A high-resolution, easy-to-build light-sheet microscope for subcellular imaging
- Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, United States
- Cecil H. and Ida Green Center for Systems Biology, UT Southwestern Medical Center, United States
- Department of Systems Biology and Engineering, Silesian University of Technology, Poland
Figures
Figure 1 with 3 supplements
Optical design of Altair light-sheet fluorescence microscopy (LSFM).
(a) Rendering of the detection arm elements. (b) Zemax Opticstudio layout and beam path of optimized illumination arm, where L1, L2, L3, and L4 are 30, 80, 75, and 250 mm achromatic doublets, respectively, and ILO is the TL20X-MPL illumination objective. (c) The simulated light-sheet beam profile in the xz plane at the focus of the illumination objective. The inset shows an enlarged region of the illumination light sheet, highlighting light-sheet thickness and uniformity. (d) The cross-sectional profile through the center of the light-sheet beam profile in (c), where the full-width-half-maximum (FWHM) of the light sheet was found to be 0.382 µm.
Figure 1—figure supplement 1
Comparison of sample- and light-sheet-scanning modes.
(a) In high-resolution light-sheet microscopy, the specimen must be positioned precisely at the intersection of the illumination and detection objective focal planes. To minimize aberrations in the excitation (blue) and detection (green) light paths, the specimen must be mounted at an angle that prevents marginal rays from interacting with the coverslip. In this configuration, only a narrow cross-section of an adherent cell is illuminated. (b) With a sample-scanning approach, the illumination beam requires a propagation length just sufficient to cover the thickest portion of the specimen—typically the nucleus—at the angle defined by the coverslip. For Altair-LSFM, the sample is mounted at ~30°, so a 6-µm-thick nucleus requires a beam propagation length of 6 µm/sin 30° ≈ 12 µm, which can be achieved with an illumination NA of ~0.285, producing a beam thickness of ~1 µm. The acquired volume is indicated by the dashed outline. (c) In contrast, a light-sheet-scanning configuration—where the light sheet and detection objective are synchronously translated in z—must generate a beam long enough to span the full cell diameter. For an adherent cell ~30 µm in diameter, the sheet must extend 30 µm/sin 30° ≈ 60 µm, requiring an illumination NA of ~0.128 and yielding a sheet thickness of ~2.3 µm. Together, these schematics illustrate how sample scanning enables the use of shorter, thinner light sheets that improve axial resolution while maintaining uniform illumination. The illumination NA required to achieve a given beam propagation length was estimated using the PSFGenerator package (Kirshner et al., 2013).
Figure 1—figure supplement 2
Educational illustration depicting the conceptual function of the resonant galvo unit.
Without pivoting of the light sheet, objects within a sample can cast shadows due to scattering or refraction. When the resonant galvo is engaged, the illumination sheet rapidly pivots at a frequency of 4 kHz such that the sample is effectively illuminated from multiple directions, and the shadows are correspondingly displaced. As a result, light effectively ‘reaches around’ objects, and the shadows are averaged out over the image acquisition period. This figure is intended as a conceptual aid to help readers visualize the role of the resonant galvo rather than act as a quantitative representation.
Figure 1—figure supplement 3
Tolerance analysis of illumination optics.
(a) Depiction of the merit function criteria used in our tolerance analysis, where we observed how the beam profile in the perturbed instances changes in both size and position. (b) Schematic of the Polaris dowel pin mounting configuration when considering machining tolerances, where in a worst-case scenario the angle offset would be 1.454°. (c) Nominal, best case, and worst-case beam profiles in xz for both coarse (+–0.005”, top row) and fine (+–0.002”, bottom row) machining tolerances.
Figure 2 with 4 supplements
Mechanical design of Altair light-sheet fluorescence microscopy (LSFM).
(a) Rendering of the completed illumination arm baseplate, with an inset showing the dowel pin holes compatible with the Polaris mounting line from Thorlabs. (b) Overhead view of the imaging configuration of our system, where our detection objective and illumination objectives are placed orthogonal to each other and the sample is scanned diagonally in the space between them in the axial direction shown by the white dashed line. (c) Rendering of our sample mounting and translation system. Here, a piezo motor is mounted onto an angled adapter to allow precise translation over the diagonal region between the objectives. Our custom 5 mm coverslip sample holder is also featured, where the inset shows an exploded assembly of the holder.
