【摘要】 目的　探讨用共聚焦激光扫描显微镜对厚样本进行光学断层时 ,扫描步距对三维重建的质量影响。方法　用醛处理法诱导大鼠主动脉弹性纤维产生强自发荧光 ,在共聚焦激光扫描显微镜下对主动脉壁进行全层光学断层。分析扫描步距等参数变化对内弹性膜构筑三维重建质量的影响。结果　①探测针孔直径等于 1个爱俐斑单位且满足离开焦点 1/ 2Z 轴分辨率距离时 ,焦平面最强荧光信号减弱 5 0 % ,此条件下获得的二维光学切片分辨率和信息的完整性达最佳平衡。②在上述条件下 ,扫描步距等于Z 轴分辨率时 ,重建结构信息完整 ;扫描步距小于Z 轴分辨率时 ,原位重建结构的信号被人为加强 ,旋转重建结构出现重影 ;扫描步距大于Z 轴分辨率时 ,重建结构出现出现信息损失。结论　以Z轴分辨率为扫描步距进行光学断层和重建 ,可获得分辨率、信息完整性和信息不重叠性达最佳三维重建效果。更多还原

【Abstract】 Objective To optimize the instrument parameters of a confocal laser scanning fluorescent microscope (CLSFM) for optic-sectioning and three-dimensional reconstruction of thick biological tissues.Method Rat aortic pieces were treated with glutaraldehyde to enhance their elastin autofluorescence and then optic-sectioned en face by CLSFM under different parameter settings of laser power, detection pinhole and Z-scanning step.Three-dimensional projections of the internal elastic lamina were reconstructed from the resultant serial optical sections to determine the optimal instrument preferences.Results ①When the detection pinhole was set to 1 Airy Disc, an optical section with the optimal balance between the resolution and the signal loss could obtained by fulfilling the condition that the fluorescence intensity in the focus plane falls to 50% maximum when the scanning plane leaves a distance of 1/2 Z-direction resolution (=2λ/NA2) from the focus. ②Under the condition above and if the scanning step was set to equal to the Z-direction resolution, an optimal 3-D reconstruction could be made from the resultant serial optical sections. A scanning step less than the Z-direction resolution resulted in superposition of false signal onto the reconstructed structure, especially when it was rotated; and a larger scanning step led to the loss of the structural signals.Conclusion when the scanning step was set to equal to the Z-direction resolution, an optimal 3-D reconstruction could be made from the resultant serial optical sections with high-resolution and intact signal.更多还原