Multiplexed Multiphoton Interrogation of Brain Connectomics

 描述(申请人提供)：多光子激光扫描显微镜使神经科学发生了革命性的变化，因为它在探测神经元突起方面比传统的电生理学方法侵入性小。在大多数情况下，这种成像仅限于在中等深度观察单一荧光物种(标准双光子显微镜在脑组织中的深度穿透限于几个微米)。最近提出的延长这种深度穿透的建议都利用了三光子激发。但为了避免因水吸收而发热，使用了长波长，只提供了访问深红色荧光标记的途径。我们建议通过提高多光子显微镜的多路复用能力和深度穿透能力来提高其通用性。为此，我们将开发一种新的激光设计，它可以同时发射不同颜色的光，选择使用非简并(与传统简并)多光子成像相反的方式来实现2光子或3光子成像。这里的关键创新之处在于一种可调谐的激光器，它可以根据需要在波长范围内发射一对颜色，避免吸收水分，但其多种能量组合加在一起，可以在整个可见光谱范围内激发各种常用的荧光传感器，如通道视紫红质、卤素紫质、先天紫质和GCaMP等。我们的激光功率和波长将实现深层组织(高达~2 mm)的成像。多路复用将通过检测我们的多色可调谐高能激光器提供的所有非简并多光子组合的荧光来执行。我们将使用标记的小鼠脑组织来演示多路复用深层组织成像的概念验证。这将创造一个机会，以最小的组织损伤成像丘脑和海马体(Depth&gt；1000μm)等深层结构，这是目前的活体成像方法所无法实现的。拟议目标的成功将为神经科学研究创造新的途径，例如，使转基因小鼠品系的大型异质神经元集合能够在基因上标记不同的神经元群体，或者促进涉及谷氨酸去除的电路询问实验，目前需要多个昂贵的超快激光。此外，由于新型激光光源本质上是全光纤的，该显微镜很容易适应未来的内窥镜活体成像。

 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.