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Ando C. Zehrer et al., 2023 eLife. https://doi.org/10.7554/eLife.89826.1 1 of 23
Cell Biology
An open-source, high resolution,
automated fluorescence microscope
Ando C. Zehrer, Ana Martin-Villalba, Benedict Diederich , Helge Ewers
Institut für Chemie und Biochemie, Freie Universität Berlin, 14195 Berlin, Germany • Department of Molecular
Neurobiology, German Cancer Research Center (DFKZ), Im Neuenheimer Feld 581, 69120 Heidelberg, Germany •
Leibniz-IPHT Jena, Albert-Einstein-Str. 9, 07745 Jena, Germany
https://en.wikipedia.org/wiki/Open_access
https://creativecommons.org/licenses/by/4.0/
Abstract
Fluorescence microscopy is a fundamental tool in the life sciences, but the availability of
sophisticated equipment required to yield high-quality, quantitative data is a major
bottleneck in data production in many laboratories worldwide. This problem has long been
recognized and the abundancy of low-cost electronics and the simplification of fabrication
through 3D-printing have led to the emergence of open-source scientific hardware as a
research field. Cost effective fluorescence microscopes can be assembled from cheaply mass-
produced components, but lag behind commercial solutions in image quality. On the other
hand, blueprints of sophisticated microscopes such as light-sheet or super-resolution systems,
custom-assembled from high quality parts are available, but require a high level of expertise
from the user.
Here we combine the UC2 microscopy toolbox with high-quality components and integrated
electronics and software to assemble an automated high-resolution fluorescence microscope.
Using this microscope, we demonstrate high resolution fluorescence imaging for fixed and
live samples. When operated inside an incubator, long-term live-cell imaging over several
days was possible. Our microscope reaches single molecule sensitivity, and we performed
single particle tracking and SMLM super-resolution microscopy experiments in cells. Our
setup costs a fraction of its commercially available counterparts but still provides a
maximum of capabilities and image quality. We thus provide a proof of concept that high
quality scientific data can be generated by lay users with a low-budget system and open-
source software. Our system can be used for routine imaging in laboratories that do not have
the means to acquire commercial systems and through its affordability can serve as teaching
material to students.
Reviewed Preprint
Published from the original
preprint after peer review
and assessment by eLife.
About eLife's process
Reviewed preprint posted
August 31, 2023 (this version)
Sent for peer review
June 23, 2023
Posted to bioRxiv
May 31, 2023
Ando C. Zehrer et al., 2023 eLife. https://doi.org/10.7554/eLife.89826.1 2 of 23
eLife assessment
This important study provides convincing evidence that the low-cost, user-friendly,
and open-hardware UC2 wide-field microscopy framework can be expanded to
enable single-molecule localization microscopy. Because the approach offers a simple
and cost-effective alternative to commercially available microscopes, which are often
expensive, this setup will be important for anyone seeking affordable solutions for
single molecule localization microscopy and single particle tracking. While the
information and data are openly accessible, the documentation could be expanded
and better structured to (further) facilitate implementation in practice.
Introduction
Fluorescence microscopy is, due to its excellent contrast, specificity and compatibility with live cell
imaging, an essential tool in the life sciences. Digital imaging, quantitative analysis and ever
higher throughput and resolution in new techniques make fluorescence microscopy more
important than ever. Nevertheless, while the development of high-end instrumentation pushes
forward our progress in understanding cellular processes, spatial and temporal resolution are not
the only challenges impeding progress in scientific discovery. This is because necessarily, the
access to cutting-edge instrumentation is reserved to a subset of scientists as a result of its cost and
technical complexity, effectively limiting data production to laboratories with access to substantial
resources. At the same time, cost of electronic or optical components, powerful processors and
high-quality cameras are at an all-time low. Together with simple prototyping through 3D-printing,
this has led to the emergence of open-source scientific hardware as a research field (Wenzel,
2023 ). The open-source microscopy community has experienced the rapid development of
different approaches aiming to make microscopy more available (Almada et al., 2019 ; Alsamsam
et al., 2022 ; Ambrose et al., 2020 ; Auer et al., 2018 ; Danial et al., 2022 ; Diederich et al.,
2019a ; Hohlbein et al., 2022 ; Sharkey et al., 2016 ; Voigt et al., 2019 ; Wenzel, 2023 ).
The first iterations of open-source microscopes came from microscopy laboratories that tried to
provide similar quality data as commercial systems while being assembled from components for
lower overall cost, however still requiring significant expertise. Solutions now exist for single
molecule localization microscopy (SMLM) (Alsamsam et al., 2022 ; Auer et al., 2018 ; Holm et
al., 2014 ; Kwakwa et al., 2016 ; Martens et al., 2019 ), they include modular platforms (H. Li et
al., 2020 ), light sources (Berry et al., 2021 ; Schröder et al., 2020 ) and even sophisticated
special solutions such as light sheet microscopy (Voigt et al., 2019 ), spinning disc microscopes
(Halpern et al., 2022 ) and high-throughput applications (Li et al., 2019 ; Walzik et al., 2015 ).
On the other hand, another group of investigators tried to provide extremely cost-effective
microscopy solutions that are accessible to everyone. The result are solutions that can be useful in
detecting e.g. parasites in inaccessible regions of the world for simple diagnosis (Li et al., 2019 ),
but are incompatible with quantitative scientific imaging (Chagas et al., 2017 ; Cybulski et al.,
2014 ; Diederich et al., 2020 , 2019b ; Sharkey et al., 2016 ; Vera et al., 2016 ). Most of these
solutions lack excitation and emission for fluorescence imaging.
Here, we propose an instrument filling the gap between the low-cost open-source instruments for
the general public and high-quality fluorescence imaging, as required for scientific research in
molecular cell biology. We provide an automated high-resolution microscope with single molecule
sensitivity that can be fully assembled and steered by lay users. Our system is based on the UC2
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toolbox (Diederich et al., 2020 , 2019a ; Wang et al., 2022 ) using common optical elements,
affordable electronic components and 3D printed parts and is combined with a high NA objective,
motorized stage and intuitive GUI-based operation.
