Supercontinuum dynamically visualizes a dividing single cell
Supercontinuum dynamically visualizes a dividing single cell
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DOI：
10.1021/ac071416z
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发表时间：
2007-12-01
影响因子：
7.4
通讯作者：
Harnaguchi, Hiro-O
中科院分区：
文献类型：
--
作者：
Kano, Hideaki;Harnaguchi, Hiro-O
在细胞分裂过程中，各种细胞器的行为是动态的。细胞器的这些动态行为的可视化是在分子水平上理解生命的有希望的一步。到目前为止，单光子或双光子激发的荧光显微镜已经被用来可视化这些细胞动力学。引入活细胞的荧光探针可以实时可视化目标分子的空间分布，从而能够在分子水平上追踪细胞动力学。然而，将荧光探针引入细胞可能会改变细胞的物理和化学条件。在这里，我们展示了一种新的方法，直接(不需要染色细胞)的活细胞过程的相干反斯托克斯拉曼散射(CARS)光谱可视化。一种新的光源，由光子晶体光纤产生的超连续谱，促进了具有高分子特异性的超宽带(>；3500 cm(-1))多路CARS光谱和成像。利用这种多重CARS技术，我们成功地跟踪了细胞分裂的整个过程，母细胞分裂成两个子细胞，隔膜的出现和消失，以及由脂膜组成的细胞器的动态分布变化。超连续谱还促进了CARS和双光子激发荧光(TPEF)光谱的同时测量，使我们所称的多重非线性光谱成像成为可能。现在，对活细胞进行高速三维图像重建，可以阐明正在分裂的活细胞内更详细的分子水平的动力学。
During cell division, various organelles behave dynamically. Visualization of these dynamic behaviors of organelles is a promising one step forward for understanding life at the molecular level. One- or two-photon excited fluorescence microscopy has so far been used for visualizing these cell dynamics. The fluorescent probe introduced into a living cell can visualize the spatial distribution of a target molecule in real time, enabling the tracing of cell dynamics at the molecular level. Introducing a fluorescent probe into a cell, however, may alter the physical and chemical conditions of the cell. Here we show a new method for direct (no need for staining cells) visualization of living cell processes with coherent anti-Stokes Raman scattering (CARS) spectroscopy. A new light source, supercontinuum generated from a photonic crystal fiber, has facilitated ultrabroadband (> 3500 cm(-1)) multiplex CARS spectroscopy and imaging with high molecular specificity. Using this multiplex CARS technique, we have been successful in tracing the whole cell division process, the splitting of a mother cell into two daughter cells, appearance and disappearance of septum, and dynamic distribution changes of organelles consisting of lipid membrane. The supercontinuum has also facilitated simultaneous measurement of the CARS and two-photon excited fluorescence (TPEF) spectra, enabling what we call multiple nonlinear spectral imaging. Three-dimensional image reconstruction of a living cell with high speed is now possible to elucidate more detailed molecular-level dynamics inside a dividing living cell.