荧光波长对共焦显微镜成像特性的影响

导出了共焦显微镜中不同荧光波长情况下的荧光功率传输函数、三维脉冲响应函数（3D－PSF）和三维光学传递函数（3D－OTF）。结果表明，不同的荧光波长对共焦显微镜的空间截止频率、分辨率、光学传递函数存在明显的影响。当激发波长与荧光波长的比值下降到一定程度时，可以看到明显的失锥现象。     

双光子和多光子共焦显微镜的成像理论

对双光子和多光子共焦扫描显微镜的成像理论作了系统的理论分析,导出了双光子和多光子共焦显微镜成像系统的三维点扩散函数和三维光学传递函数,研究结果表明：双光子共焦显微镜比单光子共焦显微镜具有更高的横向分辨率和纵向分辨率,而多光子共焦扫描显微镜又比双光子共焦扫描显微镜具有更高的空间分辨率. 关键词：     

荧光谱线分布对共焦荧光显微镜分辨率的影响

研究了荧光谱线分布对共焦荧光显微镜中分辨率的影响,导出了荧光谱线分布的荧光功率传输函数、三维脉冲响应函数.数值计算了荧光谱线均匀分布的情况,结果表明：与采用单一中心荧光波长的分辨率比较,共焦荧光显微镜的横向分辨率、纵向分辨率随着荧光谱线范围和色散的增大而下降;谱线响应范围小的探测器有利于分辨率.     

二次谐波共焦成像的分辨率

通过与双光子荧光共焦成像过程比较,研究了非线性二次谐波的强度脉冲响应函数并对它进行频域分析,获得了横向、纵向截止频率和通频带.研究结果表明：二次谐波共焦成像具有更高的分辨率.     

荧光共焦扫描系统成像特性的优化

对荧光共焦扫描系统用光强点扩散函数进行傅里变换得到系统三维传递函数的数学模型，并由此求得环形透镜和各种有限大小探测器系统的光学传递函数。用计算机模拟和光学传递九数值计算，分析了采用不同环形透镜及探测器对系统成像特性的影响     

光学仪器

眼镜、放大镜、显微镜、望远镜TH742.652006021689高斯光束荧光共焦显微镜的三维光学传递函数=Opticaltransfer function of a fluorescent confocal microscope withextended Gaussian source[刊,中]/杨初平(华南农业大学理学院.广东,广州(510642))∥激光技术.—2005,29(5).—552-554研究高斯光源的束斑半径、光源孔径和探测器孔径对荧光共焦显微镜3-D OTF的影响,获得了具有有限光源孔径、探测器孔径和束斑半径的荧光共焦显微镜的三维光学传递函数(3-D OTF)。数值计算结果表明,束斑半径影响到光源孔径和探测器孔径的选择,与采用平行光…     

双光子技术在三维成像和三维存储技术中的应用

对双光子过程的空间分辨能力进行了理论分析。阐明了双光子技术在三维成像和三维存储中的独特优势。着重介绍了双光子技术与扫描共焦显微术、近场显微术相结合进行三维成像 ,以及一种多焦点多光子显微术和用连续光源激发的双光子三维成像技术的研究和进展情况。对建立在共焦显微镜基础之上的双光子三维光存储和微细加工方面的研究也作了回顾与展望     

高斯光束共焦扫描激光显微镜的光学传递函数

考虑光源和探测器有一定大小，并引入光源的高斯分布函数，求出此接近实际的共焦扫描成像系统的光学传递函数(OTF)；运用计算机进行数值模拟并讨论光源针孔和探测针孔的大小对横向、纵向分辨率的影响.所得结果在不影响共焦扫描成像系统三维(3D)成像能力的基础上，可以有效地提高系统的信噪比. 关键词：     

小鼠卵母细胞染色体三维双光子荧光图像的轴向衰减

双光子荧光显微镜作为一种高分辨光学仪器,已经被广泛应用于生物样品的非侵入式三维光学成像中。相比共聚焦显微镜,双光子荧光显微镜拥有更深的探测深度。然而,即便如此,在对较厚的生物样品进行非侵入式光学三维成像时,样品的成像质量也往往会随着探测深度的增加而下降。在临床和生物学领域对研究母性遗传起重要作用的小鼠卵母细胞拥有较大的直径(80～100 μm),吸收和散射效应较为明显。本文研究小鼠卵母细胞染色体的三维双光子荧光图像随探测深度增加图像质量的衰减程度。通过对所得图像进行轴向衰减矫正,利用体积作为参数,将矫正前后小鼠卵母细胞内染色体三维双光子荧光图像进行对比。结果表明,由于吸收和散射效应,卵母细胞存在较严重的光学轴向衰减问题,因此,对用双光子荧光三维成像手段获得的小鼠卵母细胞图像进行衰减矫正是有必要的。这为进一步精确定量的研究卵母细胞内染色体的三维构像打下良好的基础。     

