Morgridge Institute for Research, Madison, Wisconsin, United States.
University of Wisconsin, Department of Electrical and Computer Engineering, Madison, Wisconsin, United States.
J Biomed Opt. 2023 Jun;28(6):066502. doi: 10.1117/1.JBO.28.6.066502. Epub 2023 Jun 21.
Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples.
We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM.
A single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells.
An NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times. NAD(P)H light-sheet FLIM was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a to acquisition speed advantage for the light sheet compared to the laser scanning geometry.
FLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.
荧光寿命成像显微镜（FLIM）对代谢辅酶烟酰胺腺嘌呤二核苷酸（磷酸）[NAD（P）H]的荧光寿命成像，是一种在未受干扰的活 3D 系统中监测单细胞代谢的常用方法。然而，NAD（P）H 的 FLIM 尚未在光片几何结构中进行，该结构有利于快速对活 3D 样本中的细胞进行成像。
我们旨在设计、验证和展示用于 NAD（P）H FLIM 的概念验证光片系统。
单光子雪崩二极管相机被集成到光片显微镜中，以实现光学切片，并限制对细胞 NAD（P）H FLIM 的离焦贡献。
构建并验证了 NAD（P）H 光片 FLIM 系统，该系统使用荧光寿命标准品，并对胰腺癌细胞的代谢扰动进行了 10 秒积分时间的时程成像。还使用 10 秒积分时间，对活体中性粒细胞在斑马鱼幼虫尾部伤口中的成像进行了 NAD（P）H 光片 FLIM 演示。最后，比较了激光扫描和光片几何结构中 NAD（P）H FLIM 的理论和实际成像速度，表明与激光扫描几何结构相比，光片具有 到 的采集速度优势。
NAD（P）H 的 FLIM 可在光片几何结构中实现，并且非常适合 3D 活细胞成像应用，例如在生物体中监测免疫细胞代谢和迁移。
