基于μLED的光遗传学可植入光极研究

	基于μLED的光遗传学可植入光极研究
其他题名	Implantable Optrodes based on μLED for Optogenetics
	岳森
学位类型	硕士
导师	王守岩
	2015
学位授予单位	中国科学院大学
学位专业	机械工程
关键词	神经接口光遗传学 led 神经调控
摘要	神经接口指人或动物的神经系统与外界进行信息交互的通道。随着神经工程科学的不断进步，以电生理学为基础的神经接口作为神经系统疾病或损伤的主要治疗手段，获得了较为广泛的发展与应用。光遗传学的出现与发展进一步丰富了神经接口的类别，并且解决了传统的电生理学神经接口所面临的两大问题——神经调控方面只能实现刺激作用，靶向方面刺激区域较大且目标不可控。光遗传学利用基因工程技术，实现了对特定目标类型神经的刺激或者抑制两种调控，且调控时间精度可达毫秒级别，为神经调控的作用机理、不同神经环路之间的作用与连接机制等研究创造了前提。激光技术发展较为成熟，传统光极多以激光作为光源，由光纤制作而成。但一方面激光器成本较高，光纤较为脆硬易损坏且在垂直于轴向的平面内光照面积小，并对实验体容易产生束缚影响实验结果；另一方面随着LED 技术不断发展成熟，出现了具有较大功率且尺寸微小的LED，故近年来以LED 作为光源的光极研究已经成为了新的光极研究方向。本文面向核团功能、神经环路调控等研究，以向光遗传学实验，尤其是光遗传学行为学实验提供一种更为有效、方便且成本较低易实现批量生产的全新光遗传学工具为目标，研究制作了两种基于LED 的光遗传学光极，其中一种光极具有神经刺激功能，另一种具有神经刺激与神经电信号记录功能，并对光极进行了较为全面的性能测试与功能验证。首先从光源、材料、结构尺寸这三个方面设计了光极，光极主要由聚对二甲苯覆盖膜、LED、聚酰亚胺基底这三个部分构成，且两种光极的尺寸分别控制在500μm 和600μm 之内。提出并使用了FPC 工艺与MEMS 工艺结合的光极制作方案，具有神经刺激功能的光极使用了单层板作为基底，具有神经刺激与记录功能的光极使用了三层板作为基底。制作过程中自主设计了“低温微焊接平台”。具有神经刺激与记录功能光极的镀膜过程使用了简易手工覆掩模方法，降低了生产成本，减小了制作周期。然后对制作完成的光极进行了电流电压特性曲线测试、发光功率测试、记录点阻抗测试、LED 表面温度测试、寿命测试。确定了光极连接本课题组自主研发的无线刺激器时的最大发光功率达到了3.9mW，最大有效作用面积达到了 0.98mm2。记录点阻抗在1kHz 频率下为1.14kΩ，光极LED 表面在电生理实验所用的刺激模式范围内最大的温度变化低于1.4℃，光极在生物体内的神经刺激功能有效期可以达30 天以上，神经电信号记录功能的有效期可达20 天。最后进行了三次在体实验验证了光极的神经刺激功能与神经电信号记录功能。麻醉状态下小鼠实验中本文光极诱发与激光光纤类似的小鼠皮层响应，初步验证了本文光极的神经刺激功能。自由移动状态下小鼠实验中结合无线刺激器，成功实现了对小鼠行为的无线控制，进一步更具说服力的验证了本文光极的神经刺激功能。神经电信号记录实验中本文光极成功记录到了轻微麻醉状态下小鼠次级运动皮层的局部场电位信号。本文研制的LED 光极具有光照面积大、植入方便、可一次性长期植入等特点，可实现神经核团的功能调控与神经信号记录，与无线刺激器结合形成微型可移动神经调控系统，为核团功能、神经环路调控等研究提供了更为有效、方便的全新光遗传学工具。
其他摘要	The channel been used for information exchange between human or animal’s nervous system and the outside world is called neural interface (NI). The electrophysiological neural interface (ENI), as the main treatment for nervous system diseases or injury, was applied in clinical research widely. However, there are two unavoidable problems of the ENI: in the aspect of neural regulation, ENI cannot inhibit nerve; in the aspect of target, the stimulating area of ENI is large and the nervous type is non-optional. On the other hand, optogenetics neural interface (Optrode) is a good alternative technique. Optrode is able to regulate neurons (stimulation or inhibition) through light and photosensitive protein. Furthermore, the temporal resolution of the regulation can achieve to millisecond-timescale, which provides the precondition for the research of the neural regulation mechanism and the interreaction of the different neural circuits. Most traditional optrodes use laser as light source and are made of optical fibers. There are several drawbacks of laser system. Laser devices are expensive and fibers can be fragile. Besides, the effective illumination area of the optical fibers in the plane perpendicular to the axial is small. Moreover, the fiber connecting to the laser device can restrain the experimental animals. In the recent years, along with the development of high-power and microsize LEDs, more and more optrodes are designed with usage of LEDs as light source. This study orienting neural population in nuclei and neural circuits research aims to provide the optogenetics experiments, especially the behavioral research a more efficient、easier to operate、 easier to be bulk produced and brand new optogenetics tool. Two kinds of optrodes are developed in this study. One has function of neural stimulation, the other has function of both neural stimulation and electrical recording. The optrodes’s properties are tested in the round relatively and the stimulation function of the optrodes is demonstrated. Firstly, the optrodes are designed in the aspects of light source, material and structural size. Main components of the optrodes include parylene coating layer, μLED chip and base layer. The width of the two optrodes is within 500μm and 600μm respectively. Manufacturing process of the optrodes combining FPC process and MEMS technology was proposed and applied. Single-layer substrate is used to form the base layer of the optrode with neural stimulation function and three-layer substrate is applied in the optrode with neural stimulation and electrical recording function., A “low temperature micro-welding platform” is set up to solder the μLED during the manufacturing process. Manually pasting simple mask is applied in the coating process, which keeps the cost down and reduces the production cycle. Secondly, the optrodes’s properties are tested in terms of current-voltage characteristics, optical output power, impedance of the recording contacts, thermal property and operating life. The maximum optical output power is 3.9mW and the effective illumination area is a 0.98mm2 when the optrode is driven by the wireless stimulator developed by our group. Impedance of the recording contacts is about 1.14 kΩ at 1kHz. The maximum temperature rise of the μLED surface is below 1.4℃. The neural stimulation function is still available after 30 days in vivo and the electrical recording function’s validity period is about 20 days. Moreover, the optrode’s neural stimulation and recording function is demonstrated in vivo. An electrophysiological signal recording experiment was set up and robust spiking activities of the expressing Channelrhodopsin-2 neurons in the entire cortex of a dopey mice caused by optrode stimulation were recorded. Then a behavioral experiment was set up and obvious behavior change was observed when light stimulation was applied to the expressing of Channelrhodopsin-2 neurons in the secondary motor cortex of a freely-moving mice. At last, spontaneous local field potentials in the secondary motor cortex of a slightly dopey mice were recorded by the optrode. The advantages of the optrode demonstrated in this study include large effective illumination range, flexibility of implantation and long-term implantation at one time. The optrode and wireless stimulator together form a micro-based removable neural regulation system, which provide neural population in nuclei or neural circuits research a novel optogenetics tool.
语种	中文
文献类型	学位论文
条目标识符	http://ir.ciomp.ac.cn/handle/181722/49291
专题	中科院长春光机所知识产出
推荐引用方式 GB/T 7714	岳森. 基于μLED的光遗传学可植入光极研究[D]. 中国科学院大学,2015.

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