Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs Ti: Sa laser
Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs Ti: Sa laser
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DOI：
10.1364/oe.21.007096
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发表时间：
2013-03-25
期刊：
影响因子：
3.8
通讯作者：
Borri, Paola
中科院分区：
文献类型：
--
作者：
Pope, Iestyn;Langbein, Wolfgang;Borri, Paola
我们已经开发了一种多模态多光子激光扫描显微镜的细胞成像功能的差分相干反斯托克斯拉曼散射（D-CARS），双光子荧光（TPF）和二次谐波产生（SHG）的同时收购使用一个单一的5 fs的Ti：Sa宽带（660-970 nm）激光。这些模态的光谱和时间脉冲要求通过将激光光谱分成三个部分来独立地优化：TPF/SHG激发（> 900 nm）、汽车泵浦激发（< 730 nm）和汽车斯托克斯激发（730-900 nm）。特别地，通过使用玻璃色散对泵浦脉冲和斯托克斯脉冲施加相等的线性啁啾，我们实现了10 cm（-1）的汽车光谱分辨率，并且在由泵浦和斯托克斯之间的时间延迟选择的1200-3800 cm（-1）振动范围内获得汽车图像。在TPF/SHG激发中使用棱镜脉冲压缩器以在样品处实现傅立叶限制的30 fs脉冲，以获得最佳的TPF和SHG。D-CARS用很少的无源光学元件实现，并且能够同时激发和检测两个振动频率，间隔可从20 cm（-1）到150 cm（-1）调节，用于选择性化学对比和背景抑制。使用光束扫描的激发/检测设置是围绕商业倒置显微镜支架构建的，该显微镜支架提供传统的亮场、差分干涉对比和落射荧光，用于生物样品的用户友好表征。在HeLa细胞、干细胞衍生的人脂肪细胞和小鼠组织上显示了汽车高光谱图像和在前向和外向同时采集D-CARS、TPF和SHG图像的示例。(C)2013年美国光学学会
We have developed a multimodal multiphoton laser-scanning microscope for cell imaging featuring simultaneous acquisition of differential Coherent Antistokes Raman Scattering (D-CARS), two-photon fluorescence (TPF) and second harmonic generation (SHG) using a single 5 fs Ti:Sa broadband (660-970 nm) laser. The spectral and temporal pulse requirements of these modalities were optimized independently by splitting the laser spectrum into three parts: TPF/SHG excitation (> 900 nm), CARS Pump excitation (< 730 nm), and CARS Stokes excitation (730-900 nm). In particular, by applying an equal linear chirp to pump and Stokes pulses using glass dispersion we achieved a CARS spectral resolution of 10 cm(-1), and acquired CARS images over the 1200-3800 cm(-1) vibrational range selected by the time delay between pump and Stokes. A prism pulse compressor in the TPF/SHG excitation was used to achieve Fourier limited 30 fs pulses at the sample for optimum TPF and SHG. D-CARS was implemented with few passive optical elements and enabled simultaneous excitation and detection of two vibrational frequencies with a separation adjustable from 20 cm(-1) to 150 cm(-1) for selective chemical contrast and background suppression. The excitation/detection set-up using beam-scanning was built around a commercial inverted microscope stand providing conventional bright-field, differential interference contrast and epi-fluorescence for user-friendly characterization of biological samples. Examples of CARS hyperspectral images and simultaneous acquisition of D-CARS, TPF and SHG images in both forward and epi-direction are shown on HeLa cells, stem-cell derived human adipocytes and mouse tissues. (C) 2013 Optical Society of America