任意数量离散不规则感兴趣区域的快速荧光寿命显微成像

Fast fluorescence lifetime microscopy imaging of any number of discrete irregular regions of interest

荧光寿命显微成像(fluorescence lifetime imaging microscopy,FLIM)技术在细胞微环境传感中具有特异性强、灵敏度高、可定量的优点,被广泛应用于生物医学研究.其中,基于时间相关单光子计数(time-correlated single photon counting,TCSPC)进行荧光寿命探测的方法是目前最常用的技术之一,但受成像原理和条件限制,该技术存在数据采集时间较长、成像速度不够快的不足.本文开发一种能对生物样品中任意数量离散的、形状不规则的感兴趣区域(region of interest,ROI)进行快速FLIM成像的技术.该技术利用声光偏转器(acousto-optic deflector,AOD)实现快速灵活的寻址扫描,并通过对ROI形状特征的简单在线分析,实现AOD与TCSPC同步策略的优化及寿命图像的准确重构,对于生物样品中常见的存在多个离散不规则ROI情形,可大幅节省数据采集时间,从而实现对这些ROI的快速FLIM成像.采用该技术,对氯化铵刺激下活细胞中溶酶体探针LysoSensor Green DND-189的荧光寿命变化进行了动态FLIM成像,以监测溶酶体管腔内pH值的实时变化情况.结果表明,该快速FLIM技术可用于动态监测生物样品中微环境的变化,将在活细胞微环境传感中发挥重要作用.

Fluorescence lifetime imaging microscopy(FLIM)has been widely used in biomedical research due to its high specificity,high sensitivity and quantification ability in cell microenvironment sensing.The fluorescence lifetime detection method based on time-correlated single photon counting(TCSPC)is one of the most commonly used techniques at present.However,due to the limitation of imaging principles and conditions,this technique has the disadvantages of long data acquisition time and consequently low imaging speed.In this paper,a fast FLIM technique for any number of discrete and irregular regions of interest(ROIs)in biological samples is developed.The technology uses acousto-optic deflectors(AODs)to achieve fast and flexible addressing scanning,optimize the synchronization strategy between AOD and TCSPC,and reconstruct the lifetime image through simple online feature analysis of the ROI shapes.For the case of multiple discrete irregular ROIs in biological samples,it can greatly save the time of data acquisition,thus realizing the fast FLIM imaging of these ROIs,which is benificial to the study of the heterogeneity of biological events in biological system.In particular,the fast fluorescence imaging result for 87 discrete points in the field of view shows that this method can obtain a fluorescence lifetime image in a very short acquisition time(only 52.2 ms)and thus achieving a very fast imaging speed in such a situation.Dynamic FLIM imaging of lysosome probe LysoSensor Green DND-189 in living cells stimulated by ammonium chloride is carried out to monitor the real-time change of pH value in lysosome lumen.The acquisition time for a single fluorescence lifetime image of lysosomes in two ROIs is only 200 ms.The results show that the rapid FLIM technology can be used to dynamically monitor the changes of microenvironment in biological samples,and will play an important role in the microenvironment sensing in living cells.