A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells

Brian P. English, Robert H. Singer

Research output: Chapter in Book/Report/Conference proceedingConference contribution

8 Citations (Scopus)

Abstract

Our aim is to develop quantitative single-molecule assays to study when and where molecules are interacting inside living cells and where enzymes are active. To this end we present a three-camera imaging microscope for fast tracking of multiple interacting molecules simultaneously, with high spatiotemporal resolution. The system was designed around an ASI RAMM frame using three separate tube lenses and custom multi-band dichroics to allow for enhanced detection efficiency. The frame times of the three Andor iXon Ultra EMCCD cameras are hardware synchronized to the laser excitation pulses of the three excitation lasers, such that the fluorophores are effectively immobilized during frame acquisitions and do not yield detections that are motion-blurred. Stroboscopic illumination allows robust detection from even rapidly moving molecules while minimizing bleaching, and since snapshots can be spaced out with varying time intervals, stroboscopic illumination enables a direct comparison to be made between fast and slow molecules under identical light dosage. We have developed algorithms that accurately track and co-localize multiple interacting biomolecules. The three-color microscope combined with our co-movement algorithms have made it possible for instance to simultaneously image and track how the chromosome environment affects diffusion kinetics or determine how mRNAs diffuse during translation. Such multiplexed single-molecule measurements at a high spatiotemporal resolution inside living cells will provide a major tool for testing models relating molecular architecture and biological dynamics.

Original languageEnglish (US)
Title of host publicationProceedings of SPIE - The International Society for Optical Engineering
PublisherSPIE
Volume9550
ISBN (Print)9781628417166
DOIs
StatePublished - 2015
EventBiosensing and Nanomedicine VIII - San Diego, United States
Duration: Aug 9 2015Aug 12 2015

Other

OtherBiosensing and Nanomedicine VIII
CountryUnited States
CitySan Diego
Period8/9/158/12/15

Fingerprint

Super-resolution
Microscope
Microscopes
High Speed
Camera
Cameras
cameras
microscopes
Cells
Imaging
high speed
Molecules
Imaging techniques
Cell
molecules
Illumination
Laser excitation
EMCCD
Excitation
Laser

Keywords

  • Co-movement analysis
  • Intracellular labeling
  • Living cells
  • Single particle tracking
  • Super-resolution imaging

ASJC Scopus subject areas

  • Applied Mathematics
  • Computer Science Applications
  • Electrical and Electronic Engineering
  • Electronic, Optical and Magnetic Materials
  • Condensed Matter Physics

Cite this

English, B. P., & Singer, R. H. (2015). A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells. In Proceedings of SPIE - The International Society for Optical Engineering (Vol. 9550). [955008] SPIE. https://doi.org/10.1117/12.2190246

A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells. / English, Brian P.; Singer, Robert H.

Proceedings of SPIE - The International Society for Optical Engineering. Vol. 9550 SPIE, 2015. 955008.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

English, BP & Singer, RH 2015, A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells. in Proceedings of SPIE - The International Society for Optical Engineering. vol. 9550, 955008, SPIE, Biosensing and Nanomedicine VIII, San Diego, United States, 8/9/15. https://doi.org/10.1117/12.2190246
English BP, Singer RH. A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells. In Proceedings of SPIE - The International Society for Optical Engineering. Vol. 9550. SPIE. 2015. 955008 https://doi.org/10.1117/12.2190246
English, Brian P. ; Singer, Robert H. / A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells. Proceedings of SPIE - The International Society for Optical Engineering. Vol. 9550 SPIE, 2015.
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