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Related Experiment Video

Updated: Sep 2, 2025

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Published on: June 7, 2019

Single-Photon, Time-Gated, Phasor-Based Fluorescence Lifetime Imaging through Highly Scattering Medium.

Rinat Ankri¹, Arkaprabha Basu¹, Arin Can Ulku²

¹Department of Chemistry & Biochemistry, UCLA, Los Angeles, California 90095, United States.

|August 8, 2022

Summary

This summary is machine-generated.

This study introduces a new method for fluorescence lifetime imaging (FLI) that overcomes challenges like autofluorescence and light scattering in biological tissues. The technique enables clear detection of fluorescent signals even in complex environments.

Keywords:

fluorescence lifetime imaging phasor lifetime analysis scattering medium single-photon detection time-gated camera

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Last Updated: Sep 2, 2025

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Area of Science:

Biophotonics
Cellular Imaging
Fluorescence Spectroscopy

Background:

Fluorescence lifetime imaging (FLI) is valuable for biochemical and cellular studies, including in vivo applications.
Fluorescence lifetime is an intrinsic dye property, useful for multiplexing but challenged by tissue autofluorescence and light scattering in vivo.
These factors reduce signal intensity and alter temporal profiles, complicating in vivo FLI in the visible range.

Purpose of the Study:

To demonstrate a method for overcoming autofluorescence and light scattering in in vivo FLI.
To introduce a novel time-gated camera (SwissSPAD2) and phasor analysis for robust lifetime imaging.
To enable sensitive detection of fluorescent signals in challenging biological environments.

Main Methods:

Utilized a time-gated single-photon avalanche diode array camera (SwissSPAD2).
Employed phasor analysis for a simple, fast, and visual method for lifetime imaging.
Investigated the impact of scattering and autofluorescence on phasor dispersion and signal identification.

Main Results:

Phasor dispersion was shown to increase with scattering and decreased fluorescence intensity.
Distinct fluorescence lifetimes were identifiable with background correction, provided the signal exceeded autofluorescence.
Successfully detected A459 cells expressing mCyRFP1 through scattering and autofluorescent phantom layers.

Conclusions:

The developed FLI method effectively accounts for and overcomes challenges posed by tissue autofluorescence and light scattering.
Phasor analysis combined with time-gated detection offers a powerful approach for FLI in demanding conditions.
This technique facilitates in vivo FLI using standard fluorophores and fluorescent proteins, even in highly scattering and autofluorescent tissues.