Tehrani Kayvan Forouhesh, Latchoumane Charles V, Southern W Michael, Pendleton Emily G, Maslesa Ana, Karumbaiah Lohitash, Call Jarrod A, Mortensen Luke J
Regenerative Bioscience Center, Rhodes Center for ADS, University of Georgia, Athens, GA 30602, USA.
Department of Kinesiology, University of Georgia, Athens, GA 30602, USA.
Biomed Opt Express. 2019 Jun 26;10(7):3591-3604. doi: 10.1364/BOE.10.003591. eCollection 2019 Jul 1.
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,λ,t) data sets at the speed of 4.2 volumes per second over volumes as large as 160 x 160 x 35 µm.
多光子扫描显微镜为光学切片提供了一种强大的工具，可用于捕捉快速生物事件，如血流、线粒体活动和神经元动作电位。对于许多研究而言，以类似于生物事件频率的速率可视化几个不同的焦平面非常重要。通常，显微镜配备有机械元件来移动样品或物镜以获取体积信息，但这些策略由于速度慢或存在惯性伪影而受到限制。为了克服这个问题，已经开发了远程聚焦方法，以在不移动样品或显微镜的情况下轴向移动焦平面。其中一种方法是液体透镜技术，它通过改变液体的润湿性从而改变其曲率来调整透镜的焦点。液体透镜是廉价的有源光学元件，具有进行快速多光子体积成像的潜力，因此是研究具有复杂动力学的生物系统的一种有前景且易于实现的方法。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镜，但在其实施过程中，光学像差和高能量激光脉冲的影响一直令人担忧。在本文中，我们对一种液体透镜进行了表征，并验证了其在相关生物应用中的用途。我们测量了由液体透镜引起的光学像差，并计算了其响应时间、散焦滞后和对脉冲激光的热响应。我们将这种远程聚焦方法应用于多个体内标本的成像和测量，包括间充质干细胞动力学、小鼠胫前肌线粒体电位波动和小鼠脑神经元活动。我们的系统以每秒4.2个体积的速度生成高达160 x 160 x 35 µm的5维（x、y、z、λ、t）数据集。
