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Light Sheet Fluorescent Microscopy and Automated 3D Image Analysis for Quantitative Lineage Tracing in Mouse Cornea.

Mikhail M Khandokhin1, Roman I Tsvetkov1,2, Arina V Radnaeva1,2

  • 1Centre for Regenerative Medicine, Medical Research and Education Institute, Lomonosov Moscow State University, Lomonosovskiy prospekt, 27, k 10, Moscow 119192, Russia.

Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
|February 4, 2026
PubMed
Summary

This study presents a new 3D imaging and analysis pipeline for multi-color lineage tracing using light sheet fluorescent microscopy (LSFM). The method enhances visualization of cell populations and clonal expansion in tissues like the mouse cornea.

Keywords:
confetticorneaimage analysisimmunofluorescencelight sheet microscopylineage tracing

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Area of Science:

  • Biomedical Imaging
  • Developmental Biology
  • Cell Biology

Background:

  • Light sheet fluorescent microscopy (LSFM) enables large-scale 3D imaging but faces challenges in combining tissue clearing, immunolabeling, and lineage tracing signal preservation.
  • Multi-color lineage tracing, such as Confetti tracing, requires robust methods for quantitative analysis in complex 3D environments.

Purpose of the Study:

  • To develop and validate a comprehensive methodological and analytical pipeline for multi-color lineage tracing using LSFM.
  • To optimize protocols for tissue clearing, immunolabeling, and 3D image acquisition for lineage tracing studies.
  • To enable quantitative analysis of cell distribution and clonal expansion in specific tissue compartments.

Main Methods:

  • Utilized dual-view inverted selective plane illumination (diSPIM) microscopy for high-resolution 3D imaging of mouse corneas.
  • Developed a protocol for tissue processing, immunolabeling, and Confetti lineage tracing in Prrx1-expressing corneal cells.
  • Created a 3D image analysis pipeline for morphometry, Confetti marker distribution, and clonal expansion estimation, including spectral leakage correction.

Main Results:

  • Successfully acquired detailed cellular-level 3D images of the mouse cornea.
  • Quantified cell numbers in epithelial, stromal, and endothelial layers, along with corneal dimensions.
  • The analysis pipeline accurately calculated Confetti marker distribution and clonal expansion, correcting for spectral channel leakage.

Conclusions:

  • The presented methodology and analysis pipeline facilitate advanced lineage tracing studies using LSFM and immunolabeling.
  • This approach provides a robust framework for quantitative clonal analysis in 3D.
  • The pipeline is adaptable for lineage tracing studies in various organs beyond the cornea.