入射激光对激光扫描共聚焦显微镜分辨率的影响

Effect of incident laser on resolution of LSCM

利用点扩散函数(PSF)研究了入射激光的偏振态和光束的束腰直径对激光扫描共聚焦显微镜(LSCM)分辨率的影响。根据Wolf和Richards的矢量衍射积分,建立了LSCM N-1层界面下的照明PSF模型,对零层和两层界面下的PSF进行了计算分析。结果显示:在零层界面下,圆偏振光入射的PSF在xy平面内关于焦点旋转对称,半峰全宽为0.43μm,x向线偏振光入射时PSF在x、y方向的半峰全宽分别为0.48,0.39μm;在圆偏振光入射的PSF中,高斯光束充溢系数为0,1,2,5对应的半峰全宽分别为0.43,0.47,0.62,1.49μm。在两层界面下,当探测深度为50μm时,圆偏振光PSF的半峰全宽为0.26μm,x向线偏振光入射时PSF在x、y方向的半峰全宽分别为0.28,0.24μm;在圆偏振光入射的PSF中,充溢系数为0,1,2,5对应的半峰全宽分别为0.26,0.28,0.32,0.68μm。以上计算结果表明,和线偏振光相比,圆偏振光在一个方向的分辨率优于线偏振光,在相垂直的另一个方向的分辨率弱于线偏振光,圆偏振光PSF在xy平面内关于焦点旋转对称,得到了较好的成像质量;物镜入瞳直径与激光束腰直径比值越小,LSCM的分辨率越好。

The effect of the polarization and intensity profile of an incident laser on the resolution of a Laser Scanning Confocal Microscope （LSCM） was investigated. According to the vectorial diffraction integral of Wolf and Richards, a model of the illumination Point Spread Function （PSF） of LSCM with N-1 interfaces was established to calculate and analyze the PSFs on the zero interface and two interfaces. The analysis shows that for the zero interface, the local Full Width at Half Maximum （FWHM） of the PSF with circular polarization is 0.43 μm, which is rotationally symmetric in the xy plane, and the FWHMs along ac axis and y axis of the PSF with linear polarization are 0.48, 0.39μm, respectively in the a＂ axis. As the filling parameters of a Gaussian light are 0, 1, 2, 5, the FWHMs of the PSF with circular polarization are 0.43, 0.47, 0.62 μm and 1.49μm,respectively. For two interfaces, when the probe depth is 50 /xm, the FWHM of the PSF with circular polarization is 0.26 μm, and the FWHMs along a- axis and y axis of the PSF with linear polarization are 0.28,0.24 μm, respectively in ac axis; for the filling parameters of 0,1,2,5, and the probe depth of 50 μm, the FWHMs of the PSF with circular polarization are 0.26, 0.28, 0.32 and 0.68 μm,respectively. Calculations me- tioned above show that the resolution of the circularly polarized light is better than that of the linearly polarized light in one direction, but it is worse than that of the linearly polarized light in the perpen dicular direction. The PSF with the circularly polarized light is rotationally symmetric in the a＇y plane, therefore, a better image can be obtained. The smaller the filling parameter is, the better the resolution of LSCM is.