CCD探测型共聚焦显微成像横向分辨率优化

Optimization of Lateral Resolution in Confocal Microscopic Imaging with CCD as a Detector

电荷耦合器件(CCD)探测型共聚焦显微成像是近年来共聚焦成像的新方法,其成像横向分辨率除受物镜数值孔径影响外,还与成像探测方点扩展函数分布区域中实际采用的CCD像元密切相关。用CCD取代传统共聚焦成像中的针孔和点探测器,选取点扩展函数分布区域中一定组合的CCD像元合成等效针孔,在合成针孔为不同尺寸时分别比较系统的横向分辨率,得到优化的合成针孔,通过对不同合成针孔获得的图像加权相减,实现消减成像,进一步提高横向分辨率。实验得出:当合成针孔尺寸为艾里斑直径0.8倍时(8pixel×8pixel),扫描图像同时具有较高的横向分辨率和信噪比;当用优化针孔(8pixel×8pixel)与较大针孔(10pixel×10pixel)图像加权相减时,权重系数取0.6时获得的消减图像横向分辨率相对于优化针孔图像提高了21.7%。CCD探测型共聚焦成像方法大大降低了传统共聚焦成像系统的装调难度,通过选取优化的合成针孔并进行消减成像可以提高成像的横向分辨率。

In recent years, confocal microscopic imaging with charge coupled device （CCD） as a detector is a new method of confocal imaging. In this scheme, the lateral resolution of system is not only affected by the numerical aperture of the objective, but also the pixel of CCD, which is in the point spread function area of imaging and detection part. Using CCD instead of the pinhole and point detector of conventional confocal imaging, the synthetic equivalent pinhole of CCD pixel is chosen by different sizes of the point spread function area of a certain combination. The optimized synthetic pinhole is got by comparing the lateral resolution of system with different sizes of synthetic pinhole. By weighted subtracting of the image from different synthetic pinholes, subtractive imaging is realized and the lateral resolution can be enhanced. Experimental results show that when the synthetic pinhole is 0.8 times （8 pixel×8 pixel） of Airy spot diameter, the scanning image has higher lateral resolution and signal to noise ratio. When the image with the larger pinhole （10 pixel×10 pixel） is weightedly subtracted from the image with the optimized pinhole （8 pixel×8 pixel）, the lateral resolution of subtractive image relative to optimized pinhole image with weight coefficient of 0.6 is improved by 21.7%. Using CCD as a detector can greatly reduce the complexity of the conventional confocal imaging system. By selecting the optimized synthetic pinhole and processing subtractive imaging, we can improve the lateral resolution of imaging.