Frontiers in Cell and Developmental Biology (Sep 2024)

ProDiVis: a method to normalize fluorescence signal localization in 3D specimens

  • Kyle T. Nguyen,
  • Alexandre R. Sathler,
  • Alvaro G. Estevez,
  • Alvaro G. Estevez,
  • Alvaro G. Estevez,
  • Isabelle E. Logan,
  • Maria Clara Franco,
  • Maria Clara Franco,
  • Maria Clara Franco

DOI
https://doi.org/10.3389/fcell.2024.1420161
Journal volume & issue
Vol. 12

Abstract

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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, Ki-67, 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.

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