Iyer Rishyashring R, Sorrells Janet E, Tan Kevin K D, Yang Lingxiao, Wang Geng, Tu Haohua, Boppart Stephen A
Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA.
Department of Electrical and Computer Engineering, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA.
Biomed Opt Express. 2024 Mar 4;15(4):2048-2062. doi: 10.1364/BOE.514813. eCollection 2024 Apr 1.
The dynamic range and fluctuations of fluorescence intensities and lifetimes in biological samples are large, demanding fast, precise, and versatile techniques. Among the high-speed fluorescence lifetime imaging microscopy (FLIM) techniques, directly sampling the output of analog single-photon detectors at GHz rates combined with computational photon counting can handle a larger range of photon rates. Traditionally, the laser clock is not sampled explicitly in fast FLIM; rather the detection is synchronized to the laser clock so that the excitation pulse train can be inferred from the cumulative photon statistics of several pixels. This has two disadvantages for sparse or weakly fluorescent samples: inconsistencies in inferring the laser clock within a frame and inaccuracies in aligning the decay curves from different frames for averaging. The data throughput is also very inefficient in systems with repetition rates much larger than the fluorescence lifetime due to significant silent regions where no photons are expected. We present a method for registering the photon arrival times to the excitation using time-domain multiplexing for fast FLIM. The laser clock is multiplexed with photocurrents into the silent region. Our technique does not add to the existing data bottleneck, has the sub-nanosecond dead time of computational photon counting based fast FLIM, works with various detectors, lasers, and electronics, and eliminates the errors in lifetime estimation in photon-starved conditions. We demonstrate this concept on two multiphoton setups of different laser repetition rates for single and multichannel FLIM multiplexed into a single digitizer channel for real-time imaging of biological samples.
生物样品中荧光强度和寿命的动态范围及波动很大，这就需要快速、精确且通用的技术。在高速荧光寿命成像显微镜（FLIM）技术中，以吉赫兹速率直接采样模拟单光子探测器的输出并结合计算光子计数，能够处理更大范围的光子速率。传统上，在快速FLIM中不会明确采样激光时钟；相反，检测与激光时钟同步，以便从几个像素的累积光子统计中推断出激发脉冲序列。对于稀疏或弱荧光样品，这有两个缺点：在一帧内推断激光时钟时存在不一致性，以及在对来自不同帧的衰减曲线进行平均时对齐不准确。在重复率远大于荧光寿命的系统中，由于存在大量预期无光子的静默区域，数据吞吐量也非常低效。我们提出了一种用于快速FLIM的方法，通过时域复用将光子到达时间与激发进行配准。激光时钟与光电流复用进入静默区域。我们的技术不会增加现有的数据瓶颈，具有基于计算光子计数的快速FLIM的亚纳秒死时间，可与各种探测器、激光器和电子设备配合使用，并消除了光子匮乏条件下寿命估计中的误差。我们在两种不同激光重复率的多光子设置上演示了这一概念，用于单通道和多通道FLIM复用进入单个数字化仪通道，以对生物样品进行实时成像。
