Fast fluorescence lifetime imaging microscopy using single- and multiphoton peak event detection for rapid quantification of NAD(P)H-related metabolic dynamics during apoptosis

Janet E. Sorrells, Marina Marjanovic, Rishyashring R. Iyer, Lingxiao Yang, Eric J. Chaney, Geng Wang, Haohua Tu, Stephen A. Boppart

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


Multiphoton fluorescence lifetime imaging microscopy (FLIM) is used to collect label-free metabolic information from biological samples via autofluorescence lifetime imaging of reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H). However, FLIM has traditionally been limited by slow acquisition due to the limited bandwidth of analog electronics that perform photon counting and time-tagging. This slow acquisition has restricted the applicability of multiphoton FLIM of NAD(P)H by impeding the ability to accurately study biological problems that require characterization of fast dynamics. Faster image acquisition can be achieved by directly digitizing the amplified output of a hybrid photodetector and computationally determining photon counts via the Single- and multiphoton PEak Event Detection (SPEED) algorithm. This method, bypassing the limited-bandwidth analog electronics used for photon counting and time-tagging of photons in traditional FLIM, enables fast photon counting capabilities which are well suited for fast, high-dynamic range biological processes such as metabolic changes during apoptosis. Here, we utilize this technology to examine fast dynamics of apoptosis in 2D culture of normal and cancerous human breast cell lines, rat mammary tumor tissue-derived organoids, and in vivo rat mammary tumors. Results indicate that apoptosis-related metabolic dynamics are biological model-dependent and based on local pharmacokinetics, with tumor derived organoids in Matrigel showing a significantly slower response than in vivo or in vitro 2D cell models. Future work should carefully consider these implications when determining which tumor model to use for experimentation and should improve tumor models to better represent in vivo tumor apoptosis dynamics.

Original languageEnglish (US)
Title of host publicationMultiphoton Microscopy in the Biomedical Sciences XXIII
EditorsAmmasi Periasamy, Peter T. So, Karsten Konig
ISBN (Electronic)9781510658738
StatePublished - 2023
EventMultiphoton Microscopy in the Biomedical Sciences XXIII 2023 - San Francisco, United States
Duration: Jan 29 2023Jan 31 2023

Publication series

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


ConferenceMultiphoton Microscopy in the Biomedical Sciences XXIII 2023
Country/TerritoryUnited States
CitySan Francisco


  • Fluorescence lifetime
  • breast cancer
  • cell death
  • graphics processing units
  • label-free
  • multiphoton microscopy
  • single photon

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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