An open-source, high-resolution, automated fluorescence microscope
- Institut für Chemie und Biochemie, Freie Universität Berlin, Germany
- Department of Molecular Neurobiology, German Cancer Research Cente, Germany
- Leibniz-IPHT Jena, Germany
Figures
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 …
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 …
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 hr. 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 …
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) …
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. …
Author response image 1
Diagonal cross-section of the illuminated FOV.
Pixel-size (104nm) is the same as in figure 2. Intensity has been normalized according to the maximal value.
Author response image 2
Drift Figure: a.
Drift of fluorescent TS beads on the UC2 setup positioned upon an optical table over a duration of two hours. Beads are localized and resulting displacement in i. and ii. are plotted in the graphs …
Author response image 3
Z-focus Figure: Estimation of the axial position of TS beads on the UC2 setup.
a. The change in PSF FWHM was quantified by acquiring a Z stack of a beads sample. The homebuilt high-quality setup (HQ) was used as a reference, by using the same objective and TS sample. The PSF …
Author response image 4
3D concept Figure: Two possible setup modifications to provide axial information when imaging single molecules.
a. A cylindrical lens can be placed to induce an asymmetry between the PSF FWHM in x and in y. Every Z position can be identified by two distinct PSF FWHM values in X and Y. b. By splitting the beam …
Author response image 5
Profile Figure: By moving a combination of pinhole and photometer to scan through the laser profile with a translational mount, the shape of the laser beam can be estimated.
The cling foil plays the same role as a diffuser in other setups.
Author response image 6
Basic concept of the UC2 setup: Left: Cubes (green) are connected to one another via puzzle pieces (white).
Middle: 3D printed mounts have been designed to adapt various optics (right) to the cube framework. Combined usage of cubes and design of various mounts allows to interface various optics for the …
Author response image 7
Building the UC2 widefield microscope: a.
Photograph of the complete setup. b. All pieces necessary to build the setup. A list of the components can be found in the bill of materials. c. Bottom emission layer of the microscope before …
Author response image 8
Measurements performed on the UC2 setup with lower budget objectives.
The imaged sample is HeLa cells, stably transfected to express CLC-GFP, then labelled with AF647 through immunostaining. The setup has been kept identical except for the objectives. Scale bar …
Tables
Table 1
Github repositories for hardware, electronics and software.
| Repository | Link: |
|---|---|
| General openUC2 | https://github.com/openUC2/UC2-GIT; copy archived at Diederich, 2024 |
| Specific to this setup: UC.STORM | https://github.com/openUC2/UC2-STORM-and-Fluorescence, (openUC2, 2024b) |
| ImSwitch (UC2 specific) | https://github.com/openUC2/ImSwitch/. (openUC2, 2024c) |
| UC2-Rest | https://github.com/openUC2/UC2-REST, (openUC2, 2024a) |
| UC2-ESP32 | https://github.com/youseetoo/uc2-esp32, (youseetoo, 2024) |
| UC2-ESP32 Firmware Flashintool (Online) | https://youseetoo.github.io/ |
