时间相关单光子计数记录快速荧光寿命图像(英文)

Recording fast time-series of fluorescence lifetime images by TCSPC FLIM

基于时间相关单光子计数(TCSPC)的荧光寿命成像(FLIM)的获取时间取决于成像的图像尺寸、样品寿命的精度及样品计数率.对于高密度荧光团样品,如染色组织或植物细胞,当前可用的样品计数率与TCSPC荧光寿命成像技术最高的计数率接近.该文描述了在高计数率下TCSPC性能,并估计计数损失及堆积效应.结果表明,整个体系的寿命误差比预料的要小.因此, TCSPC FLIM可在获取时间低于1s的情况下记录寿命图像.为增加FLIM时间序列记录,利用存储交换技术,在采集下一个光子期间读取正在记录的数据,使用两个平行TCSPC模块,可以每秒两幅图像记录荧光寿命时间序列.该技术可应用于活体植物组织叶绿素的瞬态测量.

The acquisition time of TCSPC FLIM depends on the number of pixels of the image, on the required lifetime accuracy, and on the count rate available from the sample. For samples with high fluorophore concentrations, such as stained tissue or plant cells the available count rates may come close to the maximum counting capability of the currently used TCSPC FLIM techniques. We describe the behaviour of TCSPC at high count rates and estimate the size of counting loss and pile-up effects. It turns out that systematic lifetime errors are smaller than previously believed. TCSPC FLIM can therefore be used to record lifetime images within acquisition times of less than 1 s. To increase the recording of FLIM time series we employed a memory swapping technique that reads out the data of one recording step during the acquisition of the next one. With parallel operation of two TCSPC modules we were able to record FLIM time series at a rate of 2 images per second. We demonstrate the technique for the measurement of chlorophyll transients in live plant tissue.