研究目的
To evaluate the performance of Halo tag and PAmCherry for single-molecule tracking in live Escherichia coli cells, comparing their fluorescence intensity, background, photostability, and results from single-molecule localization and tracking experiments.
研究成果
The study demonstrates that the Halo tag with synthetic dyes offers superior brightness and photostability for single-molecule tracking in live E. coli cells, enabling high-speed and dual-colour tracking. However, the choice between Halo tag and photoactivatable fluorescent proteins depends on the specific requirements of the microscopy experiment.
研究不足
The requirement for labelling and extensive washing of the fluorescent ligand may complicate applications that require precisely controlled growth conditions or cell perturbations. Prolonged time-lapse imaging benefits from the continuous synthesis and replenishment of fluorescent proteins.
1：Experimental Design and Method Selection:
The study compared the use of Halo tag with different fluorescent ligands against the photoactivatable fluorescent protein PAmCherry for single-molecule tracking in live E. coli cells.
2：Sample Selection and Data Sources:
Test proteins MukB and PolI were used, with endogenous C-terminal translational Halo tag fusions generated using the Lambda Red recombination technique.
3：List of Experimental Equipment and Materials:
A custom-built total internal reflection fluorescence microscope equipped with an electron-multiplying CCD camera was used for imaging.
4：Experimental Procedures and Operational Workflow:
The procedure for labelling Halo tag in live E. coli with the dye TMR was optimized, including steps for removing unbound dye to minimize fluorescent background.
5：Data Analysis Methods:
Single-molecule localization and tracking data were analyzed to evaluate fluorescence intensity, background, photostability, and the results from tracking experiments.
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