双光子共焦荧光显微系统成像分析及实验研究

双光子共焦荧光显微系统可以突破传统光学显微镜的成像分辨率极限,提高厚荧光物三维扫描的横向及轴向分辨力.基于共焦荧光显微原理和菲涅耳衍射公式,分析了反射式双光子共焦荧光显微系统的三维光学传递函数.讨论了在实验条件下,共焦模块参数对双光子共焦荧光读取分辨率的影响.双光子共焦荧光系统采用的共焦小孔半径为3.92 μm时,其层析能力比采用78.4 μm共焦小孔的系统提高了1.56倍.通过已知尺寸荧光物的双光子共焦荧光成像实验,验证了共焦小孔直径与系统横向及轴向分辨率的反比关系.

Imaging Analysis and Experimental Research on Two-Photon Confocal Fluorescent Microscope

The two-photon confocal fluorescent system can exceed the limits of imaging resolution in the traditional optical microscope and increase the lateral and axial resolutions during the three-dimensional （3D） scanning of the thick fluorescent sample.The 3D optical transfer function of the reflex two-photon confocal fluorescent system is analyzed based on the imaging principal of the confocal fluorescent microscope and the Fresnel diffraction formula.The effects of confocal parameters on the resolution of two-photon confocal fluorescent system are studied in the experimental environment.The depth-discriminating ability of the two-photon confocal fluorescent system with 3.92 μm pinhole radius increase 1.56 times compared with that of 78.4 μm pinhole radius.A two-photon confocal fluorescent imaging experiment is carried out with the given-size fluorescent object,the experimental result verifies the inverse relation between the detector size and the system resolution.