The result is a cost efficient and user-friendly high-resolution fluorescence microscope that is
compatible with long-term live-cell imaging, single particle tracking and single molecule
localization microscopy. All designs for the 3D parts, software and electronics are open-source
(Table 1 ). We here describe microscope assembly, benchmarking and a number of high-
resolution microscopy assays performed with our microscope.
Results
Assembly of a high-resolution UC2 microscope
We aimed to generate a cost-effective, high-resolution widefield fluorescence microscope that
allows us to perform a variety of common cell biological imaging experiments. These include
standard high-resolution diffraction limited immunofluorescence imaging, long-term live-cell
imaging, single particle tracking and super-resolution microscopy. To fulfill these criteria, we
reasoned that our system should have: i) excitation and detection powerful and sensitive enough
for single molecule localization, thus requiring a laser and an appropriate detector. ii) capability
to operate inside an incubator for long-term live-cell imaging, thus requiring small size and
automation. iii) an intuitive and user-friendly GUI for maximal accessibility. Lastly, the entire
system including the physical microscope, the electronics and the software must be open-source
and be possible to assemble and operate with moderate technical know-how (Figure 1a ). We
reasoned that the quality of the objective is paramount to image quality, while most other optical
parts of a light microscope can be mass-produced components. Furthermore, such a high-quality
objective allows for the swift diagnosis of problems in the assembly of the optical path due to its
high resolution and minimal chromatic and spatial aberrations. We decided to use an Olympus
1.49 NA oil objective, but any high-quality objective can be incorporated in our system, by
adapting the objective mount to the requirements of the objectives’ manufacturer.
Based on the guidelines described above and to ensure adaptability and modularity, we decided to
build our open-source automated microscope on the basis of the UC2 toolbox using its injection
molded cubes for the structural assembly. For ease of usage, we used an inverted configuration
and assembled the microscope in three layers to keep the size and design compact (Figure 1b ).
The high-precision x-y sample table and z-stage (Figure 1c ) were low-cost commercial solutions
that we motorized and implemented into the open-source, python-based microscope GUI as
automated elements of the microscope. The emission pathway at the bottom of the microscope
assembly is based on an Alvium 1800 U-158c CMOS camera from Allied Vision, which compared to
conventional scientific cameras is more affordable by a factor of 30 to 60 times at the time of
submission (Figure 1d ), more compact and consumes less energy. One important point here is
that the camera needs to be monochromatic as the Bayer pattern on a polychromatic camera
would significantly reduce the sampling as well as the quantum efficiency of a camera chip, which
is already lower compared to that of scientific cameras. The camera was connected to the
computer via USB 3.0, allowing fast data transfer and maximal compatibility. Between the
emission pathway and the sample stage, the excitation pathway contained a low-cost 635 nm
diode laser with an optional beam expander (Figure 1e ), that depending on the illumination
requirements could be inserted or removed, modulating the illumination density and
homogeneity of the excitation field (see Figure 2c ). To trigger the laser, control laser power and
to steer the x, y and z motors for focus and sample translation, we used a low-cost Arduino-coded
board that can be connected via USB3.0 to any computer. Electronics on the board and the USB-
camera are then directly controlled via an adapted GUI of the python-based open-source software
ImSwitch (Moreno et al. 2021 ), resulting in a fully automated microscope. Additionally,
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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 stars are references to help visualize the 3D structure. Top layer is the
commercially bought XY-stage. 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. 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). e. Excitation layer. Laser emission (bright red light-path) is focused by a f = 100 mm lens onto the back focal plane of the objective. The filter
cube has a excitation filter (Chroma ZET635/20x EX), a dichroic mirror (Chroma ZT640rdc) and on the bottom a (Chroma ET655lp long-pass) emission
filter (for λ > 655 nm) to separate excitation from fluorescence. Laser beam can be magnified and homogenized by using a telescope and a diffusor
(rotating cling foil) in the focus point between both lenses. Diffusor is optional and annotated with a red rectangle in the figure. All active optical
elements, e.g. mirrors and lenses are mounted to the cubes (50x50x50 mm) via custom 3D printed mounts. Cubes (green) are connected via the puzzle
pieces (white).
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ImSwitch includes the “ImScript” an automation feature, which allows to execute a sequence of
written Python commands with user defined parameters e.g. overnight time-series imaging
experiments.
Furthermore, we took advantage of the open-source (OS) nature of ImSwitch and implemented the
recently presented OS SMLM algorithms microEye (Alsamsam et al., 2022 ) to realize an online-
running localization framework that gives instant qualitative feedback if the sample preparation
works as expected. In addition, the feedback of these online-running image processing algorithms
can be looped back into the hardware control to eventually compensate possible drift (e.g.
autofocus). A large variety of available napari plugins can be used to directly work on the data
without moving them to a different computer.
All steerable electronics could alternatively be controlled with a PS4 controller for increased user-
friendliness. The assembly can be housed with 3D printed walls to block ambient light and
increase laser safety. In table 1 , a list of the different microscope and software elements and the
repository addresses to the corresponding materials and methods is displayed. A comprehensive
online repository provides the bill of materials (BOM), as well as a set of instructions on how to
setup the soft- and hardware (Table 1 ).
While assembling and testing the microscope excitation and detection, we observed a recurrent
pattern when we imaged a coverslip homogeneously covered with fluorescent molecules. Since we
only used a dichromatic mirror (for separation of excitation and emission light), we first tested,
whether a different emission (ET665lp) filter would remove the pattern. This was not the case.
Spectrometric measurement of the excitation laser beam confirmed that our low-cost laser had
minor bands in the wavelength range of 650 - 670 nm, which we could remove using an additional
band pass excitation filter (ZET635/20X). Our finding encouraged us in our approach to mix low-
budget components with high quality optics. Focusing and positioning of the sample is done with
commercial low-budget stages that offer great long-term stability. To render all three axes of
movement computer controlled, we first motorized an x-y stage via torque transmission form the
motor to the micrometer screws of the stage via timing belts. Secondly, we incorporated a
motorized lead screw-driven (NEMA11, 50mm, Amazon, 50€) z-stage holding the objective into the
modular design of the microscope.
