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

10 Scopus citations

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 publicationBiosensing and Nanomedicine VIII
EditorsMassoud H. Agahi, Hooman Mohseni, Manijeh Razeghi
PublisherSPIE
ISBN (Electronic)9781628417166
DOIs
StatePublished - 2015
EventBiosensing and Nanomedicine VIII - San Diego, United States
Duration: Aug 9 2015Aug 12 2015

Publication series

NameProceedings of SPIE - The International Society for Optical Engineering
Volume9550
ISSN (Print)0277-786X
ISSN (Electronic)1996-756X

Other

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

Keywords

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

ASJC Scopus subject areas

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

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