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Updated: Jun 18, 2026

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Published on: December 9, 2013

Three-dimensional polar representation for multispectral fluorescence lifetime imaging microscopy.

A Leray¹, C Spriet, D Trinel

¹Interdisciplinary Research Institute, Science and Technology University of Lille, USR 3078 CNRS, BCF, Villeneuve d'Ascq, France.

Cytometry. Part a : the Journal of the International Society for Analytical Cytology

|November 13, 2009

Summary

This study introduces a new method for analyzing multispectral fluorescence lifetime imaging microscopy data. The approach simplifies complex analysis, enabling precise Förster resonance energy transfer (FRET) measurements and clear identification of autofluorescence.

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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Published on: February 9, 2012

Area of Science:

Biophotonics
Microscopy
Spectroscopy

Background:

Multispectral fluorescence lifetime imaging microscopy (FLIM) is vital for analyzing complex biological samples.
Accurate analysis of FLIM data, especially in scattering tissues, is challenging and requires expertise.
Existing methods often involve complex fitting algorithms, limiting speed and accessibility.

Purpose of the Study:

To develop a simplified and visually intuitive analysis method for multispectral FLIM.
To improve the precision of Förster resonance energy transfer (FRET) measurements.
To enable robust identification of autofluorescence in biological samples.

Main Methods:

Data transformation from the time domain to the frequency domain for each spectral channel.
Calculation of a multispectral polar representation for fluorescence lifetime data.
Incorporation of emission wavelength as a third dimension in the polar representation.

Main Results:

Demonstrated simplified analysis of multiply fluorescent labeled samples.
Achieved high-precision Förster resonance energy transfer (FRET) measurements.
Successfully identified and distinguished sample autofluorescence using the 3D polar representation.

Conclusions:

The novel frequency-domain polar representation simplifies FLIM data analysis significantly.
This method enhances the accuracy and reliability of FRET measurements in complex biological environments.
The approach eliminates analysis artifacts, providing a more robust tool for biophotonics research.