Imaging experiments
We first used our microscope to image a sample of fixed mammalian cells stained against tubulin
via immunofluorescence using AF647. When we imaged these cells with moderate laser intensity,
beam expander and 50 ms integration time, we found that cells were evenly illuminated (Figure
2a ) and microtubules appeared as diffraction-limited lines inside cells (Figure 2b ). To
quantify flatness of illumination, we made use of a red auto-fluorescent benchmarking slide
(Chroma). The measured fluorescence intensity was quite homogenous across the field of view,
with variations of up to 25% of the maximal fluorescence intensity towards the edges (Figure
2c ).
These conditions are compatible with high quality fluorescence microscopy in life science
research. To test the stability of our system, we then imaged sub-diffraction fluorescent beads
repeatedly over time. When we then calculated the displacement of the beads over time, we
consistently found drift of less than 1 μm over 2 hours (Figure 2d ). We concluded that our
microscope may be suitable for imaging experiments that require repeated measurements of the
same field of view such as live-cell imaging. We thus mounted live cells at room temperature on
the optical table on our microscope and stained them with SiR-actin (Spirochrome). When we then
repeatedly illuminated the cells with moderate laser power to excite SiR-actin fluorescence and
with white light in transmission mode to image the cells, we found that the cells tolerated
acquisition conditions for several hours without damage visible in fluorescence or widefield
image (Figure 3 ). As expected, drift was negligible. Importantly, we found similar stability
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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 30 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). c. 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. Microtubule diameter can be
estimated to 340 to 420 nm according to the full width at half maximum of the fits.
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against drift over time, when we performed this experiment not on an optical table, but a wetlab
bench over a layer of foam material as a damping layer. This suggested that our microscope might
be suitable for long-term imaging in an environment that does not provide vibration damping,
such as a cell culture incubator. Given that our microscope assembly is of small size, and
connected via two flexible USB cables, we then moved on to perform live-cell imaging over longer
periods in a cell culture incubator.
To do so, we used a standard 37 °C CO2 incubator. The longer USB cables connecting the
microscope to the exterior allowed for steering from the outside. When we plated live T98G cancer
cells on coverslips and mounted them on the microscope sample holder in warm medium inside
the incubator, they exhibited normal morphology and growth over many hours. When we
performed a long-term imaging experiment using Syto deep-red fluorescence staining of the
nucleus (Thermo Fisher), we found that cells kept dividing and moving on the coverslip for several
days (Figure 4 ). We concluded that our setup was compatible with long-term live-cell imaging of
cells.
Next, we aimed to further explore the single molecule capabilities of our microscope and
performed single particle tracking (SPT) experiments on live CV-1 African green monkey cells
stably expressing GPI-GFP. When we incubated these cells with streptavidin 655 nm quantum dots
(QDs) coupled to biotin-tagged anti-GFP nanobodies (Figure 5a ), we detected QDs readily as
single diffraction limited entities on cells (Figure 5b ). When we then localized emission centers
and tracked the QDs over time, we could generate trajectories over many frames (Figure 5c,d )
and extract parameters describing particle movement such as the diffusion coefficient (D, Figure
5e ) and the moment scaling spectrum (MSS, Figure 5f (Ewers et al., 2005 )). Our
measurements were in agreement with our previous observations (J. H. Li et al., 2020 ).
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 ).
Discussion
Here we developed a cost effective, high-quality automated microscope that can be assembled by
laypeople with minimal background knowledge in optics, electronics and informatics. The
assembly guidelines including CAD files of the 3D printed parts, list of used components and
software are openly accessible on the Github repositories (see table 1 ), thus offering maximal
availability.
Our microscope is based on a simple modular assembly system called UC2 (You.See.Too.,
(Diederich et al., 2019a )). This framework is based on modular cubes that can hold different
optical components in place and allow for simplified alignment and assembly. This flexibility
furthermore facilitates the replacement of any lens, filter, laser camera or other components,
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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.
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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.
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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.
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Figure 6
dSTORM reconstruction of immuno-stained CV1 cells against tubulin (α and β -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 analysed with a double Gaussian fit (red curves are the cumulated single black fits). Distances of (38 ± 2)□μm (red box) and (43 ± 2)□μm
(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.
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allowing the user to implement already available parts into their setup, or to further trim down
the setup by removing unused parts. We made use of an improved version where the cubes are
made through injection molding, which provides a significantly higher manufacturing precision
than the same cubes fabricated using 3D-printing, resulting in greater mechanical stability and
reproducibility, while being available at low cost through mass production (Wang et al. 2022 ).
The hereby provided increased structural integrity makes the setup portable allows it to be
reproducibly built and dismantled within hours. This facilitates its use in remote areas. Improved
versions or updated parts could easily be shared digitally and printed locally, further promoting
the advantage of digital manufacturing in the laboratory. The here created assembly can easily be
replicated multiple times to increase imaging throughput still for a fraction of the cost of a single
high-end microscope.
We present data in standard immunofluorescence microscopy, live-cell microscopy and long-term
live-cell microscopy assays performed inside an incubator. Furthermore, we show single particle
tracking data of semiconductor quantum dots on live cells and single molecule localization based
super-resolution microscopy. In all cases, the quality of data we generated is comparable to those
generated by systems orders of magnitude more expensive.
Our system thus allows for sophisticated imaging assays to be performed at a professional level
with greatly reduced cost. At the same time, electronic control of components and remote steering
of the microscope can be implemented without background knowledge in electronics. This makes
the generation of high-quality image data accessible especially for life science research groups
with limited monetary means also in lower income countries. The main limitation of our system in
this direction is the requirement of a high-NA objective, the by far most expensive component of
the setup. Since for scientific imaging, high quality data and thus high-NA and corrected objectives
are essential, this component cannot be replaced. However, the quality of low-cost objectives has
recently been improving, even with objectives allowing for effective single molecule detection.
