Cheng Ji-Xin, Jia Y Kevin, Zheng Gengfeng, Xie X Sunney
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Biophys J. 2002 Jul;83(1):502-9. doi: 10.1016/S0006-3495(02)75186-2.
Laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy with fast data acquisition and high sensitivity has been developed for vibrational imaging of live cells. High three-dimensional (3D) resolution is achieved with two collinearly overlapped near infrared picosecond beams and a water objective with a high numerical aperture. Forward-detected CARS (F-CARS) and epi-detected CARS (E-CARS) images are recorded simultaneously. F-CARS is used for visualizing features comparable to or larger than the excitation wavelength, while E-CARS allows detection of smaller features with a high contrast. F-CARS and E-CARS images of live and unstained cells reveal details invisible in differential interference-contrast images. High-speed vibrational imaging of unstained cells undergoing mitosis and apoptosis has been carried out. For live NIH 3T3 cells in metaphase, 3D distribution of chromosomes is mapped at the frequency of the DNA backbone Raman band, while the vesicles surrounding the nucleus is imaged by E-CARS at the frequency of the C-H stretching Raman band. Apoptosis in NIH 3T3 cells is monitored using the CARS signal from aliphatic C-H stretching vibration.
已开发出具有快速数据采集和高灵敏度的激光扫描相干反斯托克斯拉曼散射（CARS）显微镜，用于活细胞的振动成像。通过两个共线重叠的近红外皮秒光束和一个具有高数值孔径的水浸物镜实现了高三维（3D）分辨率。同时记录前向检测CARS（F-CARS）和落射检测CARS（E-CARS）图像。F-CARS用于可视化与激发波长相当或大于激发波长的特征，而E-CARS允许检测具有高对比度的较小特征。活的未染色细胞的F-CARS和E-CARS图像揭示了微分干涉对比图像中不可见的细节。已对正在进行有丝分裂和凋亡的未染色细胞进行了高速振动成像。对于处于中期的活NIH 3T3细胞，在DNA主链拉曼带的频率下绘制染色体的3D分布图，而围绕细胞核的囊泡则通过E-CARS在C-H伸缩拉曼带的频率下成像。使用来自脂肪族C-H伸缩振动的CARS信号监测NIH 3T3细胞中的凋亡。
