Enhancement of performance in time-domain FLIM with GaAsP hybrid detectors

Kim, Dongeun; Hwang, Wonsang; Won, Young Ho; Moon, Sucbei; Lee, Sang Yoon; Kang, Min Gu; Han, Won Sub; Kim, Dug Young

doi:10.1117/12.2510959

Cited by 3 publications

(4 citation statements)

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“…To examine the differences between HPD (R10467U-40, Hamamatsu) and PMT (H10721-210, Hamamatsu) photon peak height distribution, we examined two-photon fluorescence of NADH in concentrations from 1 to 5 mM on our custom FLIM system (similar to our previously published system; , see Figure S1) using both detectors and digitizing the amplified output at 5 GS/s. The peak height distributions (Figure ) match well with previously experimental , and theoretical comparisons of HPD and PMT performance. By examining the local minima of the HPD and PMT peak height distributions, thresholds for one to five photons were set for the HPD and a threshold for a single photon was set for the PMT (dashed vertical lines, Figure ).…”

Section: Resultssupporting

confidence: 84%

“…PMTs have high gain variability due to the multiple dynode amplification, in which the randomness associated with signal amplification at each dynode is compounded. HPDs have a two-step amplification process consisting of electron bombardment and then avalanche gain, which leads to low gain variability and a more linear dependence of signal on the number of incident photons. , The signal created by a PMT when a single photon arrives at the detector is highly variable and cannot be easily distinguished from the signal created when multiple photons arrive nearly simultaneously; however, when using an HPD, single- and multiphoton responses can be distinguished. − Although this principle is known, to our knowledge, it has not previously been utilized in computational photon counting methods to count multiple photons arriving at the HPD within the detector response time.…”

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confidence: 99%

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

et al. 2022

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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.

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Section: Resultssupporting

confidence: 84%

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confidence: 99%

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

et al. 2022

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“…Hybrid photodetectors, such as GaAsP hybrid photomultipliers and InGaAs emICCD, were also used for TG imaging. 25,51,61,62 Current-assisted photonic sampler arrays combined with CMOS sensors were developed for TG detection. 63 Time-correlated single photon counting (TCSPC) technique based on SPAD or PMT detectors was used in TG imaging.…”

Section: Detectionmentioning

confidence: 99%

Time-gated fluorescence imaging: Advances in technology and biological applications

Yang

Chen

2020

J. Innov. Opt. Health Sci.

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Time-gated (TG) fluorescence imaging (TGFI) has attracted increasing attention within the biological imaging community, especially during the past decade. With rapid development of light sources, image devices, and a variety of approaches for TG implementation, TGFI has demonstrated numerous biological applications ranging from molecules to tissues. The paper presents inclusive TG implementation mainly based on optical choppers and electronic units for synchronization of fluorescence excitation and emission, which also serves as guidelines for researchers to build suited TGFI systems for selected applications. Note that a special focus will be put on TG implementation based on optical choppers for TGFI of long-lived probes (lifetime range from microseconds to milliseconds). Biological applications by TG imaging of recently developed luminescent probes are described.

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“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Sorrells¹,

Marjanović

Iyer³

et al. 2023

Multiphoton Microscopy in the Biomedical Sciences XXIII

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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 multi-photon 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.

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Enhancement of performance in time-domain FLIM with GaAsP hybrid detectors

Cited by 3 publications

References 5 publications

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

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

Time-gated fluorescence imaging: Advances in technology and biological applications

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

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