Head-mounted Photoacoustic Imaging of Deep-brain Neural Activities in Freely Behaving Animals

自由行为动物深脑神经活动的头戴式光声成像

• 批准号: 9924909
• 负责人: Vladislav Verkhusha
• 金额: $ 200.72万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9924909
• 关键词: Animal Model; Animals; Area; Behavioral; Blood; Brain; Brain imaging; Brain region; Calcium; Collaborations; Communities; Detection; Disease; Electrodes; Engineering; Fluorescence Resonance Energy Transfer; Focused Ultrasound; Functional Imaging; Goals; Head; Health; Hemoglobin; Hippocampus (Brain); Image; Imaging Techniques; Imaging technology; Knowledge; Measures; Medicine; Membrane; Microscopy; Midbrain structure; Molecular Probes; Monitor; Mus; Neurons; Neurosciences; Optical Methods; Optics; Pathologic Processes; Penetration; Performance; Phytochrome; Protein Engineering; Publications; Reporting; Research Personnel; Resolution; Scanning; Series; Signal Transduction; Speed; System; Techniques; Technology; Texas; Time; Ultrasonic Transducer; Ultrasonic wave; Universities; Variant; Water; Yang; awake; base; calcium indicator; college; design; experience; experimental study; fluorescence imaging; head mounted display; hemodynamics; imaging modality; imaging system; improved; in vivo; innovation; miniaturize; mouse model; novel; optical imaging; photoacoustic imaging; relating to nervous system; scaffold; sensor; two photon microscopy; voltage

Abstract To capture the normal brain functions, it is critically important to record the neural activities in freely-behaving animals, with high resolution, high speed, and high throughput. So far, our knowledge about neuronal activity of awake animals mainly relies on electrode recording, which, however, is invasive. Optical imaging techniques have been widely used to visualize activity of a large number of neurons in mouse models using fluorescent membrane voltage or calcium indicators. However, limited by the penetration depth (<1 mm), it is technically challenging to record the brain functions at depths beyond the cortex layer, such as in the hippocampus. A new large-scale recording technology with high resolution and deep penetration in freely-behaving animals would be of great utility for the neuroscience community. Photoacoustic microscopy (PAM) is a promising candidate for this task due to the relatively deep penetration of ultrasound waves. However, PAM has not been able to image neural activities of freely-moving animals, because (1) it is challenging to miniaturize the imaging system, (2) there lacks calcium or voltage probes that can report neural activities in deep brain, and (3) photoacoustic detection sensitivity of molecular probes is traditionally low due to the strong background signals from blood. In this proposal, we plan to overcome all of the above technical obstacles and develop head-mounted photoacoustic imaging of deep-brain neural activities in freely-behaving animals. To achieve this goal, we will follow a three-aim strategy. (1) In Aim 1, we will develop a miniaturized head-mounted PAM (HM-PAM) system. Several key innovations will reduce the system footprint to 1 cm3. HM-PAM will achieve a penetration depth of ~3.0 mm with ~10−15 µm resolution, which is deeper than that with pure optical microscopy. (2) In Aim 2, we will develop novel near-infrared photoswitchable genetically-encoded calcium indicators (NIR-PS-GECIs) as PA probes. We will engineer and optimize a new class of NIR-PS-GECIs based on photoacoustic Förster resonance energy transfer (FRET-PA). We have proven that the photoswitching, which enables differential PA imaging, is currently one of the most effective approaches to enhance the PA detection sensitivity. We will thus apply fast photoswitching of the NIR-PS-GEICs to enhance the detection sensitivity of HM-PAM. (3) In Aim 3, the optimized HM-PAM and advanced NIR-PS-GECIs will be thoroughly characterized and validated in dissociated neurons and in vivo. We will perform proof-of-concept experiments of deep-brain neural activity in freely-behaving animals. In summary, our proposal will build on the innovations of the first head-mounted PAM system, the first NIR photoswitching GECIs, and the differential FRET- PA imaging that rejects the strong background blood signals. This enabling technology will provide a powerful toolkit for studying neural activities in health, disease, and behavioral states.

摘要 为了捕捉正常的大脑功能，记录自由行为中的神经活动至关重要 动物，具有高分辨率，高速度和高通量。到目前为止，我们对神经元活动的了解 清醒的动物主要依赖于电极记录，然而，这是侵入性的。光学成像技术具有 已经广泛用于使用荧光膜在小鼠模型中可视化大量神经元的活动 电压或钙指示器。然而，受穿透深度（<1 mm）的限制， 记录大脑皮层以外的深层功能，比如海马体。新的大规模 在自由行为的动物中，高分辨率和深穿透的记录技术将具有很大的实用性 为神经科学界。光声显微镜（PAM）是这项任务的一个有前途的候选人，由于 超声波相对较深的穿透力。然而，PAM还不能对神经活动进行成像 自由移动的动物，因为（1）它是具有挑战性的成像系统，（2）缺乏钙 或电压探针，可以报告脑深部的神经活动，以及（3）光声检测灵敏度， 由于来自血液的强背景信号，分子探针传统上是低的。在这份提案中，我们计划 为了克服上述所有技术障碍并开发头戴式脑深部光声成像， 自由行为动物的神经活动。为了实现这一目标，我们将采取三个目标的战略。(1)在目标1中， 我们将开发一种小型化的头戴式PAM（HM-PAM）系统。几个关键的创新将减少 系统占地面积为1 cm 3。HM-PAM将实现约3.0 mm的穿透深度，分辨率约为10−15 µm， 比纯光学显微镜观察的更深。(2)在目标2中，我们将开发新的近红外光开关 基因编码的钙指示剂（NIR-PS-GECIs）作为PA探针。我们将设计和优化一个新的类 基于光声Förster共振能量转移（FRET-PA）的NIR-PS-GECIs。我们已经证明了 光开关技术是目前最有效的光放大成像技术之一， 提高PA检测灵敏度。因此，我们将应用NIR-PS-GEIC的快速光开关来增强NIR-PS-GEIC的性能。 HM-PAM检测灵敏度(3)在目标3中，优化的HM-PAM和先进的NIR-PS-GECIs将是 在分离的神经元和体内彻底表征和验证。我们将进行概念验证 在自由行为的动物身上进行的脑深部神经活动实验。总括而言，我们的建议将建基于 创新的第一个头戴式PAM系统，第一个近红外光开关GECIs，和差分FRET- PA成像，拒绝强背景血液信号。这项使能技术将提供强大的 研究健康、疾病和行为状态下神经活动的工具包。