Development of a Multicolor Line-Focus Microscope for Rapid Acquisitions of Excitation Spectra
Development of a Multicolor Line-Focus Microscope for Rapid Acquisitions of Excitation Spectra
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DOI：
10.1016/j.bpj.2019.11.023
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发表时间：
2020-01-07
影响因子：
3.4
通讯作者：
Shibata, Yutaka
中科院分区：
文献类型：
--
作者：
Jana, Sankar;Shibata, Yutaka
为了对光合生物的激发光谱进行快速显微镜观察，研制了一种波长色散线聚焦显微镜。在该系统中，样品上不同波长激发的多个位置的荧光信号可以在EMCCD相机上同时以二维图像的形式被检测到。利用该系统可获得635 ~ 695 nm激发波长范围内的每个像元激发光谱，覆盖了叶绿素-a和叶绿素-b的全部Q(y)波段。同时记录参考激光光谱，确保对激发激光中适度的光谱波动进行稳健的测量。采用数值孔径为0.9的物镜，获得的横向和轴向分辨率分别为0.56 μ m和1.08 μ m。激发光谱测量的理论极限和实验估计光谱分辨率分别为0.86和1.3 nm。通过对模型藻莱茵衣藻荧光珠和单细胞的检测，验证了该系统的有效性。衣藻细胞叶绿体内叶绿素-b波段的相对强度存在不均匀性。这种不均匀性反映了局部外围天线尺寸在叶绿体内的变化。
To conduct rapid microscope observations with the excitation spectral measurement for photosynthetic organisms, a wavelength-dispersive line-focus microscope was developed. In the developed system, fluorescence signals at multiple positions on a sample excited with different wavelengths can be detected as a two-dimensional image on the EMCCD camera at the same time. Using the developed system, one can obtain excitation spectra at every pixel over the excitation wavelength range from 635 to 695 nm, which covers the full range of the Q(y) bands of both chlorophyll-a and chlorophyll-b. Recording the reference laser spectra at the same time ensures robust measurement against the moderate spectral fluctuation in the excitation laser. Using an objective lens with a numerical aperture of 0.9, the lateral and axial resolutions of 0.56 and 1.08 mu m, respectively, were achieved. The theoretically limited and experimentally estimated spectral resolutions of the excitation spectral measurement were 0.86 and 1.3 nm, respectively. The validity of the system was demonstrated by measuring fluorescent beads and single cells of a model alga, Chlamydomonas reinhardtii. Intrachloroplast inhomogeneity in the relative intensity of the chlorophyll-b band could be visualized in Chlamydomonas cells. The inhomogeneity reflects the intrachloroplast variation in the local peripheral antenna size.