Abstract
Studying neural connections and activities in vivo is fundamental to understanding brain functions. Given the cm-size brain and three-dimensional neural circuit dynamics, deep-tissue, high-speed volumetric imaging is highly desirable for brain study. With sub-micrometer spatial resolution, intrinsic optical sectioning, and deep-tissue penetration capability, two-photon microscopy (2PM) has found a niche in neuroscience. However, the current 2PM typically relies on a slow axial scan for volumetric imaging, and the maximal penetration depth is only about 1 mm. Here, we demonstrate that by integrating a gradient-index (GRIN) lens and a tunable acoustic GRIN (TAG) lens into 2PM, both penetration depth and volume-imaging rate can be significantly improved. Specifically, an ∼ 1-cm long GRIN lens allows imaging relay from any target region of a mouse brain, while a TAG lens provides a sub-second volume rate via a 100 kHz ∼ 1 MHz axial scan. This technique enables the study of calcium dynamics in cm-deep brain regions with sub-cellular and sub-second spatiotemporal resolution, paving the way for interrogating deep-brain functional connectome.
Highlights
Brain is arguably the most complex organ, consisting of three-dimensional (3D) neural circuits that may span millimeter to centimeter with continuous spiking activities
Rod-like gradient-index (GRIN) lens, functions of neural circuits at several mm depth can be studied, by either single-photon [10,11] or two-photon endoscopy [12]. The former allows high-speed imaging of neural dynamics [13], the lack of optical sectioning and the vulnerability to scattering hinder its application in 3D imaging of neural circuits distributed across multiple layers
The scanned laser beam was relayed by the GRIN lens in deep mouse brain to excite two-photon fluorescence, which was epi-collected by the same GRIN lens, objective, tunable acoustic gradient-index (TAG) lens, the tube lens, and the dichroic beamsplitter to a photomultiplier tube (PMT) module (H7422A-40, Hamamatsu)
Summary
Brain is arguably the most complex organ, consisting of three-dimensional (3D) neural circuits that may span millimeter to centimeter with continuous spiking activities. Rod-like gradient-index (GRIN) lens, functions of neural circuits at several mm depth can be studied, by either single-photon [10,11] or two-photon endoscopy [12] The former allows high-speed imaging of neural dynamics [13], the lack of optical sectioning and the vulnerability to scattering hinder its application in 3D imaging of neural circuits distributed across multiple layers. Our dual GRIN lens two-photon endoscopy system allows in vivo imaging of neural circuits in centimeter-depth brain regions with high-contrast and sub-second volume rate, and has great potential for studying transient responses of 3D neural circuits with millisecond temporal resolution [24]
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