共焦扫描荧光显微镜的信噪比与信息量

在研制用于对厚的生物样品进行光学断层成像的共焦扫描荧光显微镜时，由于成像信号十分微弱及存在很强的多次散射作用，因此杂散光的抑制非常重要，而信噪比、信号背景比就成为决定能否获得高对比度、高分率图像的关键。运用光学信息量的概念，在已有的光学成像系统信息量计算、共焦扫描荧光显微镜信噪比及传递函数计算的基础上，详细分析了共焦扫描荧光显微镜信息量与信噪比等之间的定量关系。该关系表明，为了充分利用共焦扫描荧光显微镜的成像性能，必须选择适当的探测小孔。所得的结果对于共焦扫描荧光显微成像系统的研制有重要的实用价值。     

Arrangement of a 4Pi microscope for reducing the confocal detection volume with two-photon excitation

The main advantage of two-photon fluorescence confocal microscopy is the low absorption obtained with live tissues at the wavelengths of operation. However, the resolution of two-photon fluorescence confocal microscopes is lower than in the case of one-photon excitation. The 4Pi microscope type C working in two-photon regime, in which the excitation beams are coherently superimposed and, simultaneously, the emitted beams are also coherently added, has shown to be a good solution for increasing the resolution along the optical axis and for reducing the amplitude of the side lobes of the point spread function. However, the resolution in the transverse plane is poorer than in the case of one-photon excitation due to the larger wavelength involved in the two-photon fluorescence process. In this paper we show that a particular arrangement of the 4Pi microscope, referenced as 4Pi′ microscope, is a solution for obtaining a lateral resolution in the two-photon regime similar or even better to that obtained with 4Pi microscopes working in the one-photon excitation regime.     

Theoretical investigation on Raman induced kerr effect spectroscopy in nonlinear confocal microscopy

The imaging theory of Raman induced Kerr effect spectroscopy (RIKES) in nonlinear confocal microscopy is presented in this paper. Three-dimensional point spread function (3D-PSF) of RIKES nonlinear confocal microscopy in isotropic media is derived with Fourier imaging theory and RIKES theory. The impact of nonlinear property of RIKES on the spatial resolution and imaging properties of confocal microscopy have been analyzed in detail. It is proved that RIKES nonlinear confocal microscopy can simultaneously provide more information than two-photon confocal microscopy concerning molecular vibration mode, vibration orientation and optically induced molecular reorientation, etc. It is shown that RIKES nonlinear confocal microscopy significantly enhances the spatial resolution and imaging quality of confocal microscopy and achieves much higher resolution than that of two-photon confocal microscopy.     

Imaging theory of nonlinear second harmonic and third harmonic generations in confocal microscopy

ItisreportedrecentlythatnonlinearopticalphenomenonofSHGandTHGhasbeenobservedinmanybiologicaltissues[16].SHGandTHGhavebeenusedtoperformthethree-dimensionalimaginginlivingtissuesandhaveattractedmuchattentionrecently.TherearemanyadvantagesofusingSHGandTHGtoperformthethree-dimensionalimaginginlivingtissues,suchasnoninvasiveandnophotobleaching,inadditiontotheimagingpropertiesofmulti-photonfluorescenceimaging[7—9].Firstly,unlikeinthesingle-andmulti-photonfluorescenceprocesses,onlyvirtualstat…     

Resolution enhancement in a light-sheet-based microscope (SPIM)

Light-sheet-based microscopy [single-plane illumination microscope (SPIM)] performs very well at low numerical apertures. It complements conventional (FM), confocal (CFM), and two-photon fluorescence microscopy (2hnu-FM) currently used in modern life sciences. Lateral and axial SPIM point spread function (PSF) extents are measured by using fluorescent beads to determine the 3D resolution. The results are compared with values derived from an analytical theory and numerical simulations. The discrepancies are found to be less than 5%. The axial extent of a SPIM-PSF (10x/0.3 W) is approximately 5.7 microm. This value is almost a factor of 2 smaller than in CFM, more than 2.5 times smaller than in FM, and more than three times smaller than in 2hnu-FM. SPIM outperforms 2hnu-FM and FM, while CFM has a better axial resolution at NAs above 0.8.     

Two-photon fluorescence isotropic-single-objective microscopy

Two-photon excitation provides efficient optical sectioning in three-dimensional fluorescence microscopy, independently of a confocal detection. In two-photon laser-scanning microscopy, the image resolution is governed by the volume of the excitation light spot, which is obtained by focusing the incident laser beam through the objective lens of the microscope. The light spot being strongly elongated along the optical axis, the axial resolution is much lower than the transverse one. In this Letter we show that it is possible to strongly reduce the axial size of the excitation spot by shaping the incident beam and using a mirror in place of a standard glass slide to support the sample. Provided that the contribution of sidelobes can be removed through deconvolution procedures, this approach should allow us to achieve similar axial and lateral resolution.