Multiplexed Multiphoton Interrogation of Brain Connectomics

 描述（由申请人提供）：多光子激光扫描显微镜已经彻底改变了神经科学，因为它比传统的电生理学方法探测神经元过程的侵入性更小。在大多数情况下，这种成像仅限于在适度深度处观察单个荧光物质（标准双光子显微镜在脑组织中的深度穿透仅限于几百微米）。最近的建议，以扩大这一深度的渗透利用3光子激发。但为了避免因吸水而加热，采用了长波长，仅提供深红色荧光标记。我们建议通过提高多光子显微镜的复用能力和深度穿透来提高多光子显微镜的通用性。为此，我们将开发一种新型激光器设计，可以同时发射不同颜色的光，选择这些颜色以使用非简并（与传统简并相反）多光子成像实现2-或3-光子成像。这里的关键新奇是一种可调谐激光器，它可以根据需要发射一对跨越波长范围的颜色，避免水吸收，但其多种能量组合可以在整个可见光谱范围内激发各种流行的荧光传感器，如通道视紫红质，盐视紫红质，古视紫红质和GCaMP等。我们的激光器的功率和波长将能够实现深层组织（高达~ 2 mm）成像。多路复用将通过检测来自我们的多色可调谐高能激光器的所有非简并多光子组合的荧光来执行。我们将展示使用标记的小鼠脑组织的多路复用深层组织成像的概念验证。这将创造一个机会，以最小的组织损伤对深层结构如丘脑和海马（深度>1000 μm）进行成像，这是目前体内成像方法无法实现的。在拟议的目标的成功将创造新的途径神经科学研究，例如，例如，使成像的大型异构神经元集合的转基因小鼠品系，可以遗传标记不同的神经元群体，或促进电路询问实验涉及谷氨酸解开目前需要多个昂贵的超快激光。此外，由于新型激光源本质上是全光纤的，因此显微镜很容易适应于促进未来的内窥镜体内成像。

 DESCRIPTION (provided by applicant): Multiphoton laser scanning microscopy has revolutionized neuroscience since it is less invasive than traditional electrophysiology methods for probing neuronal processes. In most cases, such imaging is limited to the observation of a single fluorescent species at modest depths (the depth penetration of standard 2-photon microscopy in brain tissue is limited a few 100s of microns). Recent proposals to extend this depth penetration have made use of 3-photon excitation. But to avoid heating due to water absorption, long wavelengths were employed, providing access to only deep red fluorescent markers. We propose to improve the versatility of multiphoton microscopy by enhancing its multiplexing capacity and its depth penetration. To do this, we will develop a novel laser design that can emit light at different colors simultaneously, that are chosen to enable 2- or 3-photon imaging using non-degenerate (as opposed to traditional degenerate) multiphoton imaging. The key novelty here is a tuneable laser that emits, on demand, a pair of colors across the wavelength ranges that avoid water absorption but whose multiple combinations of energies sum up to excite a variety of popular fluorescent sensors, such as channelrhodopsins, halorhodopsins, archearhodopsin, and GCaMPs etc, across the entire visible spectrum. Our laser's power and wavelengths will enable achieving deep tissue (up to ~2mm) imaging. Multiplexing will be performed by detecting fluorescence from all the non- degenerate multiphoton combinations available from our multicolour tunable high energy laser. We will demonstrate the proof-of-concept of multiplexed deep tissue imaging using labelled mouse-brain tissue. This would create an opportunity to image deep structures such as the thalamus and hippocampus (depth>1000 μm) with minimal tissue damage, which have not been able to be achieved by current in vivo imaging methods. Success in proposed goals would create new avenues for neuroscience research, such as, for example, enabling imaging of large heterogeneous neuronal ensembles of transgenic mouse lines that can genetically label distinct neuronal populations, or facilitating circuit interrogation experiments involving glutamate uncaging that currently require multiple costly ultrafast lasers. Moreover, since the novel laser source is all-fiber in nature, the microscope is readily adaptable for facilitating future endoscopc in vivo imaging.