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

利用时空二象性的双光子荧光寿命成像显微镜

• 批准号: 10593761
• 负责人: Shu-Wei Huang
• 金额: $ 20.06万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-02 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593761
• 关键词: Acceleration; Address; Age related macular degeneration; Alzheimer' s Disease; Benchmarking; Biological; Color; Complex; Data Analyses; Detection; Development; Diagnosis; Dietary Carotenoid; Dyes; Early Diagnosis; Electrons; Environment; Family suidae; Feedback; Fiber; Fluorescence; Fundus; Future; Hydration status; Image; Imaging technology; Ions; Lasers; Light; Lutein; Machine Learning; Measures; Metabolism; Methods; Molecular Conformation; Monitor; Mus; Neurodegenerative Disorders; Neurosciences; Nicotinamide adenine dinucleotide; Noise; Ophthalmology; Optics; Phase; Photons; Phototoxicity; Physiologic pulse; Protocols documentation; Retina; Silicones; Source; Speed; System; Technology; Temperature; Time; Tissue Model; Tissues; Training; Viscosity; brain tissue; cryogenics; ex vivo imaging; experimental study; fluorescence imaging; fluorescence lifetime imaging; fluorescence microscope; fluorophore; imaging study; in vivo; innovation; insight; instrument; invention; light microscopy; light weight; machine learning framework; macula; microscopic imaging; multiphoton microscopy; nanowire; photon-counting detector; protein protein interaction; response; statistics; three dimensional structure; two-dimensional; two-photon; zeaxanthin

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.

项目摘要 荧光寿命成像显微镜（FLIM）是一种荧光成像技术 在生物医学中获得流行，因为它对分子构象最直接的见解和 荧光团的生物环境。 Flim已被应用于提供洞察力 代谢，蛋白质 - 蛋白质相互作用以及温度，粘度，pH，pH， 和离子浓度。尽管Flim提供了丰富的信息，但其广泛应用是 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， 光子计数统计数据的射击噪声是基本限制。这种限制特别主导 当反卷积为时，寿命短于Flim仪器响应函数（IRF） 准确确定荧光寿命所必需的。因此，从根本上增强了FLIM成像 速度，要么需要增加最大光子计数速率，要么需要降低FLIM IRF。 具有高光子效率的时间域FLIM可以用任何时间相关的单光子实现 计数（TCSPC）或光子计数条纹摄像头（PCSC）。最大的光子计数速率 ART时间域FLIM是每秒1-10个巨型计数（MCP），受TCSPC-FLIM中的堆积效果的限制 以及PCSC-FLIM中的读数非线性和串扰。 TCSPC-FLIM通常具有100-PS IRF，除非 实施需要低温冷却的超导纳米线单光子探测器以达到 Picsecond政权。另一方面，PCSC-FLIM可以在室温下实现Picsecond IRF，但是 需要复杂的条纹和检测光电子。使用PCSC-FLIM，这是一项关于阿尔茨海默氏症的研究 小鼠脑组织发现了一个新的30-ps寿命成分，对于将阿尔茨海默氏病与 正常的脑组织，烟酰胺腺嘌呤二核苷酸水合物（NADH）。没有PCSC-FLIM的10-PS IRF， 在合理的时间内无法观察到如此快速的荧光衰减。同样， 简短的IRF将使短寿命的非脂蛋白自动荧光团（30-70 PS）受益，这将导致更好 了解眼底自动荧光诊断，并可能为早期提供相关的视网膜信息 检测与年龄相关的黄斑变性和神经退行性疾病。 该提案将发展一个潜在的变革性flim系统，即光子thtreaking Flim（PS-FLIM），该系统 通过同时减少IRF并增加最大光子来解决成像速度挑战 计数率。基于时空双重性原理的一个新的光子条纹概念将是 在紧凑而轻巧的平台中实现了5-PS IRF和840 MCP。两光蛋白激发 将用于增加成像深度并降低光毒性。最后，机器学习框架 将合并以加速FLIM数据分析。