Label-free live-cell imaging of nucleic acids using stimulated Raman scattering microscopy.
Label-free live-cell imaging of nucleic acids using stimulated Raman scattering microscopy.
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DOI：
10.1002/cphc.201100890
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发表时间：
2012-03
期刊：
影响因子：
--
通讯作者：
Xie XS
中科院分区：
文献类型：
--
作者：
Zhang X;Roeffaers MB;Basu S;Daniele JR;Fu D;Freudiger CW;Holtom GR;Xie XS
核酸成像对于研究细胞分裂和细胞凋亡等细胞过程非常重要。非侵入性无标记技术很有吸引力。拉曼光谱根据特定的振动峰提供丰富的化学信息。然而，自发拉曼散射的信号很弱，并且需要很长的积分时间，这极大地限制了用于显微镜时的成像速度。相干拉曼散射技术，包括相干反斯托克斯拉曼散射 (CARS) 和受激拉曼散射 (SRS) 显微镜，通过将信号水平增强多达五个数量级来克服这个问题。 CARS 显微镜受到非共振背景信号的影响，这会扭曲拉曼光谱并限制灵敏度。这使得对光谱拥挤区域中的弱跃迁进行 CARS 成像具有挑战性。在指纹区域尤其如此，其中核酸显示特征峰。最近开发的 SRS 显微镜没有这些限制；激发光谱与自发拉曼光谱相同，灵敏度接近散粒噪声限制。在这里，我们展示了在指纹区域中使用 SRS 成像来绘制果蝇幼虫单个唾液腺细胞和单个哺乳动物细胞中除蛋白质和脂质外的核酸分布图。这使得与细胞分裂相关的 DNA 凝聚成像成为可能，并为体内此类过程的成像提供了可能性。
Imaging of nucleic acids is important for studying cellular processes such as cell division and apoptosis. A noninvasive label-free technique is attractive. Raman spectroscopy provides rich chemical information based on specific vibrational peaks. However, the signal from spontaneous Raman scattering is weak and long integration times are required, which drastically limits the imaging speed when used for microscopy. Coherent Raman scattering techniques, comprising coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, overcome this problem by enhancing the signal level by up to five orders of magnitude. CARS microscopy suffers from a nonresonant background signal, which distorts Raman spectra and limits sensitivity. This makes CARS imaging of weak transitions in spectrally congested regions challenging. This is especially the case in the fingerprint region, where nucleic acids show characteristic peaks. The recently developed SRS microscopy is free from these limitations; excitation spectra are identical to those of spontaneous Raman and sensitivity is close to shot-noise limited. Here we demonstrate the use of SRS imaging in the fingerprint region to map the distribution of nucleic acids in addition to proteins and lipids in single salivary gland cells of Drosophila larvae, and in single mammalian cells. This allows the imaging of DNA condensation associated with cell division and opens up possibilities of imaging such processes in vivo.
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