Fluorescence lifetime imaging of calcium flux in neurons in response to pulsed infrared light

Alex J. Walsh, Anna Sedelnikova, Gleb P. Tolstykh, Bennett L. Ibey, Hope T. Beier

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

5 Scopus citations


Pulsed infrared light can excite action potentials in neurons; yet, the fundamental mechanism underlying this phenomenon is unknown. Previous work has observed a rise in intracellular calcium concentration following infrared exposure, but the source of the calcium and mechanism of release is unknown. Here, we used fluorescence lifetime imaging of Oregon Green BAPTA-1 to study intracellular calcium dynamics in primary rat hippocampal neurons in response to infrared light exposure. The fluorescence lifetime of Oregon Green BAPTA-1 is longer when bound to calcium, and allows robust measurement of intracellular free calcium concentrations. First, a fluorescence lifetime calcium calibration curve for Oregon Green BAPTA-1 was determined in solutions. The normalized amplitude of the short and long lifetimes was calibrated to calcium concentration. Then, neurons were incubated in Oregon Green BAPTA-1 and exposed to pulses of infrared light (0-1 J/cm2; 0-5 ms; 1869 nm). Fluorescence lifetime images were acquired prior to, during, and after the infrared exposure. Fluorescence lifetime images, 64x64 pixels, were acquired at 12 or 24 ms for frame rates of 83 and 42 Hz, respectively. Accurate α1 approximations were achieved in images with low photon counts by computing an α1 index value from the relative probability of the observed decay events. Results show infrared light exposure increases intracellular calcium in neurons. Altogether, this study demonstrates accurate fluorescence lifetime component analysis from low-photon count data for improved imaging speed.

Original languageEnglish (US)
Title of host publicationMultiphoton Microscopy in the Biomedical Sciences XVII
EditorsKarsten Konig, Peter T. C. So, Ammasi Periasamy, Xiaoliang S. Xie
ISBN (Electronic)9781510605794
StatePublished - 2017
EventMultiphoton Microscopy in the Biomedical Sciences XVII - San Francisco, United States
Duration: Jan 29 2017Jan 31 2017

Publication series

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


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


  • Oregon Green BAPTA-1
  • fluorescence lifetime imaging
  • infrared neural stimulation
  • time-correlated single photon counting

ASJC Scopus subject areas

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


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