采用几何光学方法对共焦系统扫描时产生的扫描深度与厚度失真进行了理论分析,对名义扫描深度与实际扫描深度之间的关系进行了研究.以若丹明6G薄膜与玻片组成的多层样品为模型,对其进行了模拟计算,得到了扫描深度与厚度失真与系统数值孔径、折射率和样品厚度之间的关系.在实验上分别采用单光子荧光和双光子荧光作为检测信号,在反射式共焦扫描系统上进行了纵向扫描实验,并与模拟计算的结果进行了比较和分析.
Scanning depth and thickness distortion in confocal microscopy was analyzed theoretically. Multilayer modal which consists of R6G films and cover slide was constructed and simulation. Effect of objective numerical aperture, sample refraction index and thickness on the actual scanning depth were analyzed. Measurement results of the sample by confocal scanning with single-photon and two-photon fluorescence were compared with the simulation.