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等领域联系在一起。在光遗传学的帮助下，有可能通过用光照亮转基因细胞来修饰具有光敏离子通道的细胞。将所谓的视蛋白引入细胞膜(如神经元、心脏或耳蜗细胞)，使在特定波长的光刺激下激活或去激膜电位成为可能。这使得有可能选择性地打开带正电荷或带负电荷的离子的通道，以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障碍是光刺激的类型。目前，这种刺激是通过使用光纤或微LED的平面照明来实现的，这导致了对细胞群的刺激，而不是期望的单个细胞激发。因此，空间信息会丢失。另一个目前无法实现的需求是不同细胞和细胞类型的三维和同时光刺激，特别是通过使用多个波长。因此，本领域的现状表明需要一种方法来塑造定义明确的波前，以便灵活地传递三维图案化的强度分布，从而同时刺激各种具有细胞分辨率的光基因修饰细胞和细胞类型，用于体内应用。这些限制可以通过使用衍射和折射光学来克服。因此，本方案的目的是在单模光纤的尖端实现光学系统。利用这个系统，可以显示各种波长相关的强度图案，从而以细胞分辨率对单个光基因修饰细胞或细胞群进行三维刺激。为了产生这些图案而单独对波前进行整形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，利用微米和纳米加工方法在光纤尖端制造针对特定应用领域的完整系统(例如，神经元或耳蜗模拟)。通过这种波长相关的波前整形和光束图案化的方法，为光遗传微刺激探索细胞网络和加深对细胞通信的理解创造了新的机会。此外，这项研究还为开发新的生物生物治疗方法铺平了道路。

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.