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Updated: Dec 6, 2025

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Published on: February 9, 2012

Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy.

Jan Christoph Thiele¹, Dominic A Helmerich², Nazar Oleksiievets¹

¹III. Institute of Physics-Biophysics, Georg August University, Göttingen 37077, Germany.

|October 9, 2020

Summary

This summary is machine-generated.

We developed a new super-resolution imaging technique combining fluorescence lifetime and single-molecule localization microscopy (SMLM). This method achieves higher spatial resolution and provides fluorescence lifetime information for multiplexing labels with similar spectral properties.

Keywords:

DNA-PAINT FLIM confocal SMLM dSTORM multiplexed imaging super-resolution microscopy

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Last Updated: Dec 6, 2025

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

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Published on: December 9, 2013

Area of Science:

Biophysics
Optical Microscopy
Nanotechnology

Background:

Fluorescence lifetime imaging microscopy (FLIM) provides temporal information beyond intensity and color.
Current super-resolution microscopy techniques like stimulated emission depletion microscopy offer lifetime information, but single-molecule localization microscopy (SMLM) does not.
Multiplexing fluorescent labels with similar spectral properties is challenging.

Purpose of the Study:

To combine fluorescence-lifetime confocal laser-scanning microscopy with SMLM.
To achieve single-molecule localization-based fluorescence-lifetime super-resolution imaging.
To enable multiplexing of spectrally similar fluorescent labels.

Main Methods:

Integration of fluorescence-lifetime confocal laser-scanning microscopy with SMLM.
Application to direct stochastic optical reconstruction microscopy (dSTORM) and points accumulation for imaging in nanoscale topography (PAINT).
Validation on fixed cells and multiplexing capability demonstration.

Main Results:

Achieved super-resolution imaging with spatial resolution beyond the diffraction limit.
Determined the fluorescence lifetime of all localized single molecules.
Demonstrated multiplexing of two labels differing only in fluorescence lifetime, not spectral properties.

Conclusions:

The developed technique successfully integrates fluorescence lifetime information into SMLM.
This approach overcomes limitations of conventional SMLM and enables advanced multiplexing.
It offers a powerful new tool for high-resolution imaging and molecular analysis.