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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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An expectation-maximization approach to quantifying protein stoichiometry with single-molecule imaging.

Artittaya Boonkird¹, Daniel F Nino^1,2, Joshua N Milstein^1,2

¹Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, ON L5L 1C6, Canada.

Bioinformatics Advances

|January 26, 2023

Summary

This study introduces a new calibration-free method for quantitative single-molecule localization microscopy (SMLM). The approach accurately determines molecular complex stoichiometry and fluorophore properties from blinking data.

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Area of Science:

Biophysics
Microscopy
Computational Biology

Background:

Single-molecule localization microscopy (SMLM) offers nanometer-scale imaging of cellular structures.
Quantitative analysis of SMLM data requires understanding complex fluorophore photophysics.
Existing algorithms for macromolecular complex analysis in SMLM are limited by fluorophore property dependencies.

Purpose of the Study:

To develop a calibration-free quantitative SMLM method.
To determine molecular complex stoichiometry and abundance from SMLM data.
To simultaneously characterize single-fluorophore blinking properties.

Main Methods:

Utilized a statistical model based on the geometric distribution of fluorophore blinking events.
Applied an adapted expectation-maximization algorithm.
Validated the method on simulated and experimental SMLM datasets of DNA nanostructures.

Main Results:

Developed a calibration-free approach for quantitative SMLM.
Successfully determined protomer fractions (monomers, dimers, trimers) and single-fluorophore blinking distributions.
Demonstrated the method's utility on diverse SMLM datasets.

Conclusions:

The new method simplifies quantitative SMLM analysis by removing the need for calibration.
Accurate determination of molecular stoichiometry and fluorophore properties is achievable.
The approach enhances the quantitative capabilities of SMLM for biological research.