Abstract
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 µm3.
Highlights
Multi-photon microscopy enables high resolution imaging and intrinsic optical sectioning at depth due to use of near-infrared and infrared laser multi-photon excitation processes
We tested the liquid lens remote focusing on several live samples; here we present videos of 5D imaging of a single cell in suspension, mouse tibialis anterior (TA) muscle mitochondrial function, bone blood flow in a mouse skull, and neural activity in the barrel cortex
One main concern for using the liquid lens is the potential for optical aberrations due to the curvature produced by the electrical potential
Summary
Multi-photon microscopy enables high resolution imaging and intrinsic optical sectioning at depth due to use of near-infrared and infrared laser multi-photon excitation processes. The size scale of these biological efforts dictates tradeoffs between scan speed, resolution, and field of view When studying events such as cell adhesion, vascular flow patterns, or homing, rapid scanning of a relatively small volume is needed. For phenomenon such as neural firing, multiple events occur at different depths of the volume under study that need to be rapidly accessed [1,2]. In all of these situations, rapid lateral and axial scanning are needed to accurately capture biological dynamics. Rapid axial scanning can reproducibly and precisely translate that to several volumes per second depending on the number of Z positions required
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