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Fluorescence lifetime microscopy with a time- and space-resolved single-photon counting detector.

X Michalet1, O H W Siegmund2, J V Vallerga2

  • 1Department of Chemistry & Biochemistry, University of California at Los Angeles 607 Charles E. Young Drive E., Los Angeles, CA 90095, USA.

Proceedings of Spie--The International Society for Optical Engineering
|February 17, 2018
PubMed
Summary

A new H33D detector offers high-speed, high-resolution photon counting for advanced fluorescence lifetime imaging. This technology shows promise as an alternative to current methods for live-cell imaging.

Keywords:
FLIMfluorescencelifetimelive cellmicrochannel platephotocathodeposition sensitive anodequantum dotsingle-moleculesingle-photon

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Area of Science:

  • Biophysics
  • Optical Imaging
  • Biotechnology

Background:

  • Photon-counting detectors are crucial for high-resolution imaging.
  • Existing technologies for fluorescence lifetime imaging (FLIM) have limitations in speed and resolution.

Purpose of the Study:

  • To introduce and characterize the H33D detector for wide-field, photon-counting applications.
  • To demonstrate the H33D detector's utility in fluorescence lifetime imaging of live cells.
  • To compare the H33D detector's performance against established scanning confocal Time Correlated Single Photon Counting (TCSPC) systems.

Main Methods:

  • The H33D detector was developed with high temporal and spatial resolutions, capable of detecting up to 500,000 photons per second.
  • Temporal performance was assessed using fluorescent solutions with varying lifetimes.
  • Spatial resolution was evaluated using sub-diffraction limit objects like beads and quantum dots.
  • The detector was applied to live-cell fluorescence lifetime imaging and compared with a scanning confocal TCSPC setup.

Main Results:

  • The H33D detector exhibits high temporal and spatial resolutions.
  • Successful application of the H33D detector for fluorescence lifetime imaging of live cells was demonstrated.
  • Performance comparison indicated the H33D detector as a viable alternative to scanning confocal TCSPC.

Conclusions:

  • The H33D detector is a promising wide-field photon-counting technology for advanced imaging.
  • Further improvements in photocathode sensitivity and detector throughput are expected.
  • This technology offers a potential advancement over current fluorescence lifetime imaging solutions.