Nonresonant confocal Raman imaging of DNA and protein distribution in apoptotic cells
Nonresonant confocal Raman imaging of DNA and protein distribution in apoptotic cells
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DOI：
10.1016/s0006-3495(03)75124-8
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发表时间：
2003-06-01
影响因子：
3.4
通讯作者：
Otto, C
中科院分区：
文献类型：
--
作者：
Uzunbajakava, N;Lenferink, A;Otto, C
非共振共聚焦拉曼成像已被用来绘制DNA和蛋白质在单个人类细胞中的分布。这些图像是在改进的自制共聚焦拉曼显微镜上获得的。经过统计分析，利用奇异值分解，从覆盖指纹区域的光谱中重建拉曼图像。数据是以大约250纳米的步长间隔获得的，覆盖的范围从8微米到15微米的正方形。每个像素的停留时间在0.5%到2%S之间，这取决于被研究细胞的性质和状态。只有在这些条件下，使用60-120 mW的连续高功率激光才能获得高质量的非共振拉曼图像。我们将提出证据，证明这些激光功率仍然可以安全地用于恢复固定细胞中的化学分布。发展的拉曼成像方法被用来直接成像，即不需要事先标记，即在所谓的凋亡的HeLa细胞的核碎片中直接成像核苷酸缩合和蛋白质分布。在对照(非凋亡)HeLa细胞中，我们首次通过拉曼光谱显示了细胞核中存在RNA。
Nonresonant confocal Raman imaging has been used to map the DNA and the protein distributions in individual single human cells. The images are obtained on an improved homebuilt confocal Raman microscope. After statistical analysis, using singular value decomposition, the Raman images are reconstructed from the spectra covering the fingerprint region. The data are obtained at a step interval of similar to250 nm and cover a field from 8- to 15-mum square in size. Dwell times at each pixel are between 0.5 and 2 s, depending on the nature and the state of the cell under investigation. High quality nonresonant Raman images can only be obtained under these conditions using continuous wave high laser powers between 60 and 120 mW. We will present evidence that these laser powers can still safely be used to recover the chemical distributions in fixed cells. The developed Raman imaging method is used to image directly, i.e., without prior labeling, the nucleotide condensation and the protein distribution in the so-called nuclear fragments of apoptotic HeLa cells. In the control (nonapoptotic) HeLa cells, we show, for the first time by Raman microspectroscopy, the presence of the RNA in a cell nucleus.