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  

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

作　　者：陈德强[1] 周拥军[1] 黄文浩[1] 夏安东[1] 

机构地区：[1]中国科技大学精密机械和精密仪器系,合肥230026

出　　处：《光子学报》2001年第12期1486-1491,共6页Acta Photonica Sinica

基　　金：国家自然科学基金 ( 39770 1 73);教育部留学回国人员科研启动基金资助项目

摘　　要：双光子激光扫描显微镜 (TPL SM)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TPL SM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 TPL SM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 TPL SM荧光图象的畸变 ,同时还导致 TPL SM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 TPL SM荧光图象的质量 .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.

关 键 词：双光子激光扫描显微镜 TPLSM 折射率失配 水浸物镜 图象变形 

分 类 号：TH742.64[机械工程—光学工程]