Table 1:Github repositories for hardware, electronics and software.Figure 1:Schematic of the high-resolution-UC2 widefield microscope.a. Overview of the different component categories of the microscope, from the hardware, through the electronics controlling, to the software and the ImSwitch-based GUI. b. Schematic of the complete setup. Red and blue arrows (i. and ii.) are references to help visualize the 3D structure. Top layer is the commercially bought XY-stage that was motorized for this project. Timing belts convert motor torque (two grey gears top left) into stage motion (bigger black gear middle) but have been omitted in this representation for clarity. Sample holder can be printed according to the sample used, circular in this case. c. Commercially bought precision Z-stage and high-NA objective (Olympus 60x/1.49 NA TIRF) d. Excitation layer. Laser emission (bright red light-path) is focused by a f = 100 mm lens onto the back focal plane of the objective. Orange arrow (iii.) marks the filter cube which has an excitation filter (Chroma ZET635/20x EX), a dichroic mirror (Chroma ZT640rdc) and on the bottom an emission filter (Chroma ET655lp long-pass for λ > 655 nm) to separate excitation from fluorescence. Red rectangle shows a telescope build including a diffusor (rotating piece of cling foil) in the focus point between both lenses. Telescope build magnifies and homogenizes the laser beam and can be used optionally. e. Detection layer, corresponds to the bottom layer in the setup (red star as reference). Emitted fluorescence (dark red light-path) is depicted with a f = 100 mm lens on the detector (Alvium 1800 U-158c CMOS camera from Allied Vision). All active optical elements, e.g. mirrors and lenses are mounted into the injection moulded cubes (50x50x50 mm, green) via custom 3D printed mounts. Cubes are connected laterally and vertically via the puzzle shaped connection pieces. Green arrows (iv.) indicate the connecting puzzle layers between the cubes.Figure 2:Widefield imaging of immuno-stained CV1 cells against tubulin (α and β-tubulin).a. Widefield image of a tubulin stained CV1 cell. Two regions of interest are highlighted. Scale bar represents 20 µm. b. Zoomed in images of the selected ROIs in a. with respective microtubule profile plotted across the white line in the ROI images. ROI dimensions are 8x8 µm. Grey values are plotted as columns and fitted with gaussian functions (red line). Microtubule diameter can be estimated to 340 to 420 nm according to the full width at half maximum of the fits. c. Illumination profile of whole camera chip (1456x1088 px) at the sample plane as imaged on a far-red fluorescent Chroma slide. Scale bar represents 35 µm. d. Tetra-speck beads imaged over a period of three hours and color coded over time. Scale bar represents 1 µm. Bead positions are localized over time to measure their displacement to their initial position over time.Figure 3:Live-cell imaging of actin in cultured cells.Shown are still images of the same CV-1 fibroblast cells in SiR-actin fluorescence and widefield (background corrected) at different timepoints over 5 hours. Scalebar on the right represents 30 µm.Figure 4:Long-term live-cell imaging of cultured cells inside the incubator.Shown are still images of the same population of T98G glioblastoma cells over time in the same field of view. Cells are stained with SYTO nucleic-acid fluorescent dye and imaged in widefield (background corrected) every 20 minutes for over 32 hours inside an incubator. Scalebar on the right represents 30 µm.Figure 5:Single particle tracking of GPI-GFP in live CV1 cells using functionalized Quantum dots.a. Schematic of the construct used to track single GPI- molecules with quantum dots. b. Exemplary raw images of single fluorescent Quantum Dots. Image dimension is 1.6 µm x 1.6 µm. c. Trajectories of several single molecules visualized on a top-light illuminated image of CV1 cells. Scale bar represents 10 µm. d. Selected trajectory of a single particle over 167 time-points. Sampling rate is 25 ms, pixel size 104 nm. e. Histogram of the diffusion coefficient of around 5000 tracks with 20 or more detected time points each. f. MSS (moment scaling spectrum) of the same population as plotted in e. Since we were readily able to detect single quantum dots, which are single emitters of few nanometers in size, we reasoned, it might be possible to perform single molecule localization microscopy (SMLM) experiments as well. We immuno-stained CV-1 cells against tubulin using AF647 coupled secondary antibodies and incubated coverslips in BME/GLOX buffer for effective pumping into the dark state. When we then illuminated our sample with high intensity laser light (in our case ∼ 1kW/cm2), we found that the intense staining quickly faded and gave way to the typically observed blinking in SMLM experiments. We took a timeseries of 30’000 frames and localized the detected emitters using Thunderstorm (Ovesný et al., 2014). Even though we were limited to widefield illumination, we achieved a drastic improvement in resolution in reconstructed SMLM micrographs (Figure 6a). When we measured the profile of individual microtubules, we could easily detect the typical “railroad track” pattern with a spacing of 38 – 43 nm, indicative of high quality SMLM imaging (Figure 6b,c). To quantify the achieved resolution, we used Fourier ring correlation (Nieuwenhuizen et al., 2013) and found that the overall achieved resolution was 93 nm, clearly below the optical resolution limit (Figure 6d).Figure 6:dSTORM reconstruction of immuno-stained CV1 cells against tubulin (a and /1-tubulin).a. Widefield image and reconstructed super-resolution image of 30’000 widefield frames, localized and reconstructed using ThunderSTORM, applied parameters described in the methods part. Reconstruction is displayed with the average shifted histogram method, including a 25-fold magnification. Scale bar represents 5 µm. Two regions of interest in the reconstruction are marked (red square for b. and green square for c.) and zoomed in (scale bar 1[μm). Profiles within these ROIs are measured by averaging the profile of the reconstructed image over 250[nm along the microtubule (white dashed rectangles). White histogram bars are the intensity values of the pixels in the reconstruction. Two peaks arise from the circular shape of microtubules. These were analyzed with a double gaussian fit (red curves are the cumulated single black fits). Distances of (38 ± 2)[nm (red box) and (43 ± 2)[nm (green box) could be extrapolated. d. Fourier ring correlation of the complete reconstructed image. Threshold is left to the preset 1/7. Overall resolution is estimated at 93 nm.