Temporal and spatial binning of TCSPC data to improve signal-to-noise ratio and imaging speed

Alex J. Walsh, Hope T. Beier

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

1 Scopus citations


Time-correlated single photon counting (TCSPC) is the most robust method for fluorescence lifetime imaging using laser scanning microscopes. However, TCSPC is inherently slow making it ineffective to capture rapid events due to the single photon product per laser pulse causing extensive acquisition time limitations and the requirement of low fluorescence emission efficiency to avoid bias of measurement towards short lifetimes. Furthermore, thousands of photons per pixel are required for traditional instrument response deconvolution and fluorescence lifetime exponential decay estimation. Instrument response deconvolution and fluorescence exponential decay estimation can be performed in several ways including iterative least squares minimization and Laguerre deconvolution. This paper compares the limitations and accuracy of these fluorescence decay analysis techniques to accurately estimate double exponential decays across many data characteristics including various lifetime values, lifetime component weights, signal-to-noise ratios, and number of photons detected. Furthermore, techniques to improve data fitting, including binning data temporally and spatially, are evaluated as methods to improve decay fits and reduce image acquisition time. Simulation results demonstrate that binning temporally to 36 or 42 time bins, improves accuracy of fits for low photon count data. Such a technique reduces the required number of photons for accurate component estimation if lifetime values are known, such as for commercial fluorescent dyes and FRET experiments, and improve imaging speed 10-fold.

Original languageEnglish (US)
Title of host publicationMultiphoton Microscopy in the Biomedical Sciences XVI
EditorsPeter T. C. So, Karsten Konig, Ammasi Periasamy, Karsten Konig
ISBN (Electronic)9781628419467
StatePublished - 2016
EventMultiphoton Microscopy in the Biomedical Sciences XVI - San Francisco, United States
Duration: Feb 14 2016Feb 16 2016

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
ISSN (Print)1605-7422


ConferenceMultiphoton Microscopy in the Biomedical Sciences XVI
Country/TerritoryUnited States
CitySan Francisco


  • Laguerre polynomials
  • deconvolution
  • fluorescence lifetime imaging

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging


Dive into the research topics of 'Temporal and spatial binning of TCSPC data to improve signal-to-noise ratio and imaging speed'. Together they form a unique fingerprint.

Cite this