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METHODS article

Front. Cell Dev. Biol.
Sec. Cellular Biochemistry
Volume 12 - 2024 | doi: 10.3389/fcell.2024.1420161

ProDiVis: A Method to Normalize Fluorescence Signal Localization in 3D Specimens

Provisionally accepted
Kyle T. Nguyen Kyle T. Nguyen 1Alexandre R. Sathler Alexandre R. Sathler 1Alvaro G. Estevez Alvaro G. Estevez 1,2Isabelle E. Logan Isabelle E. Logan 1Maria Clara Franco Maria Clara Franco 1,2,3*
  • 1 Department of Biochemistry and Biophysics, College of Science, Oregon State University, Corvallis, Oregon, United States
  • 2 Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States
  • 3 Center for Translational Science, Florida International University, Port St. Lucie, United States

The final, formatted version of the article will be published soon.

    A common problem in confocal microscopy is the decrease in intensity of excitation light and emission signal from fluorophores as they travel through 3D specimens, resulting in decreased signal detected as a function of depth. Here, we report a visualization program compatible with widely used fluorophores in cell biology to facilitate image interpretation of differential protein disposition in 3D specimens. Glioblastoma cell clusters were fluorescently labeled for mitochondrial complex I (COXI), P2X7 receptor (P2X7R), β-Actin, and DAPI. Each cell cluster was imaged using a laser scanning confocal microscope. We observed up to ~70% loss in fluorescence signal across the depth in Z-stacks. This progressive underrepresentation of fluorescence intensity as the focal plane deepens hinders an accurate representation of signal location within a 3D structure. To address these challenges, we developed ProDiVis: a program that adjusts apparent fluorescent signals by normalizing one fluorescent signal to a reference signal at each focal plane. ProDiVis serves as a free and accessible, unbiased visualization tool to use in conjunction with fluorescence microscopy images and imaging software.

    Keywords: 3D specimen, Heatmap, confocal microscopy, fluorescence microscopy, Protein distribution, Signal Normalization, imaging, image analysis

    Received: 19 Apr 2024; Accepted: 04 Sep 2024.

    Copyright: © 2024 Nguyen, Sathler, Estevez, Logan and Franco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Maria Clara Franco, Department of Biochemistry and Biophysics, College of Science, Oregon State University, Corvallis, 97331-4003, Oregon, United States

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.