Journal: eLife

Article Title: A high-resolution, easy-to-build light-sheet microscope for subcellular imaging

doi: 10.7554/eLife.106910

Figure Lengend Snippet: ( 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 .

Article Snippet: For Altair-LSFM, the primary cost driver is the detection/illumination pair built around the Nikon N25X-APO-MP 25×/1.1 NA detection objective (≈ $33,000), which must be paired with either the Thorlabs TL20X-MPL 20×/0.6 NA illumination objective (≈$5000) or the Special Optics 54-10-7@488–910 nm (≈$15,000).

Techniques: Microscopy

Journal: Nature Communications

Article Title: Three dimensional multiscalar neurovascular nephron connectivity map of the human kidney across the lifespan

doi: 10.1038/s41467-025-60435-8

Figure Lengend Snippet: 1–4 Tissue processing and immunofluorescence staining of corticomedullary strips of tissue slices from kidney, preserving in fixative, active clearing using and performing multiplex immunofluorescence staining with antibodies targeting key functional tissue units of the kidney and / or neurovasculature. The staining was typically done for five targets across the Cy5, Cy3, 488, and 405 channels. 5 LSFM imaging. Cleared and post IF samples were imaged using Zeiss LSFM to obtain gross macroscopic view of the entire specimen at 5X followed by a series of high-resolution, 20x images to visualize subregions along cortico-medullary axis. These 20x images were typically 1 × 1 × 1 mm, although a small number were larger. 6 Data processing. Prior to any analysis, data was stitched and downsampled by 4x in both X and Y using either ZEN Blue 3.1 software or Stitchy. The purpose of this pipeline, as opposed to imaging at low resolution and skipping downsampling, was due to our access to sufficient data storage and processing resources. This enabled us to feasibly obtain high resolution data that could be stored and used for validation and display, while downsampled data could be used for analysis. 7 Data were segmented for structures of interest utilizing either Cellpose or Labkit. 8 Qualitative and quantitative data analysis for various structures and their relationships was accomplished using commercial software such as Imaris or ImageJ, in addition to in-house Python code. This included coregistration of 20x subregions onto 5x volumes, and algorithmic extraction of neural networks between glomeruli. Cartoon depictions from 1 , 4 , and 8 were obtained using Biorender. Created in BioRender. Jain, S. (2025) https://BioRender.com/z62w451 .

Article Snippet: Cleared and post IF samples were imaged using Zeiss LSFM to obtain gross macroscopic view of the entire specimen at 5X followed by a series of high-resolution, 20x images to visualize subregions along cortico-medullary axis.

Techniques: Immunofluorescence, Staining, Preserving, Multiplex Assay, Functional Assay, Imaging, Software, Biomarker Discovery, Extraction