosely fixed to the fibre. Fix vibration motor to the fibre firmly using the vibration motor mount. Steps 17, 19, 20, and 22 [Focus status box] does not display ‘Focused’ even when sample is focused. Autofocus system is not aligned. Compact diode laser is not switched on or emitting. Compact diode laser is damaged. Position aligner is not connected. Position aligner is not switched on. Position aligner is damaged. Align the focus stabilization system as described in points 240 to 245. Switch on the compact diode laser as described in point 201. Contact the supplier of the compact diode laser for advice on the root cause of the problem and to arrange for maintenance or replacement. Connect the position aligner as described in points 173 and 184. Switch on the position aligner as described in point 201. Contact the supplier of the position aligner for advice on the root cause of the problem and to arrange for maintenance or replacement. Steps 17, 19, 20, and 22 Focus is locked but not maintained during acquisition. Sample is not fixed in position on the sample holder. Thickness of Fix the sample on the sample holder using the installed clips. Use #1.5 coverslips (item 40) coverslip is not appropriate. only. Steps 17, 19, 20, and 22 Image is dim. Laser power is low. Incorrect laser line in use. Four-position slider has not moved position to the right emission filter. Sample is not properly fluorescently- labelled. Thickness of coverslip is not appropriate. Low amount of immersion oil (item 140) on the objective lens. Lasers(s) are misaligned. Sample not illuminated in TIRF. Increase the laser power using the control joystick. Switch on the laser line that matches the excitation spectrum of the fluorescent sample. Refer above for causes and solutions. Ensure sample is appropriately stained. Use #1.5 coverslips only. Squirt one or two drops of the immersion oil on the objective lens. Align the lasers appropriately as described in points 213 to 239. Steps 17, 19, 20, and 22 Focusing on sample is not possible Thickness of coverslip is not appropriate. Low amount of immersion oil (item 140) on the objective lens. Use #1.5 coverslips only. Squirt one or two drops of the immersion oil on the objective lens. Steps 17, 19, 20, and 22 Background, not from the sample, overwhelms the field of view External light sources are switched on. Front plate of the microscope box stowed away. Emission filters not properly inserted. Switch off all light sources, including the room’s main light and any other lamps. Fix the front plate to the microscope box as described in points 243 and 244. Properly insert the emission filters as described in points 121 and 122. Code availability Updated versions of the source code for NanoPro 1.0, as well as guiding instructions, can be obtained from https://github.com/jdanial/NanoPro. A compilation of NanoPro 1.0 for Windows OS is available as Supplementary Software. Contributions J.S.H.D and D.K conceived and designed the study. M.W and J.S.H.D designed the microscope. J.S.H.D, J.Y.L.L and Y.W assembled the microscope. J.S.H.D wrote the NanoPro 1.0 software with input from J.Y.L.L and Y.W. J.S.H.D, J.Y.L.L and Y.W performed the analysis. M.R.C, D.E, J.Y.L.L, Y.W and J.S.H.D revised the protocol. J.S.H.D wrote the manuscript with input from all authors. Acknowledgments We would like to thank Matthew Woolley, James Prill, and Shaun Impey from the mechanical workshop in the Yusuf Hamied Department of Chemistry at the University of Cambridge for fabricating the microscope assembly and Achini Jayasinghe (https://www.fiverr.com/achinijayasingh) for illustrating the guides. We would also like to thank Emmanouil Metzakopian and Emma Wilson (UK DRI Cambridge) for providing us with the HeLa cells. 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Shechtman, Y., Weiss, L. E., Backer, A. S., Sahl, S. J. & Moerner, W. E. Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions. Nano Lett. 15, 4194–4199 (2015). 41. Bongiovanni, M. N. et al. Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping. Nat Commun 7, 13544 (2016). 42. Zhang, Z., Kenny, S. J., Hauser, M., Li, W. & Xu, K. Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy. Nature Methods 12, 935–938 (2015). 