双光子激光扫描显微镜中折射率失配引起的图象变形研究

双光子激光扫描显微镜(TPLSM)在观察生理溶液中的生物样品的结构时,水浸物镜是最好的选择.但是TPLSM在进行生物样品的三维结构观察时,需要样品和物镜在Z轴方向上做相对移动,这种移动很容易引起样品在水溶液中飘动.为了防止这种样品的漂移,往往要在水物镜和样品之间加入盖波片.由于盖波片和水及样品间的折射率失配将会导致TPLSM荧光图象的变形,给观察结果带来误差.本文主要利用TPLSM的外源探测器(ex-ternal detection)观察荧光小球的TPLSM的荧光图象,比较插入盖波片前后小球荧光图象的变化情况,研究水浸物镜在插入盖波片后折射率失配引起TPLSM图象的变形程度及共焦小孔对改进图象变形的作用.结果显示盖波片的折射率失配不仅导致TPLSM荧光图象的畸变,同时还导致TPLSM图象的荧光强度和信噪比的下降.进一步的研究表明共焦小孔可以部分改进由于折射率失配引起畸变的TPLSM荧光图象的质量.

IMAGE DISTORTIONS OF TWO-PHOTON LASERSCANNING MICROSCOPY INDUCED BY REFRACTIVE- INDEX MISMATCH OF COVERSLIPS

It is obvious that a water immension objective is the best choice for two-photon laser scanning microscopy (TPLSM) to observe the structures of biological samples in physiological aqueous media.Unfortunately,the collection of 3D images requires moving the objective up and down with respect to the samples in Z-axis direction,which may easily result in the motion of sample.In order to avoid producing motion of the samples in aqueous,a coverslip may be used between samples and objective lens usually.The refractive-index mismatch induced by coverslips will lead to the distortion of TPLSM images.In this paper,we observed the TPLSM images of fluorescence beads with external detection of TPLSM before and after inserting coverslips.The image distortion caused by external coverslips was studied through a water immension objective.The effects of a confocal pinhole on its ability to improve the image quality were also investigated.Authors measurements showed that the refractive index mismatch caused by coverslips not only led to the TPLSM image distortion,but also resulted in the decreases of the fluorescence intensity and signal-and-noise ratio of TPLSM image,which can be partly improved by employing a suitable confocal pinhole.