Wavefront shaping by computer generated volume holograms for optogenetic applications (HoloGen)

光遗传学是一个快速发展的领域，它将遗传学和光子学联系在一起。在光遗传学的帮助下，可以通过用光照射细胞来修饰具有光敏离子通道的基因修饰细胞。将所谓的视蛋白引入细胞膜（例如在神经元、心脏或耳蜗细胞中）使得在特定波长的光刺激下激活或灭活膜电位成为可能。这使得有选择地为带正电荷或负电荷的离子打开通道成为可能，从而在细胞中诱导动作电位，从而控制细胞的电活动。在期望的目的地有效利用光的一个障碍是光刺激的类型。目前，这种刺激是通过光纤或微型led的平面照明来实现的，这导致细胞集合的刺激，而不是期望的单个细胞激发。因此，空间信息丢失。另一个目前无法实现的需求是对不同细胞和细胞类型进行三维和同步的光学刺激，特别是通过使用多个波长。因此，目前的技术状况表明需要一种方法来塑造明确定义的波前，用于三维图案强度分布的灵活光传递，用于同时刺激各种光基因修饰细胞和具有细胞分辨率的细胞类型，用于体内应用。这些限制可以通过使用衍射光学和折射光学来克服。因此，本提案的目的是在单模光纤的尖端上实现光学系统。该系统显示了各种波长相关的强度模式，导致单个光基因修饰细胞或具有细胞分辨率的细胞集合的三维刺激。为了产生这些图案，单独塑造波前的信息被编码在计算机生成的体全息图（CGVH）中。首先，对由CGVH和微透镜组成的光学系统进行了数值计算。然后，利用微纳米制造方法在光纤尖端制造出用于特定应用领域（例如神经元或耳蜗模拟）的完整系统。通过这种波长依赖波前整形和波束模式的方法，为光遗传微刺激探索细胞网络和加深对细胞通信的理解提供了新的机会。此外，这项研究为生物有机体的新治疗方法的发展铺平了道路。

Optogenetics is a rapidly evolving field, which among other topics connects the fields of genetics and photonics. With the help of optogenetics it is possible to modify genetically modified cells with light-sensitive ion channels by illuminating the cells with light. The introduction of so-called opsins into a cell membrane (e.g. in neurons, heart or cochlear cells) makes it possible to activate or deactivate membrane potentials under stimulation with light of a specific wavelength. This makes it possible to selectively opening channels for positively or negatively charged ions, to induce an action potential in the cell and thereby control the electrical activity of the cell.An obstacle to the efficient use of light at the desired destination is the type of optical stimulation. Currently, this stimulation is realized by a planar illumination with optical fibers or micro-LEDs, which leads to the stimulation of cell ensembles in contrast to a desired single cell excitation. Therefore, spatial information is lost. Another currently not realizable need is the three-dimensional and simultaneous optical stimulation of different cells and cell types, especially by using multiple wavelengths. Hence, the state of the art indicates the need of a method to shape well-defined wave fronts for a flexible light delivery of three dimensionally patterned intensity distributions for the simultaneous stimulation of various optogenetically modified cells and cell types with cellular resolution for in vivo applications. These limitations can be overcome by the use of diffractive and refractive optics.Thus, the aim of this proposal is to realize an optical system on the tip of a single mode fiber. With this system various wavelength-dependent intensity patterns are displayed resulting in a three-dimensional stimulation of single optogenetically modified cells or cell ensembles with cellular resolution. The information to individually shape the wave front in order to generate these patterns is encoded in a computer-generated volume hologram (CGVH). At first, the optical system consisting of a CGVH and microlenses is numerically calculated. Then, the complete system is fabricated for the particular field of application (e.g. neuron or cochlear simulation) on the tip of an optical fiber utilizing micro- and nanofabrication methods. By this approach of wavelength dependent wavefront shaping and beam patterning, new opportunities are generated for the optogenetic micro stimulation to explore cellular networks and to deepen the understanding of cellular communication. Furthermore, this research paves the way for the development of novel therapeutic approaches of biological organisms.