University at Buffalo, State University of New York, Buffalo, NY, USA.
Biophys J. 2010 Nov 17;99(10):3483-91. doi: 10.1016/j.bpj.2010.06.069.
Fundamental understanding of cellular processes at molecular level is of considerable importance in cell biology as well as in biomedical disciplines for early diagnosis of infection and cancer diseases, and for developing new molecular medicine-based therapies. Modern biophotonics offers exclusive capabilities to obtain information on molecular composition, organization, and dynamics in a cell by utilizing a combination of optical spectroscopy and optical imaging. We introduce here a combination of Raman microspectrometry, together with coherent anti-Stokes Raman scattering (CARS) and two-photon excited fluorescence (TPEF) nonlinear optical microscopy, to study macromolecular organization of the nucleus throughout the cell cycle. Site-specific concentrations of proteins, DNA, RNA, and lipids were determined in nucleoli, nucleoplasmic transcription sites, nuclear speckles, constitutive heterochromatin domains, mitotic chromosomes, and extrachromosomal regions of mitotic cells by quantitative confocal Raman microspectrometry. A surprising finding, obtained in our study, is that the local concentration of proteins does not increase during DNA compaction. We also demonstrate that postmitotic DNA decondensation is a gradual process, continuing for several hours. The quantitative Raman spectroscopic analysis was corroborated with CARS/TPEF multimodal imaging to visualize the distribution of protein, DNA, RNA, and lipid macromolecules throughout the cell cycle.
在细胞生物学以及生物医学领域，对分子水平细胞过程的基本理解对于早期感染和癌症疾病的诊断以及开发新的基于分子医学的疗法都具有重要意义。现代生物光子学通过结合光谱学和光学成像，提供了获取细胞内分子组成、组织和动力学信息的独特能力。在这里，我们介绍了一种结合拉曼微谱仪、相干反斯托克斯拉曼散射（CARS）和双光子激发荧光（TPEF）非线性光学显微镜的方法，用于研究整个细胞周期核内大分子的组织。通过定量共聚焦拉曼微谱仪，我们确定了核仁、核质转录位点、核斑点、组成性异染色质区域、有丝分裂染色体和有丝分裂细胞的染色体外区域中蛋白质、DNA、RNA 和脂质的局域浓度。我们的研究中得到了一个令人惊讶的发现，即在 DNA 紧缩过程中蛋白质的局部浓度不会增加。我们还证明了有丝分裂后 DNA 的去浓缩是一个逐渐的过程，持续数小时。定量拉曼光谱分析得到了 CARS/TPEF 多模态成像的证实，以可视化整个细胞周期中蛋白质、DNA、RNA 和脂质大分子的分布。
