Maximizing fluorescence collection efficiency in multiphoton microscopy.
Maximizing fluorescence collection efficiency in multiphoton microscopy.
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DOI：
10.1364/oe.19.015348
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发表时间：
2011-08-01
期刊：
影响因子：
3.8
通讯作者：
Levene MJ
中科院分区：
文献类型：
--
作者：
Zinter JP;Levene MJ
了解通过多光子显微镜的荧光传播是至关重要的，在设计高性能的系统能够深入组织成像。使用散射组织样品和Olympus 20 X 0.95NA显微镜物镜的光学模型来模拟荧光传播，作为生理相关散射参数的成像深度的函数。从这些模拟中得到的物镜后孔径处的荧光的空间角分布被用来设计一个简单的、最有效的物镜后荧光收集系统。通过来自实验组织体模的数据证实的Monte Carlo模拟表明，在大成像深度处，收集效率比传统的非优化的荧光收集几何结构提高了50% - 90%。通过对小鼠皮层中的第V层神经元成像至850 μm的深度来验证成像性能。
Understanding fluorescence propagation through a multiphoton microscope is of critical importance in designing high performance systems capable of deep tissue imaging. Optical models of a scattering tissue sample and the Olympus 20X 0.95NA microscope objective were used to simulate fluorescence propagation as a function of imaging depth for physiologically relevant scattering parameters. The spatio-angular distribution of fluorescence at the objective back aperture derived from these simulations was used to design a simple, maximally efficient post-objective fluorescence collection system. Monte Carlo simulations corroborated by data from experimental tissue phantoms demonstrate collection efficiency improvements of 50% – 90% over conventional, non-optimized fluorescence collection geometries at large imaging depths. Imaging performance was verified by imaging layer V neurons in mouse cortex to a depth of 850 μm.