Study on adaptation of single photosynthetic cells against environmental stress by using three-dimensional super-resolution microscopy with time resolution ability

用频率为7.9 kHz的共振扫描镜将近红外飞秒脉冲照射在样品的一条线上，线性区域的总多光子诱导荧光聚焦在成像多色仪的狭缝上。利用电子倍增CCD相机对不同水平像素的不同颜色的荧光和不同垂直像素的样品中不同空间位置的荧光进行解析。在644~690 nm的荧光波长范围内，X、Y和Z轴的点扩散函数的半最大宽度分别为0.39~0.40、0.33和0.56~0.59μm。用该显微镜研究了蓝藻鱼腥藻类囊体膜的亚细胞荧光光谱。研究发现，藻胆体的叶绿素分子(主要是光系统II)和藻胆素分子(叶绿素/藻胆蛋白)之间的荧光强度比随着探测的深入而变得更低。这不是由于重吸收或散射效应的位置依赖性，而是由于在亚细胞结构域的透射谱测量的帮助下，类囊体膜的局部生理状态的内在变化。类囊体膜对光条件的适应，以及它在叶绿体和蓝藻之间的变化，几十年来一直是已知的，但基于亚细胞分辨率的荧光光谱的表征一直很少。我们新开发的光谱成像系统也被应用于烟草、玉米和小球藻的叶绿体。在烟草中发现了组织依赖性，在玉米中发现了每日周期，在小球藻中发现了培养条件依赖性。

A near-infrared fs pulse was illuminated on a line in a specimen by a resonant scanning mirror oscillating at 7.9kHz, and total multiphoton-induced fluorescence from the linear region was focused on the slit of an imaging polychromator. An electron-multiplying CCD camera was used to resolve fluorescence of different colors at different horizontal pixels and fluorescence of different spatial positions in a specimen at different vertical pixels. The full widths at half maximum of the point-spread function of the system were estimated to be 0.39-0.40, 0.33 and 0.56-0.59μm for the X, Y and Z axes, respectively, at fluorescence wavelengths between 644 and 690nm. This microscope was applied to a study of the subcellular fluorescence spectra of thylakoid membranes in a cyanobacterium, Anabaena. It was found that the fluorescence intensity ratio between chlorophyll molecules mainly of photosystem II and phycobilin molecules of phycobilisome (chlorophyll/phycobilin), became lower as one probed deeper inside the cells. This was attributable not to position dependence of re-absorption or scattering effects, but to an intrinsic change in the local physiological state of the thylakoid membrane, with the help of a transmission spectral measurement of subcellular domains. Adaptation of the thylakoid membrane against light conditions, and its variation between chloroplasts and cyanobacteria, has been known for decades, but characterization based on fluorescence spectrum at a subcellular resolution has been scarce. Our newly developed spectroimaging system has also been applied to chloroplasts in tabacco, zea mays, and chlorella. Tissue dependence in the case of tobacco, daily cycle in the case of zea mays, and culture-condition dependence in the case of chlorella were found.