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Related Experiment Video

Updated: Sep 13, 2025

Optimized Workflow for Iterative Bleaching Extends Multiplexity Imaging of Highly Autofluorescent Clinical Samples
06:52

Optimized Workflow for Iterative Bleaching Extends Multiplexity Imaging of Highly Autofluorescent Clinical Samples

Published on: July 11, 2025

236

Optimized Workflow for Iterative Bleaching Extends Multiplexity Imaging of Highly Autofluorescent Clinical Samples.

Aleksandra Lunich1, Andrea J Radtke2, Margaret Williams1

  • 1Critical Care Medicine and Pulmonary Branch, National Heart, Lung and Blood Institute, National Institutes of Health.

Journal of Visualized Experiments : Jove
|July 28, 2025
PubMed
Summary

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This study optimizes imaging techniques to analyze immune responses in lung tissue affected by nontuberculous mycobacteria (NTM) disease, offering new insights into host pathology. The improved methods enable detailed analysis of challenging clinical samples for better understanding of NTM lung disease.

Area of Science:

  • Immunology
  • Pathology
  • Microbiology

Background:

  • Nontuberculous mycobacteria (NTM) pulmonary disease prevalence is rising globally.
  • Distinct lung injury patterns in NTM disease lack detailed in situ host response characterization.
  • Understanding host immune cell responses is crucial for NTM lung disease pathology.

Purpose of the Study:

  • To deeply phenotype immune cell populations in lung tissue from NTM disease patients.
  • To compare host responses in NTM lung disease with tuberculous (TB) lung disease.
  • To optimize multiplex imaging techniques for challenging FFPE lung tissue.

Main Methods:

  • Modified Iterative Bleaching Extends multi-pleXity (IBEX) imaging applied to FFPE lung tissue.
  • Developed photoirradiation and antigen retrieval protocols to reduce autofluorescence in aged samples.

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  • Designed antibody panels addressing cross-reactivity and signal-to-noise issues for multiplex imaging.

    • Main Results:

    • Successfully optimized IBEX for high-autofluorescence FFPE lung tissue.
    • Achieved high-resolution, whole-slide imaging of immune cells in NTM and TB lung disease.
    • Demonstrated a workflow for multiplexed imaging on challenging clinical samples.

    Conclusions:

    • The developed workflow enables detailed quantitative imaging of immune cells in NTM lung disease.
    • This method overcomes autofluorescence challenges in FFPE lung tissue.
    • The techniques are broadly applicable to diverse sample types for fluorescence imaging.