Orientation-independent differential interference contrast microscopy and its combination with an orientation-independent polarization system.
Orientation-independent differential interference contrast microscopy and its combination with an orientation-independent polarization system.
复制标题
方向无关的微分干涉相差显微镜及其与方向无关的偏振系统的组合。
DOI：
10.1117/1.2837406
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发表时间：
2008
影响因子：
3.5
通讯作者：
Inouè,Shinya
中科院分区：
文献类型：
--
作者：
Shribak,Michael;LaFountain,James;Biggs,David;Inouè,Shinya
我们描述了一种与方向无关的微分干涉相衬 OI-DIC 和偏振显微镜相结合的方法及其生物学应用。几张传统的 DIC 图像是通过将样本定向在不同方向来记录的，然后对图像进行数字对准和处理。然后，获得的图像用于计算相位梯度幅度和方位角分布，以及相位图像。 OI-DIC 图像是使用数值孔径 (NA) 1.4 的光学器件获得的，从而达到了以前使用相差显微镜或干涉显微镜无法达到的分辨率水平。该组合系统产生样本薄光学切片的两个互补相位图像：折射率分布和由于细胞结构的各向异性而导致的双折射分布。例如，在活的分裂细胞中，OI-DIC 图像清楚地显示染色体的详细形状，而偏振图像定量地描绘了纺锤体中双折射微管的分布，两者都不需要对细胞进行染色或其他修改。我们展示了鹤蝇精母细胞在终动期和减数分裂 I 中期的伪彩色组合图像。这些图像提供了明确的证据，表明所提出的技术可以揭示活细胞中的精细结构和分子组织，而无需与染色或荧光标记相关的扰动。
We describe a combined orientation-independent differential interference contrast OI-DIC and polarization microscope and its biological applications. Several conventional DIC images were recorded with the specimen oriented in different directions followed by digital alignment and processing of the images. Then the obtained images are used for computation of the phase gradient magnitude and azimuth distribution and, further, the phase image. The OI-DIC images were obtained using optics having numerical aperture (NA) 1.4, thus achieving a level of resolution not previously achieved with phase contrast or interference microscope. The combined system yields two complementary phase images of thin optical sections of the specimen: distribution of refractive index and distribution of birefringence due to anisotropy of the cell structure. For instance, in a live dividing cell, the OI-DIC image clearly shows the detailed shape of the chromosomes, while the polarization image quantitatively depicts the distribution of birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. We present pseudo-color combined images of a crane fly spermatocyte at diakinesis and metaphase of meiosis I. Those images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live cells without perturbation associated with staining or fluorescent labeling.