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Updated: May 15, 2026

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
07:50

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Published on: March 30, 2020

A novel immunofluorescent computed tomography (ICT) method to localise and quantify multiple antigens in large tissue

Geraint J Parfitt1, Yilu Xie, Korey M Reid

  • 1The Gavin Herbert Eye Institute, University of California Irvine, Irvine, California, United States of America.

Plos One
|January 10, 2013
PubMed

Insights

We developed immunofluorescent computed tomography (ICT) to visualize large tissue volumes in 3D at high resolution. This method allows for multi-antigen labeling and quantitative analysis of tissue structure and function.

Area of Science:

  • Biomedical Imaging
  • Histology
  • Molecular Biology

Background:

  • Current immunofluorescence methods struggle with large tissue volumes and high-resolution, multi-antigen analysis.
  • Limitations hinder reliable antibody staining, localization, and quantification in 3D tissue samples.

Purpose of the Study:

  • To develop a novel approach for high-resolution, three-dimensional visualization of large tissue volumes.
  • To enable multi-antigen labeling and quantitative analysis within intact tissue samples.
  • To create detailed 3D biological maps for enhanced tissue characterization.

Main Methods:

  • Developed immunofluorescent computed tomography (ICT) using computer reconstruction of serial sectioned and sequentially immunostained butyl-methyl methacrylate (BMMA) embedded tissue.
  • Applied ICT to reconstruct murine lower eyelid tissue, localizing cell nuclei (DAPI), Ki67, and cytokeratin 1 (CK1).
  • Integrated non-linear optical (NLO) microscopy for collagen imaging to assess cell density, proliferation, keratinization, and gland volume.

Main Results:

  • Successfully reconstructed large tissue volumes (mm³) at high resolution (<1 µm) in 3D.
  • Demonstrated preservation of antigenicity after multiple iterative stains, indicating potential for unlimited antigen labeling.
  • BMMA embedding preserved fluorescence of transgenic proteins, enhancing imaging capabilities.

Conclusions:

  • ICT offers a powerful tool for high-resolution, 3D visualization and quantitative analysis of multiple biomolecules within large tissue volumes.
  • This approach can generate valuable 3D biological maps to better characterize tissue structure and function.
  • ICT overcomes limitations of current immunofluorescence techniques for complex tissue analysis.

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