Two-photon fluorescence lifetime imaging microscopy utilizing the space-time duality

项目摘要 荧光寿命成像显微镜（FLIM）是一种荧光成像技术， 在生物医学中越来越受欢迎，因为它提供了对分子构象的最直接的洞察， 荧光团的生物环境。FLIM已被应用于提供对细胞的见解， 代谢，蛋白质-蛋白质相互作用，以及温度，粘度，pH， 和离子浓度。尽管FLIM提供了丰富的信息，但它的广泛应用仍然是 目前受到低成像速度的限制。FLIM成像速度是许多因素的复杂函数， 散粒噪声的光子计数统计是基本的限制。这一限制尤其突出， 荧光团的寿命短于FLIM仪器响应函数（IRF），当去卷积 这是精确测定荧光寿命所必需的。因此，为了从根本上增强FLIM成像， 速度，要么增加最大光子计数率或减少FLIM IRF是必要的。 利用时间相关的单光子和非相干的单光子， 计数（TCSPC）或光子计数条纹照相机（PCSC）。最大光子计数率 现有的时域FLIM是1-10兆计数每秒（Mcps），受到TCSPC-FLIM中的堆积效应的限制 以及PCSC-FLIM中的读出非线性和串扰。TCSPC FLIM通常具有100 ps IRF，除非 需要低温冷却的超导纳米线单光子探测器被实现以达到 皮秒机制。另一方面，PCSC-FLIM可以在室温下实现皮秒IRF，但 需要复杂的条纹和检测光电器件。使用PCSC-FLIM，最近对阿尔茨海默病的研究 小鼠脑组织发现了一种新的30 ps寿命成分，对于区分阿尔茨海默病和 正常脑组织，烟酰胺腺嘌呤二核苷酸水合物（NADH）。如果没有PCSC-FLIM的10 ps IRF， 在合理的时间量内不可能观察到这种快速的荧光衰减。类似地第 短IRF将有利于研究短寿命的非脂褐质自荧光团（30-70 ps），这将导致更好的 了解眼底自发荧光诊断，并可能为早期视网膜病变提供相关信息。 老年性黄斑变性和神经退行性疾病的检测。 该提案将开发一种潜在的变革性FLIM系统，光子条纹FLIM（PS-FLIM）， 通过同时降低IRF和增加最大光子数来解决成像速度挑战 计数率一个新的概念光子条纹，基于时空对偶性的原则，将 实现了在紧凑和轻量级平台中实现5 ps IRF和840 Mcps。双光子激发 将用于增加成像深度和降低光毒性。最后，机器学习框架 将被纳入加速FLIM数据分析。

PROJECT SUMMARY Fluorescence lifetime imaging microscopy (FLIM) is a type of fluorescence imaging technologies that is gaining popularity in biomedicine because it delivers the most direct insight into the molecular conformation and the biological environment of a fluorophore. FLIM has been applied to provide insights into the cellular metabolism, protein-protein interactions, and biological environment monitoring of temperature, viscosity, pH, and ion concentration. Despite the wealth of information provided by the FLIM, its widespread application is currently limited by the low imaging speed. The FLIM imaging speed is a complex function of many factors, with shot noise by the photon counting statistics being the fundamental limit. This limitation is especially dominant for fluorophores with lifetime shorter than the FLIM instrument response function (IRF) when deconvolution is necessary to accurately determine the fluorescence lifetime. Thus, to fundamentally enhance the FLIM imaging speed, either an increase of the maximum photon counting rate or a reduction of the FLIM IRF is necessary. Time-domain FLIM with high photon efficiency can be implemented with either time-correlated single-photon counting (TCSPC) or photon counting streak camera (PCSC). The maximum photon counting rate of state-of- the-art time-domain FLIM is 1-10 mega counts per second (Mcps), limited by the pile up effect in TCSPC-FLIM and the readout nonlinearity and crosstalk in PCSC-FLIM. TCSPC-FLIM generally has a 100-ps IRF, unless superconducting nanowire single-photon detectors that require cryogenic cooling are implemented to reach the picosecond regime. On the other hand, PCSC-FLIM can achieve the picosecond IRF at room temperature, but complex streaking and detection optoelectronics are required. Using PCSC-FLIM, a recent study on Alzheimer mouse brain tissue has found a new 30-ps lifetime component, critical for separating Alzheimer disease from normal brain tissue, of nicotinamide adenine dinucleotide hydrate (NADH). Without the 10-ps IRF of PCSC-FLIM, such fast fluorescence decay could not have been observed within a reasonable amount of time. Similarly, a short IRF will benefit the study of short-lived non-lipofuscin autofluorophores (30-70 ps) that will lead to a better understanding of the fundus autofluorescence diagnosis and may provide relevant retina information for the early detection of age-related macular degeneration and neurodegenerative diseases. This proposal will develop a potentially transformative FLIM system, photon-streaking FLIM (PS-FLIM), that addresses the imaging speed challenge by simultaneously reducing the IRF and increasing the maximum photon counting rate. A new concept of photon streaking, based on the principle of space-time duality, will be implemented to achieve 5-ps IRF and 840 Mcps in a compact and lightweight platform. Two-photon excitation will be utilized to increase the imaging depth and reduce the phototoxicity. Finally, machine learning framework will be incorporated to accelerate the FLIM data analysis.