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Deep tissue light delivery and fluorescence tomography with applications in optogenetic neurostimulation

机译：深层组织光传输和荧光层析成像及其在光遗传神经刺激中的应用

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摘要

Study of the brain microcircuits using optogenetics is an active area of research. This method has a few advantages over the conventional electrical stimulation including the bi-directional control of neural activity, and more importantly, specificity in neuromodulation. The first step in all optogenetic experiments is to express certain light sensitive ion channels/pumps in the target cell population and then confirm the proper expression of these proteins before running any experiment. Fluorescent bio-markers, such as green fluorescent protein (GFP), have been used for this purpose and co-expressed in the same cell population. The fluorescent signal from such proteins provides a monitory mechanism to evaluate the expression of optogenetic opsins over time. The conventional method to confirm the success in gene delivery is to sacrifice the animal, retract and slice the brain tissue, and image the corresponding slices using a fluorescent microscope. Obviously, determining the level of expression over time without sacrificing the animal is highly desirable. Also, optogenetics can be combined with cell-type specific optical recording of neural activity for example by imaging the fluorescent signal of genetically encoded calcium indicators.;One challenging step in any optogenetic experiment is delivering adequate amount of light to target areas for proper stimulation of light sensitive proteins. Delivering sufficient light density to a target area while minimizing the off-target stimulation requires a precise estimation of the light distribution in the tissue. Having a good estimation of the tissue optical properties is necessary for predicting the distribution of light in any turbid medium. The first objective of this project was the design and development of a high resolution optoelectronic device to extract optical properties of rats' brain tissue (including the absorption coefficient, scattering coefficient, and anisotropy factor) for three different wavelengths: 405nm, 532 nm and 635nm and three different cuts: transverse, sagittal, and coronal. The database of the extracted optical properties was linked to a 3D Monte Carlo simulation software to predict the light distribution for different light source configurations. This database was then used in the next phase of the project and in the development of a fluorescent tomography scanner. Based on the importance of the fluorescent imaging in optogenetics, another objective of this project was to design a fluorescence tomography system to confirm the expression of the light sensitive proteins and optically recording neural activity using calcium indicators none or minimally invasively. The method of fluorescence laminar optical tomography (FLOT) has been used successfully in imaging superficial areas up to 2mm deep inside a scattering medium with the spatial resolution of ~200micro m. In this project, we developed a FLOT system which was specifically customized for in-vivo brain imaging experiments.;While FLOT offers a relatively simple and non-expensive design for imaging superficial areas in the brain, still it has imaging depth limited to 2 mm and its resolution drops as the imaging depth increases. To address this shortcoming, we worked on a complementary system based on the digital optical phase conjugation (DOPC) method which was shown previously that is capable of performing fluorescent tomography up to 4mm deep inside a biological tissue with lateral resolution of ~50 microm. This system also provides a non-invasive method to deliver light deep inside the brain tissue for neurostimulation applications which are not feasible using conventional techniques because of the high level of scattering in most tissue samples. In the developed DOPC system, the performance of the system in focusing light through and inside scattering mediums was quantified. We also showed how misalignments and imperfections of the optical components can immensely reduce the capability of a DOPC setup. Then, a systematic calibration algorithm was proposed and experimentally applied to our DOPC system to compensate main aberrations such as reference beam aberrations and also the backplane curvature of the spatial light modulator. In a highly scattering sample, the calibration algorithm achieved up to 8 fold increase in the PBR.
机译：利用光遗传学研究大脑微电路是一个活跃的研究领域。与传统的电刺激相比，该方法具有一些优势，包括双向控制神经活动，更重要的是，在神经调节方面具有特异性。所有光遗传学实验的第一步是在靶细胞群中表达某些光敏离子通道/泵，然后在进行任何实验之前确认这些蛋白的正确表达。荧光生物标记，例如绿色荧光蛋白（GFP），已用于此目的，并在同一细胞群中共表达。来自此类蛋白质的荧光信号提供了一种监测机制，以评估随时间推移的光遗传视蛋白的表达。确认基因传递成功的常规方法是处死动物，缩回脑组织并对其切片，并使用荧光显微镜对相应的切片进行成像。显然，非常需要确定随时间的表达水平而不牺牲动物。同样，光遗传学可以与神经活动的细胞类型特异性光学记录相结合，例如通过对遗传编码的钙指示剂的荧光信号进行成像;;任何光遗传学实验中的一个挑战性步骤是向目标区域提供足够量的光以适当刺激光敏蛋白。在最小化脱靶刺激的同时向靶区域传递足够的光密度需要精确估计组织中的光分布。要预测光在任何混浊介质中的分布，必须对组织的光学特性进行良好的估算。该项目的第一个目标是设计和开发高分辨率的光电器件，以提取大鼠脑组织在三种不同波长（405nm，532nm和635nm）上的光学特性（包括吸收系数，散射系数和各向异性因子）和三种不同的切口：横向，矢状和冠状。提取的光学特性的数据库链接到3D蒙特卡洛模拟软件，以预测不同光源配置的光分布。然后，该数据库将用于项目的下一阶段以及荧光断层扫描仪的开发。基于荧光成像在光遗传学中的重要性，该项目的另一个目标是设计一种荧光层析成像系统，以确认光敏蛋白的表达并使用无钙或微创的钙指示剂光学记录神经活动。荧光层流光学层析成像（FLOT）方法已成功地用于在散射介质内部深达2mm的浅表层成像，其空间分辨率约为200微米。在此项目中，我们开发了专门针对体内脑成像实验定制的FLOT系统。虽然FLOT提供了一种相对简单且廉价的设计来对大脑浅表区域进行成像，但其成像深度限制为2 mm并且其分辨率随着成像深度的增加而下降。为了解决这个缺点，我们研究了一种基于数字光学相位共轭（DOPC）方法的互补系统，该系统先前已显示出能够在生物组织内部深达4mm的深度进行荧光层析成像，横向分辨率约为50微米。该系统还提供了一种非侵入性的方法来将光传送到脑组织内部深处，以进行神经刺激应用，由于大多数组织样本中的散射水平很高，因此使用常规技术无法实现这一目标。在已开发的DOPC系统中，系统在聚焦光通过散射介质和散射介质内部时的性能得到了量化。我们还展示了光学组件的不对准和缺陷如何极大地降低了DOPC设置的能力。然后，提出了一种系统的校准算法，并将其实验性地应用于我们的DOPC系统，以补偿主要像差，例如参考光束像差以及空间光调制器的背板曲率。在高度散射的样品中，校准算法的PBR最高可提高8倍。

著录项

  • 作者

    Azimipour, Mehdi.;

  • 作者单位

    The University of Wisconsin - Milwaukee.;

  • 授予单位 The University of Wisconsin - Milwaukee.;
  • 学科 Electrical engineering.;Engineering.
  • 学位 Ph.D.
  • 年度 2016
  • 页码 153 p.
  • 总页数 153
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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