Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research
Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research
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新型数值孔径0.9低温显微镜的研制及其在光合作用研究中的应用
DOI：
10.1016/j.bbabio.2014.03.006
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
T. Noguchi and H. Fukumura
中科院分区：
文献类型：
--
作者：
Y. Shibata;W. Katoh;T. Chiba;K. Namie;N. Ohnishi;J. Minagawa;H. Nakanishi;T. Noguchi and H. Fukumura
研制了一种新型的低温光学显微系统，该系统将物镜透镜置于低温恒温器的绝热真空空间内。在低温测量期间，物镜透镜在冷却时与样品隔离，保持在室温下。因此，作者能够使用数值孔径为0.9的色差校正物镜透镜。透镜配备有通气孔，以便与真空兼容。该系统实现了理论上预期的横向空间分辨率为沿着0.39 μm，轴向空间分辨率为沿着1.3 μm。应用该系统对绿色衣藻（Chlamydiumreinhardtii）细胞内光系统的非均匀分布进行了观测。在所有像素处的荧光光谱的高斯分解分析清楚地证明了两个光系统的非均匀分布，如分配给光系统II的荧光强度和分配给光系统I的荧光强度的可变比率所反映的。该系统也被应用于90 K下单个孤立的光系统I复合物的荧光光谱。荧光，指定从一个单一的光系统I三聚体发射，显示出间歇性的波动称为闪烁，这是典型的荧光信号从一个单一的分子。光系统I三聚体在790 nm附近出现电子振动荧光带，表明在800 nm光谱范围内色差并不严重。
A novel cryogenic optical-microscope system was developed in which the objective lens is set inside of the cryostat adiabatic vacuum space. Being isolated from the sample when it was cooled, the objective lens was maintained at room temperature during the cryogenic measurement. Therefore, the authors were able to use a color-aberration corrected objective lens with a numerical aperture of 0.9. The lens is equipped with an air vent for compatibility to the vacuum. The theoretically expected spatial resolutions of 0.39 μm along the lateral direction and 1.3 μm along the axial direction were achieved by the developed system. The system was applied to the observations of non-uniform distributions of the photosystems in the cells of a green alga,Chlamydomonas reinhardtii, at 94 K. Gaussian decomposition analysis of the fluorescence spectra at all the pixels clearly demonstrated a non-uniform distribution of the two photosystems, as reflected in the variable ratios of the fluorescence intensities assigned to photosystem II and to those assigned to photosystem I. The system was also applied to the fluorescence spectroscopy of single isolated photosystem I complexes at 90 K. The fluorescence, assigned to be emitted from a single photosystem I trimer, showed an intermittent fluctuation called blinking, which is typical for a fluorescence signal from a single molecule. The vibronic fluorescence bands at around 790 nm were observed for single photosystem I trimers, suggesting that the color aberration is not serious up to the 800 nm spectral region.