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Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Published on: September 5, 2019

A simple backscattering microscope for fast tracking of biological molecules.

Yoshiyuki Sowa¹, Bradley C Steel, Richard M Berry

¹Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, Japan.

The Review of Scientific Instruments

|December 8, 2010

Summary

We developed a novel backscattering microscope for tracking gold nanoparticles with high resolution. This technique enables precise measurement of molecular machines, even within live cells.

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Area of Science:

Biophysics
Microscopy
Molecular Biology

Background:

Advancements in single-molecule observation techniques are crucial for understanding molecular machine mechanisms.
Various markers, from single fluorophores to micron-sized ones, are used for molecular detection.
Conventional dark-field microscopy can suffer from high background scattering in biological samples.

Purpose of the Study:

To introduce a simple objective-type backscattering microscope for tracking gold nanoparticles.
To achieve nanometer and microsecond resolution for molecular movement measurements.
To evaluate the utility of backscattering microscopy for both in vitro and in vivo applications.

Main Methods:

Development of an objective-type backscattering microscope.
Utilizing gold nanoparticles as traceable markers.
Characterizing system noise and resolution (0.6 nm/axis at 55 kHz bandwidth).
Comparing background scattering with conventional dark-field microscopy.

Main Results:

The developed microscope achieves nanometer and microsecond resolution for tracking gold nanoparticles.
The system exhibits low total noise, suitable for measuring molecular movement.
Backscattering microscopy demonstrated lower background scattering from cells compared to dark-field microscopy.
The technique was successfully applied to measure the motion of the bacterial flagellar motor in live Escherichia coli.

Conclusions:

The presented backscattering microscope offers a simple yet powerful tool for high-resolution single-molecule tracking.
Its low background scattering makes it advantageous for in vivo biological imaging.
This technique provides a valuable method for studying the dynamics of molecular motors in their native environments.