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Updated: Mar 27, 2026

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Published on: February 9, 2012

Pump-probe fluorescence lifetime imaging microscopy.

Malcolm Garrow¹, Adil Haboucha², Petr Heřman¹

¹Faculty of Mathematics and Physics, Institute of Physics, Charles University, Prague, Czech Republic.

The Journal of Chemical Physics

|March 25, 2026

Summary

This summary is machine-generated.

We developed pump-probe fluorescence lifetime imaging microscopy (PP-FLIM) for high-resolution imaging of ultrafast dynamics. This technique combines spatial, temporal, and spectral information to analyze sensitive samples like chloroplasts.

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Area of Science:

Spectroscopy
Microscopy
Biophysics

Background:

Pump-probe spectroscopy is key for studying ultrafast photo-induced dynamics.
Fluorescence-detected variants link spectroscopy to microscopy, enabling spatial and temporal resolution.
Existing methods lack the combined spectral and temporal resolution for complex dynamics.

Purpose of the Study:

Introduce a novel technique, pump-probe fluorescence lifetime imaging microscopy (PP-FLIM).
Combine pump-probe spectroscopy with fluorescence lifetime imaging microscopy (FLIM).
Achieve high-resolution, non-invasive imaging of ultrafast dynamics in sensitive biological samples.

Main Methods:

Implemented fluorescence-detected pump-probe spectroscopy (F-PP) with FLIM detection.
Acquired spectrally resolved transient dynamics at each voxel.
Measured time-resolved fluorescence decay in three spatial dimensions.

Main Results:

Demonstrated PP-FLIM on a mixture of oxonol VI and cresyl violet dyes.
Successfully disentangled the transient spectra of the dyes using fluorescence lifetime.
Showcased high-resolution imaging of individual chloroplasts in intact spinach leaves.

Conclusions:

PP-FLIM provides unprecedented spatiotemporal and spectral resolution for ultrafast dynamics.
The technique is non-invasive and suitable for imaging sensitive biological structures.
PP-FLIM opens new avenues for studying complex photophysical and photochemical processes.