Douma Kim, Megens Remco T A, Reitsma Sietze, Prinzen Lenneke, Slaaf Dick W, Van Zandvoort Marc A M J
Department of Biophysics, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, Maastricht, the Netherlands.
Microsc Res Tech. 2007 May;70(5):467-75. doi: 10.1002/jemt.20424.
Fluorescence lifetime imaging (FLIM) provides a complementary contrast mechanism to fluorescence intensity and ratio imaging in intact tissue. With FLIM the time-resolved decay in fluorescence intensity of (interacting) fluorophores can be quantified by means of time correlated single photon counting (TCSPC). Here we focus on fluorescence lifetime imaging in intact blood vessels. Requisites for imaging in intact tissue are good penetration depth and limited tissue damage. Therefore, in this pilot-study, we performed TCSPC-FLIM using two-photon laser scanning microscopy to determine, with sub-cellular resolution, the fluorescence lifetime of two fluorescent probes. First, we focused on the nucleic acid dye SYTO41 in the various compartments of cells in vitro and in situ in the wall of intact mouse carotid arteries. Second, it was assessed whether the interaction of the lectin WGA-FITC with the endothelial glycocalyx affects its fluorescence lifetime. Results showed comparable mono-exponential fluorescence lifetimes of SYTO41 in the nuclei of cells in vitro and in situ. The slightly shorter fluorescence lifetime observed in the cytoplasm allowed discrimination of the nuclei. SYTO41 displayed strong mitochondrial staining, as was verified by the mitochondrion-specific probe CMXRos. In addition, mitochondrial staining by SYTO41 was accompanied by a green shift in emission. In the mitochondrial region, SYTO41 showed a highly bi-exponential and relatively fast decay, with two distinct lifetime components. It is hypothesized that the fitted bi-exponential decay can either be contributed to (1) the mathematical approximation of the fluorescence intensity decay or (2) the presence of free and DNA-bound SYTO41 in the mitochondrial compartment, leading to two lifetime components. The fluorescence lifetime of WGA-FITC decreased by approximately 25% upon binding to the endothelial glycocalyx. From this study, we conclude that FLIM offers an additional contrast mechanism in imaging intact tissue and provides information on binding status between a probe and its ligand.
荧光寿命成像（FLIM）为完整组织中的荧光强度成像和比率成像提供了一种互补的对比机制。通过FLIM，（相互作用的）荧光团的荧光强度随时间的衰减可以通过时间相关单光子计数（TCSPC）进行量化。在这里，我们专注于完整血管中的荧光寿命成像。在完整组织中成像的必要条件是良好的穿透深度和有限的组织损伤。因此，在这项初步研究中，我们使用双光子激光扫描显微镜进行TCSPC-FLIM，以亚细胞分辨率确定两种荧光探针的荧光寿命。首先，我们关注核酸染料SYTO41在体外细胞的各个区室以及完整小鼠颈动脉壁原位的情况。其次，评估凝集素WGA-FITC与内皮糖萼的相互作用是否会影响其荧光寿命。结果表明，SYTO41在体外和原位细胞的细胞核中的单指数荧光寿命具有可比性。在细胞质中观察到的稍短的荧光寿命使得能够区分细胞核。SYTO41表现出线粒体强染色，这由线粒体特异性探针CMXRos证实。此外，SYTO41的线粒体染色伴随着发射光的绿移。在线粒体区域，SYTO41显示出高度双指数且相对快速的衰减，具有两个不同的寿命成分。据推测，拟合的双指数衰减可能归因于（1）荧光强度衰减的数学近似，或（2）线粒体区室中游离和与DNA结合的SYTO41的存在，导致两个寿命成分。WGA-FITC与内皮糖萼结合后，其荧光寿命下降了约25%。从这项研究中，我们得出结论，FLIM在完整组织成像中提供了一种额外的对比机制，并提供了有关探针与其配体之间结合状态的信息。