The reduced NA diminishes the localization precision and the signal to noise ratio, thus limiting
the possible applications. Development of brighter dyes might also be an alternative to improve
the imaging quality enough to account for low budget lasers and filters.
Due to its modular assembly, extensions may be easily implemented to our microscope. Depending
on the specific research aim, multiple excitation and emission wavelengths may be desirable, but
would require a redesign of the frame to incorporate motorized filter switching and more light
sources. Alternatively, multiband filters in combination with fiber-coupled and thus combinable
laser sources are also possible. In the future, the possibilities of this system could be significantly
expanded by adding more modules specific for additional capabilities and we specifically invite
the community to do so. Especially the combination with a low-budget spinning disc (Halpern et
al., 2022 ), TIRF illumination or light-sheet imaging (Diederich et al., 2020 ) are possible future
applications.
Further steps towards user-friendliness include the replacement of the laser light-source by a
high-power LED to reduce the requirement of laser safety. Another step would be to render the
electronics assembly fully plugable, thus eliminating the soldering steps that are required in the
current assembly.
Besides the application in basic research, this cheap and easy to assemble setup would be useful
for teaching the next generation of scientists that more than ever have to be able to synthesize
capabilities spanning several disciplines. We endorse making scientific instruments available for
more scientists, students as well as the general public. Our system builds on efforts in open science
regarding software (ImSwitch,(Moreno et al., 2021 ) and microscope hardware (UC2 (Diederich et
al., 2019a )) while benefitting from a growing field of open-source microscopy and laboratory
hardware in general (Almada et al., 2019 ; Auer et al., 2018 ; Hohlbein et al., 2022 ; Martens et
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al., 2019 ; Wenzel, 2023 ). Taken together with efforts in making fluorescent dyes available
(Lavis, 2021 ), fundamental obstacles to the free diffusion of capabilities and know-how in
science are being removed. We hope that our live-cell high-resolution incubator microscope
(U.C.STORM) will catalyze these efforts.
Methods
Component fabrication
In the components list, a detailed table describes all the different pieces used for the microscope,
including the price of each component. The CAD designs as well as further information about UC2
or setup building can be found on the Github repository (see table 1 ). XY and Z stages were
commercially bought for their low price and convenient use but custom-made versions to print
and build can be found on the UC2 repository (Table 1 ). In the slicing software Prusa slicer, the
original Prusa i3 MK3S & MK3S+ printer was set. The parts were printed with the 0.15 mm SPEED
or 0.15 mm QUALITY settings. The two materials used in this project were the Prusament PLA
(Polylactic Acid) and Prusament ABS (Acrylnitril-Butadien-Styrol) filaments (both from Prusa),
which can both be selected in the slicing software ABS was used wherever heavy loads may
deform the setup in the event of temperature gradients (e.g. objective mount).
Materials were printed with PLA infill (20 - 40%) (Tprint= 215 °C; Tbed= 60 °C). Materials printed
with ABS infill (20 - 40%) (Tprint= 255 °C; Tbed= 100 °C).
UC2 setup assembly
In the UC2 system, optical components were mounted in standardized cubes. To make alignment
possible over an optical excitation or emission path over several cubes distance, the components
must experience a large enough degree of freedom within the cubes so they can be moved
manually in angle and exact position, but low enough to keep components immobile over
prolonged time periods. The challenge presented by tilted optics can on the one side be addressed
during the design process; broader mounts/adapters are less prone to tilt due to higher contact
surface with the cube. Once the optics were orthogonal to the light path, their position in relation
to the optical axis was set manually with highest possible accuracy, using appropriate mounts.
The fine adjustment was done with tiltable mirrors, as using translational mounts for every
optical element would be complex to produce or costly. A helpful tool for adjustment was to
position an aperture in the excitation pathway. The maximum of the laser spot was set to be in the
center of the aperture hole and was adjusted using the mirror. The emission path was adapted in
the same way, as imaging a fluorescent surface allows to position the maximal laser intensity spot
on the center of the detector field of view (also with a mirror).
The setup entails two imaging channels: the laser induced fluorescence (excitation path) and the
LED top-light illumination (650 nm Star LED, Cree, 3W, brightfield). The top-light light source is
mechanically detangled from the stages to remain unaltered by sample motion. As the filter cube
in the microscope is immobile, the top-light illumination requires either a high-power white LED
or an LED that emits light in a wavelength range that is not blocked by the dichromatic mirror or
the emission filter (in our case 650-660 nm).
Setup parts availability and price
The different 3D printed parts, electronics and optics used in the assembly, the corresponding CAD
files as well as an estimation of the price can be found in the corresponding Github repositories, as
listed in table 1 . General information about the UC2 system can be found on the UC2 repository.
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Image acquisition and control software
For the microscopy control, acquisition and reconstruction software, we chose ImSwitch because
its modular design adapts very well to the inherently modular nature of the cube-based optics
platform. A novel firmware “UC2-ESP32” was developed, which is fully integrated into the python-
based ImSwitch structure. Using a unified REST API-like interface, lasers, motors, LEDs, LED arrays
and readout sensors can be controlled serially via WiFi and USB. To ensure wide distribution of
this device adapter, we used a readily available hardware module that can control multiple
stepper motors and easily adapt other hardware components such as the LEDs and lasers. An
online-based flash tool loads the latest firmware onto the Wemos ESP32 D1 R32 in combination
with the CNC Shield to create a fully functional microscopy control unit. Detailed documentation
can be found in the online documentation (https://youseetoo.github.io/ ). A Python library “UC2-
REST” adapts the USB serial and integrates it into the ImSwitch software. In this project, the
strengths of open-source were fully unleashed by extending the control and acquisition algorithm
ImSwitch with an online SMLM reconstruction algorithm by (Alsamsam et al., 2022 ). The user
can select different parameters from the MicroEye framework to locate blinking events directly in
the Napari viewer (Sofroniew et al., 2022 ) to directly track possible reconstruction or sample
artefacts. The UC2-specific fork amongst other repositories can be found in Table 1 .
