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

Strategies for Achieving High-Resolution Spatially Quantitative Three-Dimensional Imaging Using Confocal Microscopy: A Practical Guide | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 6 May 2025 V1 Latest version Share on Strategies for Achieving High-Resolution Spatially Quantitative Three-Dimensional Imaging Using Confocal Microscopy: A Practical Guide Authors : Olivia A. Creasey , Bryan Chang 0009-0001-1376-6617 , Gregory L. Szot , Bo Huang , Julie B. Sneddon , and Zev J. Gartner [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174652760.02675021/v1 298 views 174 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract 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. Supplementary Material File (primer_12012024.docx) Download 7.47 MB Information & Authors Information Version history V1 Version 1 06 May 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords fluorescence microscopy islet biology laser scanning confocal microscopy quantitative 3d image processing tissue biology Authors Affiliations Olivia A. Creasey UC Berkeley UCSF Graduate Program in Bioengineering View all articles by this author Bryan Chang 0009-0001-1376-6617 University of California San Francisco Department of Pharmaceutical Chemistry View all articles by this author Gregory L. Szot University of California San Francisco Department of Surgery View all articles by this author Bo Huang University of California San Francisco Department of Pharmaceutical Chemistry View all articles by this author Julie B. Sneddon UCSF Diabetes Center View all articles by this author Zev J. Gartner [email protected] University of California San Francisco Department of Pharmaceutical Chemistry View all articles by this author Metrics & Citations Metrics Article Usage 298 views 174 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Olivia A. Creasey, Bryan Chang, Gregory L. Szot, et al. Strategies for Achieving High-Resolution Spatially Quantitative Three-Dimensional Imaging Using Confocal Microscopy: A Practical Guide. Authorea . 06 May 2025. DOI: https://doi.org/10.22541/au.174652760.02675021/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. 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