Agronskaia A V, Tertoolen L, Gerritsen H C
Utrecht University, Debye Institute, Department of Molecular Biophysics, P.O. Box 80000, 3508 TA Utrecht, The Netherlands.
J Biomed Opt. 2004 Nov-Dec;9(6):1230-7. doi: 10.1117/1.1806472.
A fast fluorescence lifetime imaging (FLIM) system is developed that can acquire images at a rate of hundreds of frames per second. The FLIM system is based on a wide-field microscope equipped with a time-gated intensified CCD detector and a pulsed laser. The time-gated detector acquires the signals from two time gates simultaneously and is therefore insensitive to movements of the specimen and photo-bleaching. The system is well suited for quantitative biological FLIM experiments and its performance is evaluated in calcium imaging experiments on beating neonatal rat myocytes. Several calcium sensitive dyes are characterized and tested for their suitability for fast FLIM experiments: Oregon Green Bapta-1 (OGB1), Oregon Green Bapta-2 (OGB2), and Oregon Green Bapta-5N (OGB5N). Overall the sensitivity range of these dyes is shifted to low calcium concentrations when used as lifetime dyes. OGB1 and OGB2 behave very similarly and can be used for FLIM-based calcium imaging in the range 1 to approximately 500 nM and OGB5N can be used up to 3 microM. The fast FLIM experiments on the myocytes could be carried out at a 100-Hz frame rate. During the beating of the myocytes a lifetime change of about 20% is observed. From the lifetime images a rest calcium level of about 65 nM is found.
开发了一种快速荧光寿命成像（FLIM）系统，该系统能够以每秒数百帧的速率采集图像。该FLIM系统基于配备了时间门控增强型电荷耦合器件（CCD）探测器和脉冲激光器的宽视场显微镜。时间门控探测器同时从两个时间门获取信号，因此对样本的移动和光漂白不敏感。该系统非常适合定量生物FLIM实验，并在新生大鼠跳动心肌细胞的钙成像实验中对其性能进行了评估。对几种钙敏感染料进行了特性分析，并测试了它们在快速FLIM实验中的适用性：俄勒冈绿钙黄绿素-1（OGB1）、俄勒冈绿钙黄绿素-2（OGB2）和俄勒冈绿钙黄绿素-5N（OGB5N）。总体而言，当用作寿命染料时，这些染料的灵敏度范围向低钙浓度偏移。OGB1和OGB2的表现非常相似，可用于1至约500 nM范围内基于FLIM的钙成像，而OGB5N可用于高达3 microM的浓度。对心肌细胞的快速FLIM实验可以以100 Hz的帧率进行。在心肌细胞跳动过程中，观察到寿命变化约20%。从寿命图像中发现静息钙水平约为65 nM。