Imaging modalities
Illumination
When implementing the telescope and beam homogenizer, the illumination density reaches up to
90 W/cm2 but could be tuned to any required value below that. In the focal point between both
lenses a rotating piece of cling film diffuses the beam. The variance in the laser density over the
field of view was measured with a fluorescent slide and is reproducibly lower than 25%.
Additionally, for long term experiments, lacking fluorescence intensity can to an extent be
compensated with longer detector exposure times. To perform SMLM i.e. dSTORM in our case, the
telescope is removed to have maximal laser density. The total available power at the sample plane
is 83 mW thus a laser density of around 520 W/cm2. But since the laser spot is smaller than the
FOV, sufficient laser density can be reached when only using a subset of the FOV (approximately a
third of the FOV) can be used with sufficient laser power (over 970 W/cm2) and a variation in laser
intensity of about 25% of the laser intensity maximum.
Objectives and pixel sizes
For the widefield imaging, the room temperature live cell imaging, the single particle tracking as
well as the dSTORM experiment, a 60x Olympus 1.49NA oil immersion objective (UPLAPO60XOHR)
is used. The setup optics have accordingly been chosen for maximal resolution thus matching the
Nyquist sampling rate on the camera. The pixel-size is determined with a calibrated grid and a
value of 104.1 nm is extrapolated. For the incubator measurements, a 20x Olympus 0.6 NA air
(MXPLFLN20X) is used. The optics in the setup are not adjusted to this objective, resulting in a
pixel-size of 319.4 nm.
Coverslip and cell seeding
Coverslip preparation
Metal holders were used in combination with circular coverslips (25 mm) which were first cleaned
with a 2% Hellmanex solution for 10 minutes then cleaned in ethanol for 10 minutes and were
finally placed in the plasma cleaner for 15 minutes after having dried. The 8-well sample-holders
(Ibidi) were washed with 1 M KOH for 10 min then rinsed with H2O and dried.
Ando C. Zehrer et al., 2023 eLife. https://doi.org/10.7554/eLife.89826.1 16 of 23
Cell culture medium
Cell culture medium was on a phenol red free DMEM base with 1% GlutaMAX and 10% FBS.
Cell seeding
Cells were detached from the cell culture dish with a 2 mM EDTA solution. 30’000 cells were
seeded on a 25 mm circular coverslip for dSTORM experiments and general wide-field imaging.
For the live cell experiments, 55’000 to 80’000 cells were seeded onto 25 mm coverslips,
alternatively 15’000 - 25’000 cells per well when using an 8 well chamber.
Microtubule staining
CV-1 cells (Wild-type Cercopithecus aethiops kidney fibroblasts) were first washed with 37 °C
warm PEM buffer. The cells were fixed with warm PEM with 4% PFA, 0.05% GA and 0.1% TX 100
for 20 minutes. After fixation, sample was quenched in 8 minutes with NH4Cl/PBS (50 mM) then
washed 3 times with PEM buffer. The cells were then permeabilized with 0.3 % TX100 in PEM for 5
minutes and washed with PEM buffer. The sample was then blocked for 30 minutes in imageIT
followed by 1 hour in a 4 % HS, 1 % BSA, 0.1 % TX100 PEM based solution.
The sample was incubated overnight (12□hours) at 4 °C with a primary mouse anti alpha- and
beta-tubulin IgG antibodies (Sigma-Aldrich (REF: T5168) and Sigma-Aldrich (REF: T5293)) diluted
by a factor of 1 to 350 in a 1% BSA and 0.1 % TX100 PEM based solution. Sample was thoroughly
washed 3 times for 5 minutes with a 1 % BSA and 0.1 % TX100 PEM-based solution. The cells were
then incubated with the secondary donkey anti-mouse antibody conjugated with the AF647
fluorophore, diluted by a factor of 1 to 250 in a 1% BSA and 0.1 % TX100 PEM based solution at
room temperature for 2 hours. The 3 washing steps were then repeated, as previously mentioned
for the primary antibody.
The sample was the post-fixated with a 4% PFA diluted in PEM solution, for 15 minutes. The cells
were then quenched with a 50 mM NH4Cl solution diluted in PBS for 8 minutes. Finally, the cells
was washed 3 times.
Single particle tracking
Anti-GFP nanobodies (LaG-16 anti-GFP, own production according to (Fabricius et al., 2018 ))
were added to a biotin (α-Biotin-ω-(succinimidyl propionate)-24(ethylene glycol)) solution at
double molar excess. After 1 hour of incubation on a shaker (300 rpm), the nanobody-biotin
construct was purified by sequential filtration through three 7 kDa MWCO desalting columns.
The biotin conjugated anti-GFP nanobodies were mixed in a 1 to 1 ratio with the streptavidin
coated quantum dots and incubated for 10 minutes at room temperature. The solution was then
diluted into 1 ml of live cell medium. The cells were then incubated at room temperature with this
solution for 15 minutes. Afterwards the supernatant was removed and replaced with regular live
cell-medium (Thermo Fischer).
SiR actin staining
The CV-1 cells were washed in 37° C warm Medium (DMEM without phenol red + FBS + glutamax).
1 μl of the original SiR Actin (Spirochrome, 100 nM) solution was diluted in 1 ml of 37° C warm
Medium (DMEM without phenol red + 10 % FBS + 1 % glutamax). To this solution, 1 μl of verapamil
was added. The sample medium was removed and exchanged with this solution. After 1 hour of
incubation in the incubator, the sample was imaged in a live cell imaging solution (Invitrogen) for
the following hours at room temperature.
Ando C. Zehrer et al., 2023 eLife. https://doi.org/10.7554/eLife.89826.1 17 of 23
Nucleic staining
T98G cells were washed in 37°□C prewarmed medium (DMEM without phenol red + 10 % FBS + 1
% glutamax). For staining, 1□μl of SYTO™ Red Fluorescent Nucleic Acid Stain (Invitrogen) was
diluted in 1□ml of complete medium and incubated with the sample for 30 minutes at 37°□C in the
incubator, then imaged over prolonged time.
