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利用带有主动轴向扫描的微型光纤耦合显微镜在自由移动的小鼠中进行三维双光子脑成像。

Three dimensional two-photon brain imaging in freely moving mice using a miniature fiber coupled microscope with active axial-scanning.

机构信息

Department of Bioengineering, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, 80045, USA.

Department of Cell & Developmental Biology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, 80045, USA.

出版信息

Sci Rep. 2018 May 25;8(1):8108. doi: 10.1038/s41598-018-26326-3.

Abstract

We present a miniature head mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. We show three-dimensional two-photon imaging of neuronal structure and record neuronal activity from GCaMP6s fluorescence from multiple focal planes in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. Additionally, dynamic control of the axial scanning of the electrowetting lens allows tilting of the focal plane enabling neurons in multiple depths to be imaged in a single plane. Two-photon imaging allows increased penetration depth in tissue yielding a working distance of 450 μm with an additional 180 μm of active axial focusing. The objective NA is 0.45 with a lateral resolution of 1.8 μm, an axial resolution of 10 μm, and a field-of-view of 240 μm diameter. The 2P-FCM has a weight of only ~2.5 g and is capable of repeatable and stable head-attachment. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research.

摘要

我们展示了一种微型头戴式双光子光纤耦合显微镜(2P-FCM),用于神经元成像,其采用微型电润湿透镜实现主动轴向聚焦。我们展示了自由移动小鼠中多个焦点平面上的神经元结构的三维双光子成像,并记录了 GCaMP6s 荧光的神经元活动。还演示了 GFP 和 tdTomato 荧光的双色同时成像。此外,电润湿透镜的轴向扫描的动态控制允许倾斜焦平面,从而可以在单个平面上对多个深度的神经元进行成像。双光子成像允许在组织中增加穿透深度,工作距离为 450μm,外加 180μm 的主动轴向聚焦。该物镜的数值孔径为 0.45,横向分辨率为 1.8μm,轴向分辨率为 10μm,视场直径为 240μm。2P-FCM 的重量仅约 2.5g,可重复且稳定地附着在头部。具有动态轴向扫描的 2P-FCM 提供了从功能上不同的神经元层进行记录的新能力,为神经科学研究开辟了新的机会。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/92ad/5970169/c708b500eef7/41598_2018_26326_Fig1_HTML.jpg

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