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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

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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Autocorrelation function of finite-length data in fluorescence correlation spectroscopy.

John Kohler¹, Kwang-Ho Hur¹, Joachim Dieter Mueller²

¹School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, USA.

Biophysical Journal

|October 21, 2022

Summary

This study introduces a new theory for fluorescence correlation spectroscopy, enabling unbiased analysis of finite-length data. This method improves accuracy for live-cell imaging, expanding its applications.

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Area of Science:

Biophysics
Spectroscopy
Data Analysis

Background:

Experimental autocorrelation functions in fluorescence correlation spectroscopy (FCS) are biased due to finite data length.
Conventional FCS analysis struggles with data from living cells, often affected by signal instabilities.

Purpose of the Study:

To develop a theoretical framework for unbiased analysis of experimental autocorrelation functions in FCS.
To extend FCS applicability to live-cell measurements with intensity variations and instabilities.

Main Methods:

Formulated a new theoretical framework accounting for data length in FCS analysis.
Validated the theory using experiments and simulations of diffusion.
Applied short data segmentation techniques to analyze unstable fluorescence signals.

Main Results:

The new framework provides unbiased estimates of theoretical correlation functions.
Accuracy and precision of parameter estimates were characterized for diffusion processes.
Reformulated theory allows analysis with segment times approaching diffusion time, enhancing robustness.

Conclusions:

The developed theoretical framework significantly improves FCS analysis for finite-length data.
This approach enhances the robustness and applicability of FCS, particularly for challenging live-cell experiments.
The study expands the range of experimental systems accessible to fluorescence correlation spectroscopy.