Five-dimensional two-photon volumetric microscopy of in-vivo dynamic activities using liquid lens remote focusing
Five-dimensional two-photon volumetric microscopy of in-vivo dynamic activities using liquid lens remote focusing
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DOI：
10.1364/boe.10.003591
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发表时间：
2019-07-01
影响因子：
3.4
通讯作者：
Mortensen, Luke J.
中科院分区：
文献类型：
--
作者：
Tehrani, Kayvan Forouhesh;Latchoumane, Charles, V;Mortensen, Luke J.
多光子扫描显微镜为光学切片提供了一种强大的工具，可用于捕获快速生物事件，如血流，线粒体活动和神经元动作电位。对于许多研究，重要的是以类似于生物事件频率的速率可视化几个不同的焦平面。通常，显微镜配备有机械元件来移动样品或物镜透镜以捕获体积信息，但是这些策略由于其缓慢的速度或惯性伪影而受到限制。为了克服这个问题，远程聚焦方法已被开发，以轴向移动焦平面，而不需要样品或显微镜的物理移动。在这些方法中有液体透镜技术，它通过改变液体的润湿性从而改变其曲率来调节透镜的焦点。液体透镜是一种廉价的有源光学元件，具有快速多光子体积成像的潜力。尽管使用液体透镜技术的远程聚焦可用于体积点扫描多光子显微术，但光学像差和高能激光脉冲的影响在其实施中一直是关注的问题。在本文中，我们描述了液体透镜，并验证其在相关生物应用中的用途。测量了液体透镜引起的光学像差，并计算了其响应时间、离焦滞后。和对脉冲激光的热响应。我们应用这种方法的远程聚焦成像和测量多个体内标本，包括间充质干细胞动力学，小鼠胫骨前肌线粒体电位波动，小鼠脑神经活动。我们的系统产生5维（x，y，z，lambda，t）数据集的速度为每秒4.2卷，体积大到160 × 160 × 35 μ m（3）。(C)根据OSA开放获取出版协议的条款，2019年美国光学学会
Multi-photon scanning microscopy provides a robust tool for optical sectioning, which can be used to capture fast biological events such as blood flow, mitochondrial activity, and neuronal action potentials. For many studies, it is important to visualize several different focal planes at a rate akin to the biological event frequency. Typically, a microscope is equipped with mechanical elements to move either the sample or the objective lens to capture volumetric information, but these strategies are limited due to their slow speeds or inertial artifacts. To overcome this problem, remote focusing methods have been developed to shift the focal plane axially without physical movement of the sample or the microscope. Among these methods is liquid lens technology, which adjusts the focus of the lens by changing the wettability of the liquid and hence its curvature. Liquid lenses are inexpensive active optical elements that have the potential for fast multi-photon volumetric imaging. hence a promising and accessible approach for the study of biological systems with complex dynamics Although remote focusing using liquid lens technology can be used for volumetric point scanning multi-photon microscopy, optical aberrations and the effects of high energy laser pulses have been concerns in its implementation. In this paper, we characterize a liquid lens and validate its use in relevant biological applications. We measured optical aberrations that are caused by the liquid lens, and calculated its response time, defocus hysteresis. and thermal response to a pulsed laser. We applied this method of remote focusing for imaging and measurement of multiple in-vivo specimens, including mesenchymal stem cell dynamics, mouse tibialis anterior muscle mitochondrial electrical potential fluctuations, and mouse brain neural activity. Our system produces 5 dimensional (x,y,z,lambda,t) data sets at the speed of 4.2 volumes per second over volumes as large as 160 x 160 x 35 mu m(3). (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement