Muscle fine structure and microtubule birefringence measured with a new pol-scope.
Muscle fine structure and microtubule birefringence measured with a new pol-scope.
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使用新型偏振镜测量肌肉精细结构和微管双折射。
DOI：
10.1086/bblv187n2p244
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Oldenbourg,R
中科院分区：
文献类型：
--
作者：
Tran,PT;Inoué,S;Salmon,ED;Oldenbourg,R
偏振光显微镜传统上是研究活细胞精细结构的重要工具（1，2）。然而，常规设计仅允许在窄范围的取向中进行延迟成像。我们之一（RO）通过开发一种新型偏光显微镜（pol-scope）改进了传统设计，该显微镜可以测量由于样本各向异性而产生的阻滞，而无论方向如何。新型偏光显微镜的设计是基于传统的偏光显微镜，但有两个基本的修改：（a）样品是近圆偏振光照明，和（B）传统的补偿器是由两个液晶可变延迟器。液晶可变延迟器形成了一个“通用补偿器”，由电力驱动，消除了对光学元件的任何机械重新调整的需要。摄像机和计算机辅助图像分析提供了构成视场（3）的图像的所有点的样本各向异性的测量。为了验证新型偏振镜的可靠性和成像效率，我们用偏振镜测量了横纹肌的延迟。青蛙缝匠肌用戊二醛和四氧化锇溶液固定并包埋在树脂中，然后切成薄片并安装在无应变显微镜载玻片上的匹配索引介质（Euparal）中。我们分别对约180 nm和360 nm薄的金色和紫色切片进行成像。图1a示出了在不同角度取向下测量的薄肌肉部分的相同区域的双折射。无论取向如何，我们都清楚地观察到肌原纤维精细结构，包括各向同性Z线、弱双折射I带和各向异性A带（图1 B）。我们测量了在肌节长度上平均的180 nm肌肉切片的0.22 ± 0.03 nm [n= 71]延迟和360 nm肌肉切片的0.35 ± 0.02 nm [n= 71]延迟。这些数值与先前的测量结果一致（4）。
The polarized light microscope has traditionally been an important tool for studying fine structure in living cells (1, 2). However, conventional design allows for retardance imaging only in a narrow range of orientations. One of us (RO) has improved upon conventional design by developing a new type of polarizing microscope (pol-scope), which can measure the retardance due to specimen anisotropy irrespective of orientation. The design of the new pal-scope is based on the traditional polarizing microscope but has two essential modifications:(a) the specimen is illuminated with nearly circularly polarized light, and (b) the traditional compensator is replaced by two liquid-crystal variable retarders. The liquid-crystal variable retarders form a “universal compensator” that is driven electrically, eliminating the need for any mechanical readjustment o the optical components. A video camera and computer-assisted image analysis provide measurements of specimen anisotropy for all points of the image constituting the field of view (3). We used the pol-scope to measure the retardance of striated muscle for testing the reliability and imaging efficiency of the new pol-scope. Frog sartorious muscles were fixed with glutaraldehyde and osmium tetroxide solutions and embedded in resin, then sliced into thin sections and mounted in a matching index medium (Euparal) on a strain-free microscope slide. We imaged gold and purple sections which are about 180 nm and 360 nm thin, respectively. Figure la shows the birefringence of the same region of a thin muscle section measured at different angular orientations. Irrespective of orientation, we clearly observe the myofibrillar fine structures, consisting of isotropic Z lines, weakly birefringent I bands, and anisotropic A bands (Fig. 1 b). We measured retardance of 0.22+ 0.03 nm [n= 71 for the 180 nm muscle section, and retardance of 0.35+ 0.02 nm [n= 71 for the 360 nm muscle section averaged over the length of the sarcomeres. These values are consistent with earlier measurements (4).