Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in...

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.