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A Protocol for Real-time 3D Single Particle Tracking

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DrSTAR: Tracking real-time nanometer axial changes.

Tomasz J Nawara¹, William F Dean¹, Alexa L Mattheyses¹

¹Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama.

Biophysical Journal

|January 20, 2023

Summary

This summary is machine-generated.

We developed dynamic reference STAR (DrSTAR), an automated software package that simplifies and accelerates the analysis of simultaneous two-wavelength axial ratiometry (STAR) microscopy data. This tool enhances the study of protein dynamics and plasma membrane interactions.

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Area of Science:

Cell Biology
Biophysics
Microscopy

Background:

Protein interactions with the plasma membrane are crucial for cell functions like migration and endocytosis.
Understanding how proteins sense or induce plasma membrane shapes is limited.
Simultaneous two-wavelength axial ratiometry (STAR) microscopy offers high temporal and axial resolution but has complex data processing.

Purpose of the Study:

To overcome the limitations of STAR microscopy data processing.
To present dynamic reference STAR (DrSTAR), an automated, user-friendly software package.
To facilitate the study of nanometer axial changes in protein distribution at the plasma membrane.

Main Methods:

Development of dynamic reference STAR (DrSTAR), an open-source MATLAB-based package.
Implementation of a novel local background referencing algorithm.
Automation of data processing for multiple experimental conditions and biological replicates.

Main Results:

DrSTAR significantly accelerates the processing time for STAR microscopy data.
The software enables efficient analysis of axial (Δz) information.
DrSTAR overcomes previous challenges in quantitative data processing for STAR microscopy.

Conclusions:

DrSTAR provides a user-friendly and efficient solution for analyzing STAR microscopy data.
The package broadens the applicability of STAR microscopy for studying protein-plasma membrane interactions.
This advancement will aid in investigating the influence of biological and biophysical factors on membrane dynamics.