(d)STORM imaging
Imaging buffer
Imaging buffer was made by adding 400 μl Beta-mercaptoethanol to 80 μl GLOX (2.5 mg/ml glucose
oxidase, 0.2 mg/ml catalase, 200 mM Tris-HCl pH 8.0, 50 % glycerol).
Image acquisition
The 30’000 frames of the (d)STORM data set have been acquired with ImSwitch on the full camera
chip (1456 x 1088 px). The exposure time was 20 ms, the gain was set to 20 (maximal value), the
black-level and offset to 0 and no time delay was set between two frames. Laser power was set to
1024 (maximal value). Setup was positioned on a regular table with a layer of foam material to
dump vibrations. The room was left during the measurement.
The images were acquired within around 15 minutes and saved directly on the computer disc in a
.hdf5 file format for time efficiency and data handling advantages.
Analysis and reconstruction
The (d)STORM data-set was imported as a whole into ImageJ as a hdf5 file. Acquiring the full CMOS
chip 30’000 times generated enormous datasets (95.6 Gb). To load the file, a Fiji plugin to load the
stack as virtual stack was used. (N5, (Saalfeld et al., 2022 )). The stack of images was then
cropped to retain the region of interest, which has been positioned to match the region where
sufficient laser density induces blinking of single molecules during the acquisition. The analysis
was done using ThunderSTORM (Ovesný et al., 2014 ), a publicly available SMLM reconstruction
plugin on ImageJ. The camera parameters mentioned in the image acquisition section were used
for the analysis. Image filtering was done with a Difference-of-Gaussian filter σl =1.1 px; σ2 = 1.7
px. The approximate localization of molecules was done using the local maximum method with
default settings (std(Wave.F1) as peak intensity threshold and 8-neighborhood connectivity). Sub-
pixel localization of molecules was done with the PSF: Integrated Gaussian (Fitting radius 3px) and
maximum likelihood fitting method (initial sigma 1.7 px). Reconstruction of localized the raw-data
was visualized using the average shifted histograms method (Magnification: 25x; lateral shifts: 3).
Author contributions
HE and AMV acquired funding and conceptualized the study. BD developed the UC2 platform and
adapted the electronics and software. ACZ and BD build and improved the microscope and its
application. ACZ conducted the experiments and data analysis. ACZ, HE and BD wrote the paper.
Acknowledgements
This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research
Foundation) as part of TRR 186 (Project Number 278001972) and by Freie Universität Berlin. The
authors would like to thank all members of the Ewers laboratory for helpful discussions. We thank
Ando C. Zehrer et al., 2023 eLife. https://doi.org/10.7554/eLife.89826.1 19 of 23
References
Almada P, Pereira PM, Culley S, Caillol G, Boroni-Rueda F, Dix CL, Charras G, Baum B, Laine RF,
Leterrier C, Henriques R. (2019) Automating multimodal microscopy with NanoJ-Fluidics
Nat Commun 10 https://doi.org/10.1038/s41467-019-09231-9
Alsamsam MN, Kopūstas A, Jurevičiūtė M, Tutkus M. (2022) The miEye: Bench-top
superresolution microscope with cost-effective equipment HardwareX 12 https://doi.org
/10.1016/j.ohx.2022.e00368
Ambrose B, Baxter JM, Cully J, Willmott M, Steele EM, Bateman BC, Martin-Fernandez ML, Cadby
A, Shewring J, Aaldering M, Craggs TD (2020) The smfBox is an open-source platform for
single-molecule FRET Nat Commun 11 https://doi.org/10.1038/s41467-020-19468-4
Auer A, Schlichthaerle T, Woehrstein JB, Schueder F, Strauss MT, Grabmayr H, Jungmann R.
(2018) Nanometer-scale Multiplexed Super-Resolution Imaging with an Economic 3D-
DNA-PAINT Microscope Chemphyschem 19:3024–3034 https://doi.org/10.1002/cphc
.201800630
Berry K, Taormina M, Maltzer Z, Turner K, Gorham M, Nguyen T, Serafin R, Nicovich PR (2021)
Characterization of a fiber-coupled EvenField illumination system for fluorescence
microscopy Opt Express, OE 29:24349–24362 https://doi.org/10.1364/OE.430440
Chagas AM, Prieto-Godino LL, Arrenberg AB, Baden T. (2017) The €100 lab: A 3D-printable
open-source platform for fluorescence microscopy, optogenetics, and accurate
temperature control during behaviour of zebrafish, Drosophila, and Caenorhabditis
elegans PLOS Biology 15 https://doi.org/10.1371/journal.pbio.2002702
Cybulski JS, Clements J, Prakash M. (2014) Foldscope: Origami-Based Paper Microscope PLOS
ONE 9https://doi.org/10.1371/journal.pone.0098781
Danial JSH, Lam JYL, Wu Y, Woolley M, Dimou E, Cheetham MR, Emin D, Klenerman D. (2022)
Constructing a cost-efficient, high-throughput and high-quality single-molecule
localization microscope for super-resolution imaging Nat Protoc 17:2570–2619 https://doi
.org/10.1038/s41596-022-00730-6
Diederich B, Diederich B, Richter R, Carlstedt S, Uwurukundo X, Uwurukundo X, Wang H, Mosig
A, Heintzmann R, Heintzmann R. (2019) UC2 – A 3D-printed General-Purpose Optical Toolbox
for Microscopic ImagingImaging and Applied Optics 2019 (COSI, IS
MATH, PcAOP (2019) Paper ITh3B.5. Presented at the Imaging Systems and Applications
https://doi.org/10.1364/ISA.2019.ITh3B.5
Diederich B, Lachmann R, Carlstedt S, Marsikova B, Wang H, Uwurukundo X, Mosig AS,
Heintzmann R. (2020) A versatile and customizable low-cost 3D-printed open standard for
microscopic imaging Nat Commun 11 https://doi.org/10.1038/s41467-020-19447-9
Diederich B, Then P, Jügler A, Förster R, Heintzmann R. (2019) cellSTORM—Cost-effective