43. Cnossen, J., Cui, T. J., Joo, C. & Smith, C. Drift correction in localization microscopy using entropy minimization. bioRxiv 2021.03.30.437682 (2021) doi:10.1101/2021.03.30.437682. Figures Figure 1 Schematic diagrams of open-source microscopies developed for super-resolution imaging. (a) miCube (excluding laser combiner, sample stage and sample enclosure). (b) K2 TIRF. (c) liteTIRF. (d) WOSM (excluding sample enclosure). (e) Unnamed low-cost microscope. (f) cellSTORM (including cell phone show in grey). Centrale module and sample holder shown in red, light tools and glass shown in yellow, enclosure and mounting pillars shown in green and Optomechanics shown in black or light grey. Figure and relative sizes are not to scale. Figure 2 Schematic diagram of the NanoPro 1.0 assembly and its individual components. (a) Full microscope assembly (excluding optics, electrical connections, small screws and some optomechanical components). (b) laser combiner showing the red, green, blue, and ultra violet lasers from left to right as well alignment mirrors and fibre launch system (excluding optical fibre). (c) Autofocus system showing infrared laser and photosensitive detector on the right. (d) X, Y and Z slip-stick positioners (including sample holder and sample). (e) Four-position slider. (f) sCMOS camera. (g) Perspex sample enclosure (including LED and excluding handle). (h) Dualshock PS4 control joystick. Figure 3 Assessing the performance of NanoPro 1.0 on ground-truth nanoruler samples. Drift- corrected DNA-PAINT images of (a) 40 nm, scale bar = 10 µm (100 nm for inset) and (b) 20 nm, scale bar = 10 µm (100 nm for inset), nanorulers (see materials). (b) exemplary drift correction curves as produced by a cross-correlation algorithm (X shown in green and Y shown in blue). (d) localization precision histogram under typical imaging conditions (see procedure). Counts (x10000). Figure 4 Images from good- and poor-quality alignment. Full field-of-view diffraction-limited images of the ground-truth 40 nm nanorulers’ sample acquired with (a) good alignment conditions, (b) excitation path misaligned, (c) emission path misaligned, and (d) sampled holder titled. Cropped-view super-resolved images of the 40 nm nanorulers’ sample acquired with (e) good fibre agitation showing homogeneous illumination and (f) no, or poor, fibre agitation showing dark and bright patches with varying localization densities. Scale bar (a – f), 10 µm, and insets, 1 µm. Figure 5 Exemplary images of cellular and recombinant macromolecular complexes using NanoPro 1.0. (a) Microtubules immunostained with a primary / secondary antibody system and imaged using dSTORM. (b) Clathrin pits immunostained with a primary / secondary antibody system and imaged using dSTORM. (c) Multi-target imaging of Microtubules and Clathrin pits using exchange DNA-PAINT. (d) Multi- sample imaging of recombinant alpha-Synuclein aggregates using aptamer-based DNA-PAINT at different concentrations: (1) 2.8 µM, (2) 1.4 µM, (3) 700 nM, (4) 350 nM, (5) 175 nM, (6) 87.5 nM, (7) 43.75 nM, and (8) 21.875 nM. Scale bar (a – c), 10 µm, and (d), 1 µm. Preparation protocols can be found in the supplementary information. Tables Attribute miCube K2TIRF liteTIRF WOSM Unnamed low cost microscope cellSTORM ONI Nano Imager NanoPro 1.0 Spatial resolution < 50 nm Unknown < 10 nm (with origami- based drift correctio) Unknown ~110 nm (with computational drift correction) ~ 100 nm (without drift correction) < 20 nm [listed] (with computational drift correction) < 20 nm (with computational drift correction) Temporal resolution (lowest exposure time at full frame) 10 ms 50 ms 25 ms 50 ms 20 ms 30 ms (depending on mobile phone used) 10 ms 10 ms Active drift stabilization No Yes No No No No Yes Yes Flat field illumination No Yes No No No No Yes Yes Construction complexity Low (partially Illustrated) High Medium Medium Medium Low (fully illustrated) Pre- assembled Low (fully illustrated) Multi sample imaging Yes, but not drift stable Yes, medium range (30 mm X/Y stage movement) No No No No Yes, medium range (30 mm X/Y stage movement) Yes, long range (50 mm X/Y stage movement) Multi color imaging No Yes No No No No Yes Yes 3D imaging No Yes Yes No No No Yes Possible, not demonstrated Analysis and post processing No No No No No No Yes No Illumination modes Epi HILO TIRF Epi HILO TIRF Epi HILO TIRF Epi HILO TIRF Epi HILO Epi Epi HILO TIRF Epi HILO TIRF Temperature control No Yes No No No No Yes Possible, not demonstrated Environment requirements None listed None listed None listed None listed None listed None listed None Ideally located in a vibration isolated room Setup cost for full features (not including maintenance or service) ~ 127,000 USD ~ 137,000 USD 24,000 USD ~ 70,000 – 125,000 USD ~ 3,800 USD 1,000 USD N/A 68,000 USD Table 1 Full comparison between NanoPro 1.0, open-source and commercial microscopes. Data is either published or obtained from quotations by consent. Cost category Costs (USD) Year 1 Costs (USD) Year 2 Costs (USD) Year 3 Costs (USD) Year 4 Costs (USD) Year 5 Direct costs Equipment Fibre (items 1 – 2) 1400 0 0 