【摘要】 目的:探讨共聚焦系统各参数对厚样本光学断层、三维重建的影响。方法:用共聚焦系统对醛诱导后的大鼠主动脉作光学断层。分析激光强度、探测针孔直径、扫描步距等参数对内弹性膜三维重建的影响。结果和结论:(1)针孔为1Airy单位,调整激光强度以满足离开焦点1/2Z-轴分辨率距离时,焦平面最强荧光信号减弱50%,此条件下获得的光学切片分辨率和信息完整性达最佳平衡,切片厚度可经验地定视为等于系统Z轴分辨率。(2)在上述条件下,扫描步距为Z轴分辨率时,重建结构完整。(3)当扫描步距不变,针孔小于1Airy单位时,重建结构部分缺损;针孔大1Airy单位时,重建结构模糊。(4)样本经透明处理后,断层深度可增大4倍。更多还原

【Abstract】 Objective: To determine the influence of optimal instrument parameters of the confocal laser scanning fluorescent microscope (CLSFM) for optic-sectioning and three-dimensional reconstruction of thick biological tissues.Methods: Rat aortic specimens were treated with glutaraldehyde to enhance the elastin autofluorescence and then optic-sectioned en face by CLSFM under different 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. Results and Conclusions: (1) When the detection pinhole was set to 1 Airy unit, an optical section with the optimal compromise between the resolution and the signal loss could be obtained if the fluorescence intensity in the focus plane falls to 50% maximum when the scanning plane leaves it a distance of 1/2 Z-direction resolution (=2λ/NA~2). (2) Under the above condition and if the scanning step was set as to the Z-direction resolution, an optimal 3-D reconstruction could be produced from the resultant serial optical sections. A scanning step less than the Z-direction resolution resulted in a superposition of false signals and an elongation of the reconstructed structure in Z-direction, especially when it was rotated. A larger scanning step led to the loss of the structural signals and a Z-direction flattening of the reconstructed structure. (3) Under the optimized settings defined above, any slight changes of the detection pinhole may affect the quality of the reconstructed images. (4) The maximal detection depth into a thick tissue could be considerably increased by transparentizing the tissue.更多还原