Figure 2—figure supplement 1
Baseplate post-installation procedure.
Process of affixing a post to the baseplate, where one first places the post onto dowel pins inserted into the corresponding holes and then fixes the post to the baseplate with a screw.
Figure 2—figure supplement 2
Computer-aided design (CAD) model of the Altair light-sheet fluorescence microscopy (LSFM) system.
CAD rendering of our full system consisting of an illumination path, a detection path, and a dedicated sample positioning assembly.
Figure 2—figure supplement 3
Custom sample chamber in Altair light-sheet fluorescence microscopy (LSFM).
(a) Computer-aided design (CAD) rendering of our custom sample chamber, featuring three possible objective ports, each with two sets of O-rings to ensure a liquid-proof seal. (b) Top-down rendering of the traditional imaging configuration for the system, where the illumination and detection objectives are placed orthogonal to one another. (c) The second transmissive imaging configuration of the system used to image the beam itself, where the illumination objective is placed directly in front of the detection objective.
Figure 2—figure supplement 4
System wiring diagram for optoelectronic control.
General wiring diagram of the system showing all the optoelectrical and optomechanical components used, and an inset showing how these components are wired into the NI DAQ.
Figure 3
Experimental characterization of light-sheet thickness and resolution.
(a) Experimental light-sheet beam profile at the focus. The light sheet was visualized in a transmission geometry with fluorescence derived from a fluorescein solution. (b) The center cross-section profile of (a), showing both raw data and a fitted curve with a full-width-half-maximum (FWHM) of ~0.415 µm. (c–e) Maximum-intensity projections for an isolated 100 nm fluorescent bead. All three orthogonal perspectives are provided to reveal any potential optical aberrations. A slight degree of coma is observable in the XZ view. (f) Gaussian-fitted distribution of the FWHM of beads imaged in a z-stack in each dimension both before (solid) and after (dashed) deconvolution.
Figure 4 with 1 supplement
Multicolor subcellular imaging with Altair light-sheet fluorescence microscopy (LSFM).
Lateral maximum-intensity projections of mouse embryonic fibroblasts (MEFs) fluorescently labeled with nuclei (cyan), tubulin (gray), actin (gold), and the Golgi apparatus (magenta). (a) Maximum-intensity projection of the full z-stack in the xy plane. (b–e) Individual channels corresponding to (a): (b) nuclei, (c) microtubules, (d) actin, and (e) Golgi apparatus. (f) Maximum-intensity projection of a second z-stack in the xy plane. (g–j) Individual channels corresponding to (f): (g) nuclei, (h) microtubules, (i) actin, and (j) Golgi apparatus. Nuclei were labeled with DAPI, actin filaments with phalloidin, and both microtubules and the Golgi apparatus were stained using indirect immunofluorescence.
Figure 4—figure supplement 1
Computer-aided design (CAD) renderings of custom heated sample chamber design.
This updated design utilizes thermocouples and heating pad elements for temperature regulation, enabling live-cell imaging capabilities for Altair light-sheet fluorescence microscopy (LSFM).
Figure 5
Live-cell volumetric imaging of intermediate filament and microtubule dynamics.
Selected time points from a time-lapse sequence of actively migrating retinal pigment epithelial (RPE) cells, showing vimentin (blue) and microtubules (orange). The series comprises 50 frames; representative time points are displayed.
Videos
Animation 1
Time-lapse of retinal pigment epithelial immortalized with human telomerase reverse transcriptase (RPE-hTERT) cells with endogenously tagged microtubules and vimentin.
Time-lapse sequence of RPE-hTERT cells with endogenously tagged cytoskeletal markers: microtubules (red) and vimentin (blue).
Tables
Table 1
Light-sheet fluorescence microscopy (LSFM) variants and their associated illumination and detection optics.
