Two-photon microscopy in brain tissue: parameters influencing the imaging depth
Two-photon microscopy in brain tissue: parameters influencing the imaging depth
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DOI：
10.1016/s0165-0270(01)00438-1
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发表时间：
2001-10-15
影响因子：
3
通讯作者：
Charpak, S
中科院分区：
文献类型：
--
作者：
Oheim, M;Beaurepaire, E;Charpak, S
组织的光散射限制了双光子显微镜的成像深度及其在活体脑功能成像中的应用。我们研究了散射对荧光激发和收集的影响，并确定了限制大脑成像深度的组织和仪器参数。(i)在脑切片中，我们测量到λ = 800 nm处的散射长度在幼年皮质组织（P14-P18）中比在成年组织（P90）中高2倍。(ii)在典型的体内成像的检测几何形状，我们表明，收集的部分荧光下降在大的深度，并且它是成比例的平方的有效角接受的检测光学器件。将显微镜的角度接受度与物镜透镜的角度接受度相匹配可以导致在大深度（> 500 μ m）处的收集效率的类似于3的增益。与标准的60 x-63 x物镜相比，低放大倍数（20 x）、高数值孔径物镜（0.95）进一步增加了荧光收集，增加了10倍，而不影响分辨率。这种改进应该允许与神经元或血管脑活动相关的荧光测量比标准物镜更深> 100 μ m。(C)2001 Elsevier Science B. V.保留所有权利。
Light scattering by tissue limits the imaging depth of two-photon microscopy and its use for functional brain imaging in vivo. We investigate the influence of scattering on both fluorescence excitation and collection, and identify tissue and instrument parameters that limit the imaging depth in the brain. (i) In brain slices, we measured that the scattering length at lambda = 800 nm is a factor 2 higher in juvenile cortical tissue (P14-P18) than in adult tissue (P90). (ii) In a detection geometry typical for in vivo imaging, we show that the collected fraction of fluorescence drops at large depths, and that it is proportional to the square of the effective angular acceptance of the detection optics. Matching the angular acceptance of the microscope to that of the objective lens can result in a gain of similar to 3 in collection efficiency at large depths ( > 500 mum). A low-magnification (20 x), high-numerical aperture objective (0.95) further increases fluorescence collection by a factor of similar to 10 compared with a standard 60 x -63 x objective without compromising the resolution. This improvement should allow fluorescence measurements related to neuronal or vascular brain activity at > 100 mum deeper than with standard objectives. (C) 2001 Elsevier Science B.V. All rights reserved.