Sorrells Janet E, Iyer Rishyashring R, Yang Lingxiao, Martin Elisabeth M, Wang Geng, Tu Haohua, Marjanovic Marina, Boppart Stephen A
Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
ACS Photonics. 2022 Aug 17;9(8):2748-2755. doi: 10.1021/acsphotonics.2c00505. Epub 2022 Jul 12.
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 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.
时间分辨光子计数方法具有有限的带宽,这限制了诸如荧光寿命成像显微镜(FLIM)等技术的采集速度。为了实现更快的成像,当探测器的输出以高采样率直接数字化时,可以采用计算方法来计数光子。在此,我们展示了使用混合光电探测器结合多阈值峰值检测的计算光子计数,以计数在探测器响应时间内一个或多个光子到达探测器的情况。该方法可用于区分每个数字化点多达五个光子计数,而之前在使用光电倍增管采集的数据上进行的计算光子计数演示一次仅计数一个光子。我们在自由移动的动物以及正在经历凋亡的人乳腺癌细胞系中均证明,这种新型的多阈值峰值检测方法能够准确表征产生高达223%光子率的样品的强度和荧光寿命,这一光子率高于之前展示的光子计数FLIM系统。