High-Speed, wide field fluorescent imaging of cortex in freely moving animals

描述（由申请人提供）：对清醒，自由运动的动物的神经元活动的研究是“正常”神经元功能的最具代表性的观点。麻醉药和约束对神经生理学和相关动物行为有深远的影响。实时神经元事件处理的研究需要具有高时间带宽（> 500hz）的记录方法和检测生理相关事件（即电压变化）的能力。目前，只有单个神经元单元、多单元活动和场电位的微电极记录具有足够的时间分辨率和便携性，可以记录自由运动动物的神经元活动。该项目将生产三个微型显微镜/成像系统，可以头戴，并允许光学记录自由运动大鼠神经元膜电压的变化。在资助的为期两年的ARRA项目中，开发了两个原型设备和两个图像传感器，用于收集荧光电压染料信号的宽视场图像序列。目前的应用程序将完成制造和操作两种不同的风格，头挂，高速，荧光显微镜。这包括设计和制造两种不同的最终确定的成像传感器，每个传感器的定制光学元件，稳定的照明光源和形状适合头骨支架的显微镜体。本文提出的研究还将建立和测试一种新的荧光显微镜设计，该设计将通过消除45°二向色镜来降低传统显微镜的高度。我们还将缩小我们目前的相机控制和记录系统，使其完全包含在老鼠的背包中。这个背包将包含一个现场可编程门阵列，热交换器/冷却剂泵，SD存储器和一个完全自主运行显微镜的电池。最后，这些仪器将在大鼠体感皮层（桶状皮质）的成像研究中进行测试。将在自由运动的大鼠身上研究皮层对物体辨别的反应。这些研究将系统地设计每个组件，以最大限度地提高激发光传输、荧光灯收集和记录速度，同时最大限度地减少重量体积、能源使用和热量产生。该设备将能够记录快速（1000帧/秒）荧光图像序列的bbbb2平方毫米的皮质区域，并检测微小的变化？/ F(0.1%)。该装置体积小（头戴式小于1平方厘米），重量轻（5克），可以安装在自由移动的老鼠头上。该设备旨在作为能够将人类神经元活动实时转化为行动的设备的先驱，光学大脑机器假肢。

DESCRIPTION (provided by applicant): The study of neuronal activity in awake, freely moving animals is the most representative view of 'normal' neuronal function. Anesthetics and restraint have profound effects on neuronal physiology and the correlate animal behavior. The study of real time neuronal event processing requires recording methods with high temporal bandwidth (>500 hz) and the ability to detect physiologically relevant events (i.e. voltage changes). Currently only microelectrode recording of single neuronal units, multiunit activity and field potentials have sufficient temporal resolution and portability to allow recording of neuronal activity in freely moving animals. The proposed project will produce three miniature microscope/imaging systems which can be head-mounted and will allow the optical recording of changes in membrane voltage of neurons in freely moving rats. During the funded two-year ARRA project, two prototype devices and two image sensors were developed that collect wide field image sequences of fluorescent voltage dye signals. The current application will complete the fabrication and operationalize two different style, head-mountable, high-speed, fluorescence microscopes. This includes the design and fabrication of two different finalized, imaging sensors, custom optics for each, a stabilized illumination source and form fit microscope bodies with skull mounts. The studies proposed herein will also build and test a novel fluorescence microscope design that will reduce the height of the traditional microscope by eliminating the 45� dichroic mirror. We will also miniaturize our current camera control and recording system to be fully contained in a back pack on the rat. This back pack will contain a field programmable gate array, heat exchanger/coolant pump, SD memory and a battery to run the microscope fully autonomously. Finally, the instruments will be tested in imaging studies of the rat somatosensory cortex (barrel cortex). Cortical responses to object discrimination will be studied in freely moving rats. These studies will systematically engineer each component to maximize excitation light delivery, fluorescent light collection and recording speed while minimizing weight volume, energy usage and heat generation. The device will be capable of recording rapid (1000 fps) fluorescent image sequences of a >2 mm2 area of cortex and detect small changes in ?F/F (0.1%). The device will be small (<1 cm2 head mounted) and light weight enough (5 g) to be mounted on the head of a freely moving rat. The device is intended as a precursor to devices that can translate neuronal activity in humans into actions in real time, optical brain machine prosthetic.