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Protein Dynamics in Living Cells

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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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Related Experiment Video

Updated: Aug 23, 2025

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Published on: January 18, 2017

Advanced Data Analysis for Fluorescence-Lifetime Single-Molecule Localization Microscopy.

Jan Christoph Thiele¹, Oleksii Nevskyi¹, Dominic A Helmerich²

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

Frontiers in Bioinformatics

|October 28, 2022

Summary

This summary is machine-generated.

Fluorescence-lifetime single molecule localization microscopy (FL-SMLM) enhances super-resolution imaging by adding fluorescence lifetime data. This study analyzes lifetime determination methods and offers software for super-resolved FL-SMLM image generation.

Keywords:

CRLB (Cramér-Rao lower bound) analysis FLIM (fluorescence lifetime imaging microscopy)SMLM (single molecule localisation microscopy)fluoresence lifetime fitting lifetime uncertainty super-resolution microscopy

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Published on: September 5, 2019

Area of Science:

Biophysical imaging techniques
Super-resolution microscopy
Fluorescence spectroscopy

Background:

Single Molecule Localization Microscopy (SMLM) provides spatial super-resolution.
Fluorescence lifetime, independent of intensity and spectrum, offers additional information.
Lifetime can quantify Förster Resonance Energy Transfer (FRET), probe local environments, and enable image multiplexing.

Purpose of the Study:

To perform a theoretical analysis of fluorescence lifetime determination in FL-SMLM.
To compare different lifetime-fitting approaches.
To provide guidelines for optimized FL-SMLM procedures and introduce new software.

Main Methods:

Theoretical analysis of fluorescence lifetime determination in FL-SMLM.
Investigation of the impact of background and noise on lifetime measurements.
Development and presentation of the public-domain software 'Fluorescence-Lifetime TrackNTrace'.

Main Results:

Established clear guidelines for optimized FL-SMLM procedures.
Demonstrated the utility of lifetime information for FRET, environmental probing, and multiplexing.
The 'Fluorescence-Lifetime TrackNTrace' software converts fluorescence microscopy movies into super-resolved FL-SMLM images.

Conclusions:

FL-SMLM effectively integrates lifetime information for advanced super-resolution imaging.
The presented theoretical analysis and guidelines improve FL-SMLM data processing.
The 'Fluorescence-Lifetime TrackNTrace' software facilitates the generation of super-resolved FL-SMLM images.