Abstract
3D microscopy of large biological samples (>0.5 cm3) is transforming biological research. Many existing techniques require trade‐offs between image resolution, sample size, and method complexity. A simple robust instrument with the potential to conduct large‐volume 3D imaging currently exists in the form of the optical high‐resolution episcopic microscopy (HREM). However, the development of the instrument to date is limited to single‐fluorescent wavelength imaging with nonspecific eosin staining. Herein, developments to realize the potential of the HREM to become multifluorescent high‐resolution episcopic microscopy (MF‐HREM) are presented. MF‐HREM is a serial‐sectioning and block‐facing wide‐field fluorescence imaging technique, which does not require tissue clearing or optical sectioning. Multiple developments are detailed in sample preparation and image postprocessing to enable multiple specific stains in large samples and show how these enable segmentation and quantification of the data. The application of MF‐HREM is demonstrated in a variety of biological contexts: 3D imaging of whole tumor vascular networks and tumor cell invasion in xenograft tumors up to 7.5 mm3 at resolutions of 2.75 μm, quantification of glomeruli volume in the adult mouse kidney, and quantification of vascular networks and white‐matter track orientation in adult mouse brain.
Highlights
( for large organs) verses stain preservation, morphological changes, time, and complexity.[6,7,8]
Microscopy techniques aiming to image large samples must In contrast, the second approach, serial-section block-face overcome the opacity of the tissue to visible light caused by scat- (SSBF) imaging, produces inherently aligned images, thereby ter and absorption; tissue clearing and serial-sectioning are two overcoming the slice alignment challenge and preventing the of the most common approaches
Each stage of the pipeline requires optimization for a specific experiment and we have conducted these optimizations for a variety of adult mouse organs and experimental conditions
Summary
The pipeline for MF-HREM is straightforward and does not require specialist equipment (Figure 1C). Each stage of the pipeline requires optimization for a specific experiment (organ and stain combination) and we have conducted these optimizations for a variety of adult mouse organs and experimental conditions. These optimized protocols can serve as a starting point for other experimental conditions and demonstrate the breadth of potential applications for MF-HREM
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