Strategies for Achieving High-Resolution Spatially Quantitative Three-Dimensional Imaging Using Confocal Microscopy: A Practical Guide

Le résumé fourni par la source

Cells operate and make decisions within complex, three-dimensional (3D) tissue environments which profoundly influences cell behavior compared to two-dimensional (2D) studies. Therefore, understanding cell biology within tissues necessitates accurate 3D imaging of cell morphology, protein and organelle localization, tissue structure, and cell-cell interactions. Achieving spatially quantitative 3D (SQ3D) images requires careful consideration of numerous parameters to avoid spatial inaccuracies that could lead to incorrect biological conclusions. Here, we address practical decisions and optimizations necessary for producing SQ3D images of fixed tissue using commercially available laser scanning confocal (LSC) microscopes. We focus on LSC microscopy due to its accessibility and utility in acquiring high-resolution images of small tissues like organoids and islets. We discuss the importance of appropriate microscope hardware, sample preparation, labeling, clearing, and image acquisition settings to generate information-dense datasets. Special attention is given to selecting imaging parameters for technical consistency, optimal resolution, and signal-to-noise ratio, considering factors such as voxel size, pinhole settings, and excitation light intensity. This detailed workflow guides researchers in acquiring high-quality, reproducible 3D images, which are critical for advancing our understanding of cell and tissue biology and leveraging emerging machine learning technologies for analysis.

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