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Title: Active-Feedback 3D Single-Molecule Tracking Using a Fast-Responding Galvo Scanning Mirror
Real-time three-dimensional single-particle tracking (RT-3D-SPT) allows continuous detection of individual freely diffusing objects with high spatiotemporal precision by applying closed-loop active feedback in an optical microscope. However, the current tracking speed in RT-3D-SPT is primarily limited by the response time of control actuators, impeding long-term observation of fast diffusive objects such as single molecules. Here, we present an RT-3D-SPT system with improved tracking performance by replacing the XY piezoelectric stage with a galvo scanning mirror with an approximately five-time faster response rate (~5 kHz). Based on the previously developed 3D single-molecule active real-time tracking (3D-SMART), this new implementation with a fast-responding galvo mirror eliminates the mechanical movement of the sample and allows more rapid response to particle motion. The improved tracking performance of the galvo mirror-based implementation is verified through simulation and proof-of-principle experiments. Fluorescent nanoparticles and ~ 1 kB double-stranded DNA molecules were tracked via both the original piezoelectric stage and new galvo mirror implementations. With the new galvo-based implementation, notable increases in tracking duration, localization precision, and the degree to which the objects are locked to the center of the detection volume were observed. These results suggest faster control response elements can expand RT-3D-SPT to a broader range of chemical and biological systems.  more » « less
Award ID(s):
1847899
PAR ID:
10548149
Author(s) / Creator(s):
; ; ; ; ;
Publisher / Repository:
ACS
Date Published:
Journal Name:
The Journal of Physical Chemistry A
Volume:
127
Issue:
30
ISSN:
1089-5639
Page Range / eLocation ID:
6320 to 6328
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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