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Related Experiment Video

Updated: Oct 26, 2025

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model
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Method for Multiplexed Dynamic Intravital Multiphoton Imaging.

Asylkhan Rakhymzhan1, Andreas Acs2, Ruth Leben3

  • 1Biophysical Analytics, Deutsches Rheumaforschungszentrum - a Leibniz Institute, Berlin, Germany. asylkhan.rakhymzhan@drfz.de.

Methods in Molecular Biology (Clifton, N.J.)
|July 31, 2021
PubMed
Summary
This summary is machine-generated.

This study introduces a new method for multiplexed dynamic intravital two-photon imaging. It allows distinguishing seven fluorophore signals using only four detector channels for better in vivo immune system research.

Keywords:
Fluorescing proteinsIntravital imagingMultiphoton microscopyNear-infrared fluorescent dyesOptical parametric oscillatorSimilarity unmixingTwo-color two-photon excitation

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Area of Science:

  • Immunology
  • Microscopy
  • Biophysics

Background:

  • Intravital two-photon microscopy is crucial for studying cellular dynamics in living organisms.
  • Its application in immunology provides unique insights into in vivo pathophysiologic processes.

Purpose of the Study:

  • To develop a method for multiplexed dynamic intravital two-photon imaging.
  • To enhance the ability to monitor cellular dynamics and communication within the immune system in vivo.

Main Methods:

  • A synergistic strategy combining spectrally broad fluorophore emissions.
  • Utilizing a wave-mixing concept for simultaneous excitation of multiple fluorophores.
  • Employing an effective unmixing algorithm based on spectral similarity calculations.

Main Results:

  • Successfully distinguished seven distinct fluorophore signals.
  • Achieved multiplexed imaging using only four detector channels.
  • Enabled detailed monitoring of cellular and tissue compartments in vivo.

Conclusions:

  • The developed method significantly advances intravital two-photon imaging capabilities.
  • This technique offers improved resolution for studying complex biological systems, particularly the immune system.
  • Facilitates deeper understanding of in vivo pathophysiologic processes through enhanced cellular observation.