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A Protocol for Real-time 3D Single Particle Tracking
Published on: January 3, 2018
Localizing and tracking single nanoscale emitters in three dimensions with high spatiotemporal resolution using a
Michael A Thompson1, Matthew D Lew, Majid Badieirostami
1Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
Nano Letters
|December 17, 2009
Summary
This study introduces a double-helix point spread function (DH-PSF) for precise 3D nanoscale localization and tracking of faint single molecules. The method achieves approximately 10 nm precision for weak emitters, enabling real-time biological imaging.
Area of Science:
- Microscopy
- Biophysics
- Nanotechnology
Background:
- Accurate three-dimensional (3D) nanoscale localization and tracking of single molecules are crucial for understanding biological processes.
- Conventional widefield fluorescence microscopy faces limitations in achieving high precision, especially for dim emitters.
Purpose of the Study:
- To demonstrate the capability of a double-helix point spread function (DH-PSF) widefield fluorescence microscope for 3D nanoscale localization and tracking of dim single emitters.
- To quantitatively analyze the factors influencing localization precision, including photon count and emitter position.
Main Methods:
- Utilizing a widefield fluorescence microscope equipped with a DH-PSF.
- Characterizing localization precision based on photon detection and axial (z) position.
- Comparing experimental results with Fisher information calculations for theoretical precision limits.
Main Results:
- Achieved approximately 10 nm localization precision along x, y, and z axes for weak emitters.
- Demonstrated quantitative dependence of localization precision on photon number and z position.
- Successfully tracked single quantum dots in solution and quantum dot-labeled structures within living cells in 3D.
Conclusions:
- The DH-PSF enables high-precision 3D nanoscale localization and tracking of dim single emitters.
- This technique offers significant potential for advanced biological imaging and nanoscale investigations.
- The DH-PSF provides a powerful tool for real-time observation of molecular dynamics in biological systems.
