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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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Updated: Aug 3, 2025

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Published on: February 9, 2012

Single-shot time-folded fluorescence lifetime imaging.

Valentin Kapitany¹, Vytautas Zickus^1,2, Areeba Fatima¹

¹Extreme Light, School of Physics & Astronomy, University of Glasgow, Glasgow G12 8QQ, UK.

Proceedings of the National Academy of Sciences of the United States of America

|April 12, 2023

Summary

This summary is machine-generated.

We developed time-folded fluorescence lifetime imaging microscopy (TFFLIM), a robust single-shot method for bioimaging. TFFLIM overcomes limitations of current techniques, enabling better detection of cell dynamics and environmental changes.

Keywords:

FLIM physics-inspired neural network single-shot time-folded cavity

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Area of Science:

Bioimaging
Microscopy
Biophysics

Background:

Fluorescence lifetime imaging (FLIM) is crucial for observing cellular dynamics.
Current time-domain FLIM methods face challenges with dynamic scenes and uncertainty.
Single-shot techniques can have significant uncertainties depending on lifetime sampling.

Purpose of the Study:

To introduce a novel, robust, single-shot fluorescence lifetime imaging microscopy technique.
To address the limitations of existing time-domain FLIM approaches.
To provide a method for imaging dynamic biological scenes with reduced uncertainty.

Main Methods:

Developed a time-folding cavity for fluorescence lifetime imaging microscopy (TFFLIM).
The cavity generates multiple, temporally shifted, spatially sheared replicas of the fluorescence lifetime.
Utilized a fixed time gate to capture these multiple lifetime replicas in a single shot.

Main Results:

Demonstrated a robust, single-shot fluorescence lifetime imaging microscopy (TFFLIM) approach.
Experimentally validated TFFLIM across a wide range of fluorescence lifetimes.
Successfully imaged fluorescent beads and Convallaria samples using the TFFLIM technique.

Conclusions:

Time-folded fluorescence lifetime imaging microscopy (TFFLIM) offers a significant advancement in bioimaging.
This single-shot method enhances the ability to study dynamic cellular processes.
TFFLIM provides a reliable tool for analyzing subtle changes in cell dynamics and microenvironments.