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3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles
Published on: October 1, 2014
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Orbital Single Particle Tracking on a commercial confocal microscope using piezoelectric stage feedback
Luca Lanzanò1, Enrico Gratton1
1Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA 92697, United States.
Methods and Applications in Fluorescence
|November 25, 2014
Summary
This study presents a novel single particle tracking (SPT) setup using orbital tracking and feedback. This method achieves high-precision 3D tracking of fluorescent particles, demonstrated with live cell imaging.
Area of Science:
- Biophysics
- Optical Microscopy
- Cell Biology
Background:
- Single Particle Tracking (SPT) enables nanometer-precision localization of fluorescent particles.
- Orbital tracking analyzes intensity distribution along a scanned circle for particle positioning.
- Active feedback integration allows for high temporal resolution (millisecond) 2D and 3D tracking.
Purpose of the Study:
- To describe a novel SPT setup utilizing orbital tracking with active feedback.
- To implement this system with minimal modifications to a commercial confocal laser scanning microscope (Zeiss LSM 510).
- To evaluate the advantages and limitations of this feedback-based orbital tracking approach.
Main Methods:
- Implementation of an orbital tracking method with active feedback.
- Integration with a commercial confocal laser scanning microscope (Zeiss LSM 510).
- Utilizing an external piezoelectric stage scanner for feedback control.
Main Results:
- Successful implementation of a user-friendly SPT setup with feedback.
- Demonstration of 3D tracking capabilities with millisecond temporal resolution.
- Identification of mechanical response limitations associated with the external piezo-scanner.
Conclusions:
- The described SPT setup offers a practical approach for high-resolution particle tracking.
- The system is suitable for live cell experiments, as shown by tracking acidic vesicles.
- Further optimization may be needed to overcome limitations of external scanning components.