super-resolution on a cellphone using dSTORM PLOS ONE 14 https://doi.org/10.1371/journal
.pone.0209827
Ando C. Zehrer et al., 2023 eLife. https://doi.org/10.7554/eLife.89826.1 20 of 23
Ewers H, Smith AE, Sbalzarini IF, Lilie H, Koumoutsakos P, Helenius A. (2005) Single-particle
tracking of murine polyoma virus-like particles on live cells and artificial membranes Proc
Natl Acad Sci USA 102:15110–15115 https://doi.org/10.1073/pnas.0504407102
Fabricius V, Lefebre J, Geertsema H, Marino SF, Ewers H. (2018) Rapid and efficient C-terminal
labeling of nanobodies for DNA-PAINT J Phys D Appl Phys 51 https://doi.org/10.1088/1361
-6463/aae0e2
Halpern AR, Lee MY, Howard MD, Woodworth MA, Nicovich PR, Vaughan JC (2022) Versatile,
do-it-yourself, low-cost spinning disk confocal microscope Biomed Opt Express, BOE
13:1102–1120 https://doi.org/10.1364/BOE.442087
Hohlbein J, Diederich B, Marsikova B, Reynaud EG, Holden S, Jahr W, Haase R, Prakash K. (2022)
Open microscopy in the life sciences: quo vadis? Nat Methods 19:1020–1025 https://doi.org
/10.1038/s41592-022-01602-3
Holm T, Klein T, Löschberger A, Klamp T, Wiebusch G, van de Linde S, Sauer M. (2014) A
Blueprint for Cost-Efficient Localization Microscopy ChemPhysChem 15:651–654 https://doi
.org/10.1002/cphc.201300739
Kwakwa K, Savell A, Davies T, Munro I, Parrinello S, Purbhoo MA, Dunsby C, Neil MAA, French
PMW (2016) easySTORM: a robust, lower-cost approach to localisation and TIRF
microscopy Journal of Biophotonics 9:948–957 https://doi.org/10.1002/jbio.201500324
Lavis LD (2021) What if we just give everything away? eLife 10 https://doi.org/10.7554/eLife
.74981
Li H, Krishnamurthy D, Li E, Vyas P, Akireddy N, Chai C, Prakash M. (2020) Squid: Simplifying
Quantitative Imaging Platform Development and Deployment https://doi.org/10.1101
/2020.12.28.424613
Li H, Soto-Montoya H, Voisin M, Valenzuela LF, Prakash M. (2019) Octopi: Open configurable
high-throughput imaging platform for infectious disease diagnosis in the field https://
doi.org/10.1101/684423
Li JH, Santos-Otte P, Au B, Rentsch J, Block S, Ewers H. (2020) Directed manipulation of
membrane proteins by fluorescent magnetic nanoparticles Nat Commun 11 https://doi.org
/10.1038/s41467-020-18087-3
Martens KJA, van Beljouw SPB, van der Els S, Vink JNA, Baas S, Vogelaar GA, Brouns SJJ, van
Baarlen P, Kleerebezem M, Hohlbein J. (2019) Visualisation of dCas9 target search in vivo
using an open-microscopy framework Nat Commun 10 https://doi.org/10.1038/s41467-019
-11514-0
Moreno XC, Al-Kadhimi S, Alvelid J, Bodén A, Testa I. (2021) ImSwitch: Generalizing
microscope control in Python Journal of Open Source Software 6https://doi.org/10.21105/joss
.03394
Nieuwenhuizen RPJ, Lidke KA, Bates M, Puig DL, Grünwald D, Stallinga S, Rieger B. (2013)
Measuring image resolution in optical nanoscopy Nat Methods https://doi.org/10.1038
/nmeth.2448
Ando C. Zehrer et al., 2023 eLife. https://doi.org/10.7554/eLife.89826.1 21 of 23
Ovesný M, Křížek P, Borkovec J, Švindrych Z, Hagen GM (2014) ThunderSTORM: a
comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution
imaging Bioinformatics 30:2389–2390 https://doi.org/10.1093/bioinformatics/btu202
Saalfeld S, Pisarev I, Hanslovsky P, Champion A, Rueden C, Bogovic J, Kittisopikul M jakirkham
(2022) saalfeldlab/n5: n5-2.5.1 https://doi.org/10.5281/zenodo.6578232
Schröder D, Deschamps J, Dasgupta A, Matti U, Ries J. (2020) Cost-efficient open source laser
engine for microscopy Biomed Opt Express, BOE 11:609–623 https://doi.org/10.1364/BOE
.380815
Sharkey JP, Foo DCW, Kabla A, Baumberg JJ, Bowman RW (2016) A one-piece 3D printed
flexure translation stage for open-source microscopy Review of Scientific Instruments
87 https://doi.org/10.1063/1.4941068
Sofroniew N, Lambert T, Evans K, Nunez-Iglesias J, Bokota G, Winston P, Peña-Castellanos G,
Yamauchi K, Bussonnier M, Doncila Pop D, Can Solak A, Liu Z, Wadhwa P, Burt A, Buckley G,
Sweet A, Migas L, Hilsenstein V, Gaifas L, Bragantini J, Rodríguez-Guerra J, Muñoz H, Freeman J,
Boone P, Lowe A, Gohlke C, Royer L, PIERRÉ A, Har-Gil H, McGovern A. (2022) napari: a multi-
dimensional image viewer for Python https://doi.org/10.5281/zenodo.7276432
Vera RH, Schwan E, Fatsis-Kavalopoulos N, Kreuger J. (2016) A Modular and Affordable Time-
Lapse Imaging and Incubation System Based on 3D-Printed Parts, a Smartphone, and Off-
The-Shelf Electronics PLOS ONE 11 https://doi.org/10.1371/journal.pone.0167583
Voigt FF, Kirschenbaum D, Platonova E, Pagès S, Campbell RAA, Kastli R, Schaettin M, Egolf L,
van der Bourg A, Bethge P, Haenraets K, Frézel N, Topilko T, Perin P, Hillier D, Hildebrand S,
Schueth A, Roebroeck A, Roska B, Stoeckli ET, Pizzala R, Renier N, Zeilhofer HU, Karayannis T,
Ziegler U, Batti L, Holtmaat A, Lüscher C, Aguzzi A, Helmchen F. (2019) The mesoSPIM
initiative: open-source light-sheet microscopes for imaging cleared tissue Nat Methods
16:1105–1108 https://doi.org/10.1038/s41592-019-0554-0
Walzik MP, Vollmar V, Lachnit T, Dietz H, Haug S, Bachmann H, Fath M, Aschenbrenner D,
Abolpour Mofrad S, Friedrich O, Gilbert DF (2015) A portable low-cost long-term live-cell
imaging platform for biomedical research and education Biosensors and Bioelectronics
64:639–649 https://doi.org/10.1016/j.bios.2014.09.061
Wang H, Lachmann R, Marsikova B, Heintzmann R, Diederich B. (2022) UCsim2: two-
dimensionally structured illumination microscopy using UC2 Philosophical Transactions of
