多变视场的多焦点多光子激发荧光显微技术

发展了一种新型的多焦点多光子激发荧光显微技术，通过软件控制空间光调制器，在所需要的成像区域产生相应的激发光点阵，通过扫描入射角实现激发点阵快速并行扫描激发，通过CCD并行记录所产生的荧光信号，获得任意视场图像。分别实现了10×10和50×50点阵激发下的全视场双光子荧光图像，并且实现了同时多个区域寻址的多焦点激发成像。对比其他多焦点显微技术，该技术具有明显的灵活性，不但保持了多焦点激发的快速成像的优点，而且还增加了任意数量成像区域同时寻址和阵列点密度变化，所有这些变化只需要通过软件加载相应的相位图案给空间光调制器，而不需要任何硬件更改。

Abstract

A novel multifocal multiphoton excited fluourescence microscopy (MMM) is presented. The required focus array can be produced in multiple interesting subregions through uploading the accurate phase pattern to the spatial light modulator (SLM) with the software. The focus array can be scanned with a two-mirror galvo scanner by scanning the single beam that is incident on the SLM. The two photon fluorescence signal is recorded directly with an electron-multiplying CCD in parallel. The two photon fluorescence images in the whole field-of-view are obtained under the different modes such as 10×10 and 50×50 arrays, and the multiple subregions of interest are addressably excited simultaneously. Compared with the other MMMs, our MMM has a good flexibility without sacrificing of the imaging speed and spatial resolution and also without hardware changes.

参考文献

[1] W. Denk, J. H. Strickler, W. W. Webb. Two-photon laser scanning fluorescence microscopy［J］. Science, 1990, 248(4951): 73～76

[2] Chen Wang, Lingling Qiao, Zhengle Mao et al.. Reduced deep-tissue image degradation in three-dimensional multiphoton microscopy with concentric two-color two-photon fluorescence excitation［J］. J. Opt. Soc. Am. B, 2008, 25(6): 976～982

[3] 陈德强, 夏安东. 双光子激光扫描荧光显微镜及其应用［J］. 物理, 2000, 29(4): 232～236

Chen Deqiang, Xia Andong. Two-photon laser scanning fluorescence microscopy and its applications［J］. Physics, 2000, 29(4): 232～236

[4] 张春阳, 贡宜萱, 马辉等. 双光子及共聚焦激光扫描显微术研究天花粉蛋白对人绒癌细胞的作用机制［J］. 生物化学与生物物理进展, 2001, 28(5): 717～721

Zhang Chunyang, Gong Yixuan, Ma Hui et al.. Studying the effect of trichosanthin on choriocarcinoma cells with both two-photon and confocal laser scanning microscopy［J］. Progress in Biochemistry and Biophysics, 2001, 28(5): 717～721

[5] 魏勋斌, 郭进, 李延等. 光学活体成像技术进展［J］. 激光与光电子学进展, 2009, 46(8): 41～47

Wei Xunbin, Guo Jin, Li Yan et al.. Progress of in vivo optical imaging［J］. Laser & Optoelectronics Progress, 2009, 46(8): 41～47

[6] 李政, 张镇西. 纳米金标记活细胞双光子荧光成像研究［J］. 深圳大学学报理工版, 2008, 25(3): 248～251

Li Zheng, Zhang Zhenxi. Two-photon fluorescence imaging of cationic gold nanoparticles labeled fibroblasts and its application［J］. J. Shenzhen University Science and Engineering, 2008, 25(3): 248～251

[7] 吕晓华, 占成, 张红民等. 随机扫描多光子荧光显微成像系统［J］. 光学学报, 2006, 26(12): 1823～1828

Lü Xiaohua, Zhan Cheng, Zhang Hongmin et al.. Construction of random-access scanning multiphoton fluorescence microscope system［J］. Acta Optica Sinica, 2006, 26(12): 1823～1828

[8] A. Bullen, S. S. Patel, P. Saggau. High-speed, random-access fluorescence microscopy: high-resolution optical recording with voltage-sensitive dyes and ion indicators［J］. Biophys. J., 1997, 73(1): 477～491

[9] Vijay Iyer, Bradley E. Losavio, Peter Saggau. Compensation of spatial and temporal dispersion for acousto-optic multiphoton laser-scanning microscopy［J］. J. Biomed. Opt., 2003, 8(3): 460～471

[10] Shaoqun Zeng, Xiaohua Lü, Chen Zhan et al.. Simultaneous compensation for spatial and temporal dispersion of acousto-optical deflectors for two-dimensional scanning with a single prism［J］. Opt. Lett., 2006, 31(8): 1091～1093

[11] J. M. Larson, S. A. Schwartz, M. W. Davidson. Resonant Scanning in Laser Confocal Microscopy [OL]. MicroscopyU: The Source for Microscopy Education. http://www.microscopyu.com/articles/confocal/resonantscanning.html

[12] J. Bewersdorf, R. Pick, S. W. Hell. Multifocal multiphoton microscopy［J］. Opt. Lett., 1998, 23(9): 655～657

[13] A. H. Buist, M. Muller, J. Squier et al.. Real time two-photon absorption microscopy using multipoint excitation［J］. J. Microsc., 1998, 192(2): 217～226

[14] Y. Shao, J. Qu, H. Li et al.. High-speed spectrally resolved multifocal multiphoton microscopy［J］. Appl. Phys. B, 2010, 99(4): 633～637

[15] L. Sacconi, E. Froner, R. Antolini et al.. Multiphoton multifocal microscopy exploiting a diffractive optical element［J］. Opt. Lett., 2003, 28(20): 1918～1920

[16] Z. Zalevsky, D. Mendlovic. Gerchberg-Saxton algorithm applied in the fractional Fourier or the Fresnel domain［J］. Opt. Lett., 1996, 21(12): 842～844

李震, 屈军乐, 邵永红, 高志. 多变视场的多焦点多光子激发荧光显微技术[J]. 光学学报, 2012, 32(10): 1018001. Li Zhen, Qu Junle, Shao Yonghong, Gao Zhi. Multiple Variational Field-of-View Multifocal Multiphoton Fluorescence Microscope[J]. Acta Optica Sinica, 2012, 32(10): 1018001.

多变视场的多焦点多光子激发荧光显微技术

关于本站 Cookie 的使用提示

全站搜索

多变视场的多焦点多光子激发荧光显微技术

相关论文

相关资讯

关于本站 Cookie 的使用提示

全站搜索