0 0 Light (items 3 – 9) 7600 1000 (laser replacement) 2000 (laser replacement and maintenance) 1000 (laser replacement) 2000 (laser replacement and maintenance) Light analysis (items 10 – 14) 13800 0 0 0 0 Motion control (items 15 – 18) 10400 0 0 0 0 Optics (items 19 – 40) 16300 0 0 0 0 Optoelectronics (items 41 – 62) 4300 100 (vibration motor replacement) 100 (vibration motor replacement) 100 (vibration motor replacement) 100 (vibration motor replacement) Optomechanics (items 63 – 134) 13700 0 0 0 0 Sample (Items 125 – 136) 2900 0 0 0 0 Safety (item 137) 200 0 0 0 0 Software (items 138 – 139) 0 0 0 0 0 Other (items 140 – 144) 200 0 (replacements to lens cleaning tissue [item 142] and immersion oil [item 140] are not included being lab consumables) 0 (replacements to lens cleaning tissue [item 142] and immersion oil [item 140] are not included being lab consumables) 0 (replacements to lens cleaning tissue [item 142] and immersion oil [item 140] are not included being lab consumables) 0 (replacements to lens cleaning tissue [item 142] and immersion oil [item 140] are not included being lab consumables) Total Indirect costs (USD) 70800 1100 2100 1100 2100 Indirect costs Personnel Step 1 (Procurement and space preparation) [6 months at 0.05% FTE of 1 PhD student] 2200 0 0 0 0 Step 2 (Fabrication) 0 (included in direct costs) 0 0 0 0 Step 3 (Unpacking and installation) [2 days at 100% FTE of 4 PhD students] 1900 0 0 0 0 Step 4 (Assembling the laser combiner) [3 hours at 100% FTE of 1 PhD student] 30 0 0 0 0 Step 5 10 0 0 0 0 (First partial assembly of the microscope box) [1 hour at 100% FTE of 1 PhD student] Step 6 (Assembling the excitation module and focus stabilization system) [2 hours at 100% FTE of 1 PhD student] 20 0 0 0 0 Step 7 (Second partial assembly of the microscope box [1 hour at 100% FTE of 1 PhD student] 10 0 0 0 0 Step 8 (Assembling the emission module) [1 hour at 100% FTE of 1 PhD student] 10 0 0 0 0 Step 9 (Assembling the sample stage) [2 hours at 100% FTE of 1 PhD student] 20 0 0 0 0 Step 10 (Setting up electrical connections) [2 hours at 100% FTE of 1 PhD student] 20 0 0 0 0 Step 11 (Assembling the laser combiner enclosure) [0.5 hours at 100% FTE of 1 PhD student] 5 0 0 0 0 Step 12 (Assembling the sample enclosure) [2 hours at 100% FTE of 1 PhD student] 20 0 0 0 0 Step 13 (Assembling and aligning the sample holder) [0.5 hours at 100% FTE of 1 PhD student] 5 0 0 0 0 Step 15 (Setting up the computer and 20 0 0 0 0 installing the NanoPro software) [2 hours at 100% FTE of 1 PhD student] Step 16 (Aligning the laser combiner) [4 hours at 100% FTE of 1 PhD student] 40 0 0 0 0 Step 17 (Aligning the excitation and emission paths) [0.5 hours at 100% FTE of 1 PhD student] 5 0 0 0 0 Step 18 (Aligning the focus stabilization system) [0.5 hours at 100% FTE of 1 PhD student] 5 0 0 0 0 Step 19 (Measuring the camera pixel size) [0.5 hours at 100% FTE of 1 PhD student] 5 0 0 0 0 Step 20 (Imaging ground- truth samples to establish performance) [1 hour at 100% FTE of 1 PhD student] 10 0 0 0 0 Step 21 (Data processing) [1 hour at 100% FTE of 1 PhD student] 10 0 0 0 0 Maintenance and troubleshooting [50 hours at 100% FTE of 1 PhD student] 0 500 500 500 500 Total indirect costs (USD) 4300 500 500 500 500 Total costs [Direct + Indirect] (USD) 75100 1600 2600 1600 2600 Table 2 Detailed cost breakdown of NanoPro 1.0. Table includes direct and indirect costs as well as projected maintenance costs up to five years. Full Time Equivalent (FTE) for PhD students is calculated at the highest published salary worldwide (https://ethz.ch/en/the-eth-zurich/working-teaching-and- research/welcome-center/employment-contract-and-salary/salary.html) which is 80,320 CHF (equivalent to 87,000 USD). FTE for technicians is calculated at 1.5 times the FTE for PhD students which amounts to 130,500 USD. Figures above 100 USD are approximated to the nearest 100 USD. Inflation and time value of money are not included. Component (item number) Alternative component Vendor (catalogue number) Price difference (USD) Effect on localization precision Apochromatic, 100x magnification, 1.49 numerical aperture, TIRF objective (item 19) 100 x magnification, 1.3 numerical aperture, objective Thorlabs (N100X-PFO) 5600 0.5 nm (theoretical calculations). Effect on TIRF imaging unquantified. Effect on multi-colour imaging unquantified. Prime BSI Express, sCMOS camera (item 14) Chameleon3 CM3-U3- 31S4M, PointGrey, CMOS camera Edmund Optics (36-075) 11,000 0.5 nm – 1 nm (theoretical calculations). Effect on multi-colour imaging unquantified. Table 3 Alternative components, their pricing, and their effect on the localization precision. Data obtained from18.