the Royal Society A: Mathematical, Physical and Engineering Sciences 380 https://doi.org/10.1098
/rsta.2020.0148
Wenzel T. (2023) Open hardware: From DIY trend to global transformation in access to
laboratory equipment PLOS Biology 21 https://doi.org/10.1371/journal.pbio.3001931
Author information
Ando C. Zehrer
Institut für Chemie und Biochemie, Freie Universität Berlin, 14195 Berlin, Germany
Ana Martin-Villalba
Department of Molecular Neurobiology, German Cancer Research Center (DFKZ), Im
Neuenheimer Feld 581, 69120 Heidelberg, Germany
Ando C. Zehrer et al., 2023 eLife. https://doi.org/10.7554/eLife.89826.1 22 of 23
Benedict Diederich
Leibniz-IPHT Jena, Albert-Einstein-Str. 9, 07745 Jena, Germany
For correspondence: benedict.diederich@leibniz-ipht.de
Helge Ewers
Institut für Chemie und Biochemie, Freie Universität Berlin, 14195 Berlin, Germany
For correspondence: helge.ewers@fu-berlin.de
Editors
Reviewing Editor
Felix Campelo
ICFO-Institut de Ciencies Fotoniques, Spain
Senior Editor
Tony Ng
King's College London, United Kingdom
Reviewer #1 (Public Review):
The authors have developed an open-source high-resolution microscope that is easily
accessible to scientists, students, and the general public. The microscope is specifically
designed to work with incubators and can image cells in culture over long periods. The
authors provide detailed instructions for building the microscope and the necessary software
to run it using off-the-shelf components. The system has great potential for studying cell
biology and various biological processes.
The authors' work will make scientific instruments more accessible and remove obstacles to
the free diffusion of capabilities and know-how in science. This important contribution will
enable more people to conduct scientific research.
Reviewer #2 (Public Review):
Making state-of-the-art (super-resolution) microscopy widely available has been the subject
of many publications in recent years as correctly referenced in the manuscript. By advocating
the ideas of open-microscopy and trying to replace expensive, scientific-grade components
such as lasers, cameras, objectives, and stages with cost-effective alternatives, interested
researchers nowadays have a number of different frameworks to choose from. In the
iteration of the theme presented here, the authors used the existing modular UC2 framework,
which consists of 3D printable building blocks, and combined a cheapish laser, detector and
x,y,(z) stage with expensive filters/dichroics and a very expensive high-end objective (>15k
Euros). This particular choice raises a first technical question, to which extent a standard NA
1.3 oil immersion objective available for <1k would compare to the chosen NA 1.49 one.
The choice of using the UC2 framework has the advantage, that the individual building blocks
can be 3D printed, although it should be mentioned that the authors used injection-molded
blocks that will have a limited availability if not offered commercially by a third party. The
strength of the manuscript is the tight integration of the hardware and the software (namely
the implementations of imSwitch as a GUI to control data acquisition, OS SMLM algorithms
for fast sub-pixel localisation and access to Napari).
Ando C. Zehrer et al., 2023 eLife. https://doi.org/10.7554/eLife.89826.1 23 of 23
The presented experimental data is convincing, demonstrating (1) extended live cell imaging
both using bright-field and fluorescence in the incubator, (2) single-particle tracking of
quantum dots, and (3) and STORM measurements in cells stained against tubulin.
In the following I will raise two aspects that currently limit the clarity and the potential
impact of the manuscript.
First, the manuscript would benefit from further refinement. Elements in Figure 1d/e are not
described properly. Figure 2c is not described in the caption. GPI-GFP is not introduced. MMS
(moment scaling spectrum) could benefit from a one sentence description of what it actually
is. In Figure 6, the size of the STORM and wide-field field of views are vastly different, the
distances between the peaks on the tubuili are given in micrometers rather than nanometers.
(more in the section on recommendations for the author)
Second, and this is the main criticism at this point, is that although all the information and
data is openly available, it seems very difficult to actually build the setup due to a lack of
proper documentation (as of early July 2023).
1. The bill of materials (https://github.com/openUC2/UC2-STORM-and-Fluorescence#bill-of
-material) should provide a link to the commercially available items. Some items are named
in German. Maybe split the BoM in commercially available and 3D printable parts (I first
missed the option to scroll horizontally).
2. The links to the XY and Z stage refer to the general overview site of the UC2 project (https://
github.com/openUC2/) requiring a deep dive to find the actual information.
3. Detailed building instructions are unfortunately missing. How to assemble the cubes (pCad
files showing exploded views, for example)? Trouble shooting?
4. Some of the hardware details (e.g. which laser was being used, lenses, etc) should be
mentioned in the manuscript (or SI)
I fully understand that providing such level of detail is very time consuming, but I hope that
the authors will be able to address these shortcomings.
