Computational Photon Counting Using Multithreshold Peak Detection for Fast Fluorescence Lifetime Imaging Microscopy

Abstract

Time-resolved photon counting methods have a finite bandwidth that restricts the acquisition speed of techniques like fluorescence lifetime imaging microscopy (FLIM). To enable faster imaging, computational methods can be employed to count photons when the output of a detector is directly digitized at a high sampling rate. Here, we present computational photon counting using a hybrid photodetector in conjunction with multithreshold peak detection to count instances where one or more photons arrive at the detector within the detector response time. This method can be used to distinguish up to five photon counts per digitized point, whereas previous demonstrations of computational photon counting on data acquired with photomultiplier tubes have only counted one photon at a time. We demonstrate in both freely moving C. elegans and a human breast cancer cell line undergoing apoptosis that this novel multithreshold peak detection method can accurately characterize the intensity and fluorescence lifetime of samples producing photon rates up to 223%, higher than previously demonstrated photon counting FLIM systems.

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

“…Photon counts are determined after digitization (instead of before) to bypass the limited bandwidth analog electronics of TCSPC (Fig. 1) [8,9]. Photons are counted using the Single-and multi-photon PEak Event Detection (SPEED) algorithm which uses a simple thresholded local maxima detection to enable fast and accurate FLIM for photon count rates up to 180 Megacounts-per-second per channel [8].…”

“…1) [8,9]. Photons are counted using the Single-and multi-photon PEak Event Detection (SPEED) algorithm which uses a simple thresholded local maxima detection to enable fast and accurate FLIM for photon count rates up to 180 Megacounts-per-second per channel [8]. Digitized data points with a larger magnitude than the points immediately before and after it are considered "peaks".…”

“…These thresholds are determined based on calibration data for a hybrid photodetector (HPD) under constant gain conditions. HPDs, unlike PMTs, have a very linear gain, and have been shown to have semi-discrete peak height distributions that represent different numbers of photons incident on the detector [8,10]. This principle is used by creating multiple thresholds in SPEED to resolve more than one temporally-encoded photon count per digitized point, allowing for high photon rate capacity [8].…”

“…HPDs, unlike PMTs, have a very linear gain, and have been shown to have semi-discrete peak height distributions that represent different numbers of photons incident on the detector [8,10]. This principle is used by creating multiple thresholds in SPEED to resolve more than one temporally-encoded photon count per digitized point, allowing for high photon rate capacity [8]. Photon rate is the average number of photons per laser pulse, given as a percentage (1 photon per pulse is 100% photon rate).…”

“…Photon rate is the average number of photons per laser pulse, given as a percentage (1 photon per pulse is 100% photon rate). The maximum acceptable photon rate of most TCSPC systems to around 5-10%, and 100% for the best single-detector systems [6,7], whereas SPEED using an HPD has shown accurate performance at photon rates up to 226% [8].…”