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Related Experiment Video

Updated: May 12, 2026

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation
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Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in

Jan Leo Rinnenthal1, Christian Börnchen, Helena Radbruch

  • 1German Rheumatism Research Center, Berlin, Germany.

Plos One
|April 25, 2013
PubMed
Summary

We developed a faster fluorescence imaging technique for deep tissues. This new method, parallelized time-correlated single-photon counting (p-TCSPC), enables real-time molecular analysis of cellular responses in living animals, aiding disease research.

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Area of Science:

  • Biomedical optics
  • Cellular imaging
  • Neuroscience

Background:

  • Two-photon microscopy advances live cell imaging in deep tissues.
  • Current limitations in speed hinder molecular analysis of cellular responses in disease.
  • Förster resonant energy transfer (FRET) and fluorescence lifetime imaging (FLIM) offer molecular insights but face acquisition speed challenges.

Purpose of the Study:

  • To develop a faster FLIM technique for deep-tissue 3D and 4D imaging.
  • To enable quantification of molecular mechanisms in cellular responses in vivo.
  • To advance the study of neuroinflammation and other immune-induced pathologies.

Main Methods:

  • Introduction of a parallelized time-correlated single-photon counting (p-TCSPC) detector.
  • Implementation of dynamic single-beam scanning FLIM for large 3D areas (hundreds of milliseconds).
  • Implementation of ultrafast 2D FLIM for cell physiology (tens of milliseconds).

Main Results:

  • p-TCSPC achieves dynamic 3D FLIM of large areas and ultrafast 2D FLIM.
  • Demonstrated dynamic deep-tissue intravital imaging capabilities.
  • Acquired 256x256 pixel FLIM maps in 468 ms and 131x131 pixel maps every 82 ms at 115 µm depth.
  • Enabled time-lapse 4D FLIM in mouse models of neuroinflammation.

Conclusions:

  • p-TCSPC significantly enhances FLIM acquisition speed for deep-tissue imaging.
  • The technology allows for unprecedented quantification of neuronal dysfunction in neuroinflammation.
  • This advancement is crucial for developing new therapeutic strategies for diseases affecting cellular responses.