Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells. Here, we describe the theoretical background that justifies the developments of high-speed single photon counting systems. We show that systems with low dead-times not only result in faster acquisition throughputs but also improved dynamic range and spatial resolution. We also share the implementation of hardware and software as an open platform, show applications of fast FLIM biochemical imaging on living cells and discuss strategies to balance precision and accuracy in FLIM. The recent innovations and commercialisation of fast time-domain FLIM systems are likely to popularise FLIM within the biomedical community, to impact biomedical research positively and to foster the adoption of other FLIM techniques as well. While supporting and indeed pursuing these developments, with this work we also aim to warn the community about the possible shortcomings of fast single photon counting techniques and to highlight strategies to acquire data of high quality.

中文翻译：

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快速单细胞生物化学：理论、开源显微镜和应用。

荧光寿命传感使研究人员能够探测荧光团的物理化学环境，为我们观察细胞复杂的分子组成提供了一个窗口。荧光寿命成像显微镜 (FLIM) 可量化和绘制细胞生物化学（动态过程的复杂整体）。不幸的是，典型的高分辨率 FLIM 系统的采集速度相当有限，通常不足以捕获活细胞中生化过程的时间演变。在这里，我们描述了证明高速单光子计数系统发展合理性的理论背景。我们表明，死区时间短的系统不仅可以提高采集吞吐量，还可以提高动态范围和空间分辨率。我们还分享了作为开放平台的硬件和软件的实现，展示了快速 FLIM 生化成像在活细胞上的应用，并讨论了平衡 FLIM 精度和准确度的策略。快速时域 FLIM 系统的最新创新和商业化可能会在生物医学界普及 FLIM，对生物医学研究产生积极影响，并促进其他 FLIM 技术的采用。在支持并确实追求这些发展的同时，通过这项工作，我们还旨在警告社区快速单光子计数技术可能存在的缺点，并强调获取高质量数据的策略。