All-Optical Histology for 3-D Cortical Reconstructions

描述(申请人提供)：超短激光脉冲作为实验室工具的出现为探索和操纵神经系统的解剖和功能开辟了新的机会。超短脉冲是驱动生物分子对光的非线性吸收的基本手段，它导致了一个局域的激发区域，构成了双光子扫描显微镜的基础。最近，非线性光学吸收已被用作一种可靠和可重复地在脑组织中创建微米级消融的手段，同时最大限度地减少侧枝和热损伤。这些消融可以用作全光学组织学的驱动技术，使整个大脑以微米分辨率进行解剖成像。这些消融也可以用来扰乱新皮质血流，作为探测正常和病变组织的一种手段。然而，还需要更多的努力来使用和推进多光子消融和成像技术的混合，作为一种能够研究神经元和血管结构的手段。 我们提出的研究涉及非线性光学和解剖学的融合。 。推进多光子消融与成像的结合，建立以全光学为基础的组织学作为标准的解剖学工具。这包括参数的优化和联合消融和成像软件的进步。 。重建大鼠大脑皮质触觉感觉区的细胞体和血管位置。这些信息将被用来评估基本的结构参数，包括横向和径向细胞密度之间的相关性，以及基本的代谢参数，如血管系统内的相互连接和体细胞相对于毛细血管的分布。 这两个目标，一个是技术的，一个是科学的，内在地联系在一起，将并行进行。 这些拟议的进展将为组织学自动化提供一种新的工具，这是理解大脑功能的基础。我们将向生物医学界提供这一工具。所提出的模型系统可能会大大提高我们对大脑建筑学的大尺度结构的理解。

DESCRIPTION (provided by applicant): The advent of ultrashort laser light pulses as laboratory tool has opened up new opportunities to probe and manipulate anatomy and function in nervous systems. Ultrashort pulses are the essential means to drive the nonlinear absorption of light by biomolecules, which leads to a localized region of excitation and forms the basis of two-photon scanning microscopy. More recently, nonlinear optical absorption has been exploited as a means to reliably and reproducibly create micron-sized ablations in brain tissue with a minimum of collateral and thermal damage. These ablations can be use as the driving technology in an all-optical histology, which allows anatomy to be imaged with micrometer resolution throughout the entire brain. These ablations may also be used to perturb neocortical blood flow as a means to probe normal and diseased tissues. Yet much additional effort is required to use and advance the mixture of multi-photon ablation and imaging techniques as a means to enable studies of neuronal and vascular architectonics. Our proposed research concerns the confluence of nonlinear optics and anatomy. . Advance the mixture of multi-photon ablation and imaging to establish all-optical based histology as a standard anatomical tool. This includes the optimization of parameters and the advancement of software for combined ablation and imaging. . Reconstruct cell soma and vasculature positions throughout the vibrissa sensory areas in rat cortex. This information will be used to evaluate essential architectonic parameters, including correlations among cell densities in lateral as well as radial directions, as well as essential metabolic parameters, such as the interconnectivity within the vasculature and the distribution of somata relative to capillaries. These two goals, one technical and the other scientific, are intrinsically linked and will proceed in parallel. The proposed advancements will provide a novel tool for the automation of histology, which underlies an understanding of brain function. We will make this tool readily available to the biomedical community. The proposed model system may substantially improve upon our understanding of the large-scale structure of brain architectonics.