Multi-photon excitation microscopy in intact animals
Multi-photon excitation microscopy in intact animals
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DOI：
10.1111/j.1365-2818.2006.01570.x
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发表时间：
2006-04-01
影响因子：
2
通讯作者：
Balaban, Robert S.
中科院分区：
文献类型：
--
作者：
Rothstein, Emily C.;Nauman, Michael;Balaban, Robert S.
双光子激发荧光显微镜和背散射二次谐波发生显微镜允许活动物内的亚细胞事件的调查，但这些实验的许多方面需要进行优化，以克服传统的显微镜的几何形状，运动和光学耦合的主题。本报告描述了一个稳定的系统，用于支持一个活的仪器小鼠或兔内源性还原烟酰胺腺嘌呤二核苷酸和外源性染料双光子激发荧光显微镜测量，和背散射二次谐波发生显微镜测量。该系统是改良的倒置LSM 510显微镜（Carl Zeiss，Inc.，Thornwood，NY，U.S.A.）使用旋转潜望镜将倒置的观察镜转换为直立的形式，物镜位于距显微镜底座中心约15 cm处，允许轻松放置仪器动物。将Olympus 20x水浸物镜通过盐水浴或定制的水合透明凝胶光学耦合到组织，而不使用盖玻片。将装有仪器的动物固定在专门设计的保持器上，该保持器使动物在物镜下保持平衡，并允许不同的通风方案以最大限度地减少运动。使用这种方法，从外周和体腔器官常规收集活体动物的高质量图像。使用这种方法进行生理学研究的其余最重要的问题是微米尺度上的运动。运动补偿的几种策略进行了描述和讨论。
Two-photon excitation fluorescence microscopy and backscattered-second harmonic generation microscopy permit the investigation of the subcellular events within living animals but numerous aspects of these experiments need to be optimized to overcome the traditional microscope geometry, motion and optical coupling to the subject. This report describes a stable system for supporting a living instrumented mouse or rabbit during endogenous reduced nicotinamide adenine dinucleotide and exogenous dye two-photon excitation fluorescence microscopy measurements, and backscattered-second harmonic generation microscopy measurements. The system was a modified inverted LSM510 microscope (Carl Zeiss, Inc., Thornwood, NY, U.S.A.) with a rotating periscope that converted the inverted scope to an upright format, with the objective located approximately, 15 cm from the centre of the microscope base, allowing easy placement of an instrumented animal. An Olympus 20x water immersion objective was optically coupled to the tissue, without a cover glass, via a saline bath or custom hydrated transparent gel. The instrumented animals were held on a specially designed holder that poised the animal under the objective as well as permitted different ventilation schemes to minimize motion. Using this approach, quality images were routinely collected in living animals from both the peripheral and body cavity organs. The remaining most significant issue for physiological studies using this approach is motion on the micrometre scale. Several strategies for motion compensation are described and discussed.