The table lists the type of microscope, the illumination, and detection optics—including numerical aperture (NA) where available and immersion type in parentheses—as well as the overall design architecture (e.g. rail carrier, cage system, etc.).
| Microscope | Illumination optics | Detection optics | Design |
|---|---|---|---|
| OpenSPIM (Pitrone et al., 2013) | Olympus UMPLFLN 10× W NA 0.3 (Water) | UMPLFLN 20× W NA 0.5 (Water) | Rail Carrier |
| X-OpenSPIM (Girstmair et al., 2022) | Nikon CFI Plan Fluor 10× NA 0.3 (Water) | Nikon CFI Apochromat NIR 40× W NA 0.8 (Water) | Rail Carrier |
| EduSPIM (Jahr et al., 2016) | Zeiss LSFM 5× NA 0.1 (Air) | Zeiss LD Epiplan 5× NA 0.13 (Air) | Cage System |
| OpenSPIN (Gualda et al., 2013) | Nikon CFI Plan Fluor 4× NA 0.13 (Air) Nikon Plan Fluor 10× NA 0.3 (Air) | Nikon CFI Plan Fluor 4× NA 0.13 (Air) Nikon CFI75 LWD 16× NA 0.8 (Water) | Rail Carrier |
| UC2 (Diederich et al., 2020) | Generic 4× NA 0.14 (Air) | Generic 4× NA 0.14 (Air) Generic 10× NA 0.3 (Air) | 3D Printed Components |
| pLSM (Chen et al., 2024) | Mitutoyo Plan Apo 10× NA 0.28 (Air) | Mitutoyo Plan Apo 10× NA 0.28 (Air) ASI 54-10-12 16.67× NA 0.4 (Multi-Immersion) | Cage System |
| descSPIM (Otomo et al., 2024) | 500 and 150 mm cylindrical lenses (Air) | Thorlabs TL2X-SAP 2× NA 0.1 (Air) | Cage System |
| mesoSPIM (Voigt et al., 2019) | Nikon 50 mm f/1.4 G (Air) | Olympus MVPLAPO 1× NA 0.15 (Air) | Rail Carrier |
| BT-mesoSPIM (Vladimirov et al., 2024) | Nikon 50 mm f/1.4 G (Air) | Variable. Magnification 2–20×, NA 0.1–0.28 (Air) | Cage System |
| diSPIM (Wu et al., 2013) | Nikon CFI Apochromat NIR 40× W NA 0.8 (Water) | Nikon CFI Apochromat NIR 40× W NA 0.8 (Water) | Dovetail Tube System |
| CompassLSM (Liu et al., 2021) | Olympus XLFLUOR 4× NA 0.28 (Air/Water) | Olympus MVX PLAPO 1× NA 0.25 (Air) Olympus MVX PLAPO 2× C NA 0.5 (Air) Olympus UPlanFL 4× NA 0.13 (Air) Nikon CFI Plan Apo 10× C NA 0.5 (Water) | Rail Carrier |
| Lattice Light-Sheet Microscopy (LLSM) (Chen et al., 2014) | Special Optics 54-10-7 28.6× NA 0.67 (Water) | Nikon CFI75 Apochromat 25× C NA 1.1 (Water) | Free Space Optics |
| Altair-LSFM | Thorlabs TL20X-MPL 20× NA 0.6 (Water) | Nikon CFI75 Apochromat 25× C NA 1.1 (Water) | Custom Baseplate & Dovetail Tube System |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/106910/elife-106910-mdarchecklist1-v1.docx
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Supplementary file 1
Detailed equipment list for Altair light-sheet fluorescence microscopy (LSFM).
- https://cdn.elifesciences.org/articles/106910/elife-106910-supp1-v1.docx
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Supplementary file 2
Approximate cost to build an Altair light-sheet fluorescence microscopy (LSFM).
- https://cdn.elifesciences.org/articles/106910/elife-106910-supp2-v1.docx
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Supplementary file 3
Electrical pinouts used on National Instruments PCIe-6738 data acquisition card.
All analog and digital connections were made using a National Instruments SCB-68A shielded terminal block.
- https://cdn.elifesciences.org/articles/106910/elife-106910-supp3-v1.docx
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Supplementary file 4
Acquisition performance specifications.
- https://cdn.elifesciences.org/articles/106910/elife-106910-supp4-v1.docx
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Supplementary file 5
Approximate time to assemble Altair light-sheet fluorescence microscopy (LSFM).
- https://cdn.elifesciences.org/articles/106910/elife-106910-supp5-v1.docx
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Supplementary file 6
Detailed documentation for the assembly and operation of Altair light-sheet fluorescence microscopy (LSFM).
- https://cdn.elifesciences.org/articles/106910/elife-106910-supp6-v1.pdf
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A high-resolution, easy-to-build light-sheet microscope for subcellular imaging
eLife 14:RP106910.
https://doi.org/10.7554/eLife.106910.3
