一种实现显微荧光寿命图的测量方法

将脉宽120fs、重复率76MHz激光引入激光扫描显微镜的激发光路,利用其扫描系统对荧光标记样品激发扫描,将激发出的荧光从荧光探测光路引入备用的外部探测口;在探测口接一快速光电倍增管,将光电倍增管的信号送给时间相关单光子计数器,获得时间相关的荧光强度图;最后通过计算机处理获得荧光寿命图。应用此系统对青色荧光蛋白(CFP)、黄色荧光蛋白(YFP)荧光寿命进行了测量,并应用CFP、YFP实现荧光共振能量转移的测量。通过实验看出利用已有的激光扫描显微镜,配合较先进的寿命测量方法,可以很好地实现显微荧光寿命图的测量。

Method achieving fluorescence lifetime micro-image

Laser pulses of 120fs duration and 76MHz repetition rate were brought in exciting light pathway of laser scanning microscope and the sample of fluorescent tags was excited, using the scanning system of laser scanning microscope. The fluorescence excited by exciting light was brought in external detected port from fluorescence detection pathway. The fast photomultiplier was connexted on the port. The signal of the fast photomultiplier was sent to time_correlated single photon counting and time_correlated fluorescence intensity image was got. Than, the computer processes the image and the fluorescence lifetime image was obtained. The fluorescence lifetime of cyan fluorescent protein(CFP) and yellow fluorescent protein(YFP) with the system were measured. The measurement of fluorescence resonance energy transfer with CFP and YFP was achieved. With the experiments that the fluorescence lifetime micro_image measurement can be well done by combining laser scanning microscope with advanced method of fluorescence lifetime measurement is showed.