Single-molecule tracking in living microbial cells

Key Findings:

Sample Preparation: The protocol emphasizes the importance of fluorescent labeling of proteins of interest (POIs) through genetic insertion of fluorescent proteins (FPs) or self-labeling protein tags (SLPs) like Halo-tag. It provides detailed instructions for culturing E. coli and yeast cells, optimizing labeling conditions, and preparing microscope slides for imaging.

Data Acquisition: The protocol utilizes total internal reflection fluorescence microscopy (TIRF) with highly inclined and laminated optical sheet (HILO) illumination to enhance signal-to-noise ratio. It details the settings for laser power, exposure time, and image acquisition parameters to ensure optimal tracking of single molecules.

Data Processing: The protocol briefly describes the analysis steps involved in sptPALM, including identification of fluorophores using Gaussian fitting, trajectory extraction using nearest-neighbor algorithms, and analysis of diffusion behavior through mean-squared displacement (MSD) calculations.

Significance:

This protocol allows researchers to directly observe and quantify the spatiotemporal dynamics of individual molecules in living microbial cells, providing insights into essential biological processes such as transcription, replication, and repair.

By understanding the motion and interactions of individual molecules, researchers can establish precise models of molecular and cellular reactions, leading to a deeper understanding of fundamental biological mechanisms.

The work entitled “Single-molecule tracking in living microbial cells”was published on Biophysics Reports (published on Feb, 2025).