Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging
Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging
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DOI：
10.1016/s0006-3495(98)77643-x
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发表时间：
1998-10-01
影响因子：
3.4
通讯作者：
White, JG
中科院分区：
文献类型：
--
作者：
Centonze, VE;White, JG
多光子激发荧光成像通过将荧光团激发限制在焦平面来生成样品的光学截面。仅在物镜的焦点体积中实现的高光子密度足以通过密度依赖的多光子激发过程来激发荧光探针分子。我们提出了比较共焦与多光子激发成像相同的光学部分内的样品。这些并排的比较成像模式证明了多光子成像的显着优势，可以从更深的生物标本内获得的数据。对各种生物样品的观察表明，在所有情况下，相对于共焦成像，用多光子激发获得的成像穿透深度至少有两倍的改善。在共焦成像的样品内的图像对比度的更明显的退化，主要是由于散射的发射光子，这减少了信号，并增加了局部背景的点扩散函数的测量表明，分辨率不显着改变与增加深度的任一模式的显微镜。多光子成像不遭受信号对背景的退化到与共焦成像几乎相同的程度，因为该方法对发射信号的散射不敏感。使用安装在物镜附近的外部光电检测器（仅在多光子成像系统中可能）直接检测发射的光子提高了系统灵敏度和用于成像的散射发射光子的利用率。我们证明，这种技术提供了更多的改进，多光子激发成像的能力，以产生良好的质量图像，从更深的组织内相对于共焦成像。
Multiphoton excitation fluorescence imaging generates an optical section of sample by restricting fluorophore excitation to the plane of focus. High photon densities, achieved only in the focal Volume of the objective, are sufficient to excite the fluorescent probe molecules by density-dependent, multiphoton excitation processes. We present comparisons of confocal with multiphoton excitation imaging of identical optical sections within a sample. These side-by-side comparisons of imaging modes demonstrate a significant advantage of multiphoton imaging; data can be obtained from deeper within biological specimens. Observations on a variety of biological samples showed that in all cases there was at least a twofold improvement in the imaging penetration depth obtained with multiphoton excitation relative to confocal imaging. The more pronounced degradation in image contrast deep within a confocally imaged sample is primarily due to scattered emission photons, which reduce the signal and increase the local background as measurements of point spread functions indicated that resolution does not significantly change with increasing depth for either mode of microscopy. Multiphoton imaging does not suffer from degradation of signal-to-background to nearly the same extent as confocal imaging because this method is insensitive to scatter of the emitted signal. Direct detection of emitted photons using an external photodetector mounted close to the objective (possible only in a multiphoton imaging system) improves system sensitivity and the utilization of scattered emission photons for imaging. We demonstrate that this technique provides yet further improvements in the capability of multiphoton excitation imaging to produce good quality images from deeper within tissue relative to confocal imaging.