Advanced observation of brain and nerve cells using two-photon microscopy with novel techniques
Advanced observation of brain and nerve cells using two-photon microscopy with novel techniques
复制标题
DOI：
10.1093/jmicro/dfac047
复制
发表时间：
2022-09-21
期刊：
影响因子：
1.8
通讯作者：
Nemoto, Tomomi
中科院分区：
文献类型：
--
作者：
Ishii, Hirokazu;Takahashi, Taiga;Nemoto, Tomomi
双光子激发荧光显微镜是一种从细胞到组织水平理解生理现象的可靠技术，可归因于近红外超短激光脉冲诱导的非线性激发过程。最近，我们一直在推广使用半导体激光器、自适应光学、矢量束和纳米材料来提高观测深度或空间分辨率。研制的半导体激光光源成功地显示了增强的黄色荧光蛋白(EYFP)在海马齿状回表达的神经元的结构，而不需要以视频速率切除新皮质和海马角(CA1)区的神经元活动。我们还提出了使用100 nm厚度的含氟聚合物纳米片进行活体成像，并实现了1 mm深度的麻醉小鼠脑成像的大视野。此外，开发的自适应光学2 PM显示了次级运动皮质V层中表达EYFP的神经元的单个树突，这是由于大脑表面的曲率而难以观察到的。此外，我们结合2 PM和受激发射耗尽显微镜来提高空间分辨率。这种组合显微镜是非侵入性的，具有优越的空间分辨率，超过了传统光的衍射极限。在这篇综述中，我们描述了我们最新的成果，并讨论了2 PM的未来。
Two-photon excitation fluorescence microscopy [two-photon microscopy (2PM)] is a robust technique for understanding physiological phenomena from the cellular to tissue level, attributable to the nonlinear excitation process induced by near-infrared ultrashort laser light pulses. Recently, we have been promoting the use of semiconductor lasers, adaptive optics, vector beams and nanomaterials to improve the observation depth or spatial resolution. The developed semiconductor-based laser light source successfully visualized the structure of the enhanced yellow fluorescent protein (EYFP)-expressing neurons at the hippocampal dentate gyrus without resecting the neocortex and neuronal activity in the hippocampal cornu ammonis (CA1) region in anesthetized mice at video rates. We also proposed using fluoropolymer nanosheets of 100-nm thickness for in vivo imaging and realized a wide field of view during anesthetized mouse brain imaging of 1-mm depth. Furthermore, the developed adaptive optical 2PM visualized single dendritic spines of EYFP-expressing neurons in cortical layer V of the secondary motor cortex, which had been difficult to observe due to the curvature of the brain surface. In addition, we combined 2PM and stimulated emission depletion microscopy to improve spatial resolution. This combined microscopy is noninvasive and has a superior spatial resolution, exceeding the diffraction limit of the conventional light. In this review, we describe our recent results and discuss the future of 2PM.