A versatile CARS microscope for biological imaging
A versatile CARS microscope for biological imaging
复制标题
DOI：
10.1002/jrs.2249
复制
发表时间：
2009-07-01
影响因子：
2.5
通讯作者：
Elfick, Alistair
中科院分区：
文献类型：
--
作者：
Downes, Andrew;Mouras, Rabah;Elfick, Alistair
我们已经建立了一个新的，多功能的相干反斯托克斯拉曼散射（汽车）显微镜的生物成像。该系统采用Nd：YVO 4激光器作为斯托克斯（6 ps，1064 nm），可调谐光学参量振荡器（OPO）（5 ps，700-1000 nm）作为泵浦激发，产生的可观测范围为600-4000 cm（-1）。第二个OPO可以用作低波数激发（低至100 cm（-1））的替代泵。泵浦和斯托克斯激发被聚焦到单模光纤上以便于对准，该单模光纤连接到共焦倒置光学显微镜的输入端。使用光谱仪过滤epi检测的汽车信号，并使用雪崩光电二极管对光子进行计数。这种检测优于使用带通滤波器和光电倍增管，因为它允许所有波数成像，并确保拒绝不需要的荧光和散射光。我们的显微镜包括一个加热器阶段和灌注细胞成像活细胞，并允许互补的生物成像技术（微分干涉对比度（DIC），二次谐波产生，和频产生，单光子和双光子荧光，荧光寿命成像）。此外，我们的系统具有原子力显微镜（AFM），使汽车成像在10 nm的分辨率。模块化成像系统的细节和优点进行了更充分的描述，并提出了最新的结果。版权所有（C）2009约翰威利父子有限公司
We have constructed a novel, versatile Coherent anti-Stokes Raman scattering (CARS) microscope for biological imaging. The system employs a Nd:YVO4 laser as Stokes (6 ps, 1064 nm), and a tunable optical parametric oscillators (OPOs) (5 ps, 700-1000 nm) as pump excitation, resulting in an observable range of similar to 600-4000 cm(-1). A second OPO can be used as a replacement pump for low wavenumber excitation (down to similar to 100 cm(-1)). Pump and Stokes excitations are focused onto a single-mode fibre for ease of alignment, which is connected to the input of a confocal inverted optical microscope. The epi-detected CARS signal is filtered using a spectrometer, and the photons counted with an avalanche photodiode. This detection is preferable to using bandpass filters and photomultipliers, as it allows all wavenumbers to be imaged and ensures rejection of unwanted fluorescent and scattered light. Our microscope comprises a heater stage and perfusion cell for imaging live cells, and allows complementary biological imaging techniques (differential interference contrast (DIC), second harmonic generation, sum frequency generation, one- and two-photon fluorescence, fluorescence lifetime imaging). In addition, our system features an atomic force microscope (AFM) which enables CARS imaging at 10-nm resolution. The details and advantages of the modular imaging system are described more fully, and the latest results are presented. Copyright (C) 2009 John Wiley & Sons, Ltd.