Image enhancement of the in vivo leukocyte-endothelium contact zone using optical sectioning microscopy.
Image enhancement of the in vivo leukocyte-endothelium contact zone using optical sectioning microscopy.
复制标题
使用光学切片显微镜对体内白细胞-内皮接触区进行图像增强。
DOI：
10.1007/bf02684192
复制
发表时间：
1997
影响因子：
3.8
通讯作者：
Lipowsky,HH
中科院分区：
文献类型：
--
作者：
Shen,Z;Lipowsky,HH
白细胞[白细胞(WBC)]与内皮[内皮细胞(EC)]黏附强度的一个主要决定因素是两个细胞之间形成的接触面积，这通常被活体显微镜固有的焦外信息所掩盖。为了改善WBC-EC接触区的可视化，发展了光学切片显微镜技术来增强WBC-EC粘连在大鼠肠系膜毛细血管后小静脉中的明亮图像。将50×/1.0nA的物镜固定在计算机驱动的压电式支架上，通过在16μm的深度内沿1μm步长沿垂直方向聚焦的方式数字化来自ccd摄像机的图像，获得视频图像。通过测量显微镜的光学传递函数，使用奇异值分解技术和Tikhonov-Miller规则在傅立叶域内对中心图像进行解卷积，以去除散焦信息。测量原始图像中白细胞-EC接触区(LC)的长度，得到的数值为4.32±1.08μm(平均值±SD)。局部应用化学诱导剂f-Met-Leu-Phe可使Lc增加26%至3.49±0.72μm，提示白细胞膜上黏附分子的上调导致额外的膜面积从表面皱纹向黏附区募集。去卷积的其他优点是更详细地显示微血管壁和实质组织的结构特征。因此，明场光学切片显微镜可以为微血管的活体研究提供一个有价值的工具，并作为一种有用的替代荧光显微镜，而没有外源荧光团和暴露在紫外线辐射下的不良影响。
A major determinant of the strength of leukocyte [white blood cell (WBC)] to endothelium [endothelial cell (EC)] adhesion is the contact area formed between the two cells, which is often obscured by out-of-focus information inherent to intravital microscopy. To improve visualization of the WBC-EC contact zone, techniques of optical sectioning microscopy were developed to enhance brightfield images of WBC-EC adhesion in postcapillary venules of the mesentery of the rat. A 50×/1.0 NA objective was held in a piezoelectric mount that was computer-driven, and video images were obtained by digitizing images from a CCD camera while focusing through the vertical direction in 1 μm steps over a depth of 16 μm. Using measurements of the microscope's optical transfer function, deconvolution of the central image was performed in the Fourier domain using the technique of singular value decomposition with Tikhonov-Miller regulation to remove out-of-focus information. Measurement of the length of the WBC-EC contact zone (LC) in the original images yielded values on the order of 4.32±1.08 μm (mean±SD). The enhanced images showed a significantly 35% smallerLCequal to 2.78±0.70 μm. Topical application of the chemoattractant f-met-leu-phe resulted in a 26% increase inLCto 3.49±0.72 μm, thus suggesting that upregulation of adhesion molecules on the WBC membrane results in the recruitment of additional membrane area from surface ruffles into the zone of adhesion. Other advantages of the deconvolution were to visualize structural characteristics of the microvascular wall and parenchymal tissue in greater detail. Thus, brightfield optical sectioning microscopy may provide a valuable tool forin vivostudies of the microvasculature, and serves as a useful alternative to fluorescence microscopy without the undesirable effects of exogenous fluorophores and exposure to ultraviolet radiation.