The Development and Use of Elastic Resonators and Optogenetics to Study Locomotion in Small Soft-bodied Animals

具有流体静力骨骼的陆生动物通过协调它们柔软的身体如何压缩和变形以与基质相互作用来解决复杂的运动问题。以前的研究通过对Manduca sexta毛虫等大型动物的研究，在了解这些动物如何在各种各样的基质上灵活地进行这一动作方面取得了进展。然而，由于没有足够的工具来同时记录动物的行为，从神经肌肉、行为和驱动的角度，通过高时空分辨率的力，进展受到限制。最近，一种技术，弹性谐振腔应力干涉显微镜（ERISM），为了允许在细胞水平上的迁移力成像利用光的干涉在一个可变形的弹性腔。这项技术在两个金镜子之间使用弹性体，其长度决定了腔内共振光的波长。当细胞产生力时，腔体变形，从而减少了总长度，从而改变了局部共振波长。因此，对于每个给定的空腔长度，基底上的推力和拉力可以通过检查局部共振中的这种位移来确定。直到最近，这项技术还没有解决超过0.5 Hz区域的必要时间分辨率，然而，这项技术的发展是为了使用更少的波长，以便允许在10 Hz时间范围内的力成像-尽管这项技术仍然局限于记录细胞力。该项目旨在开发ERISM，利用弹性模量较大（20-60千帕）的弹性体记录小动物的力，并将其与荧光显微镜相结合，同时进行力测量和荧光活性测量。发展这项技术将使我们能够观察软体动物产生的力。其中一种动物，黑腹果蝇幼虫，通过使用Gal-4无人机系统，具有几乎无与伦比的遗传控制历史。这种动物将允许我们通过基因工具来研究肌肉的同时收缩，例如在其每块肌肉中表达荧光蛋白，或者直接在其神经系统中表达遗传编码的钙指标（GECIs）来观察神经肌肉活动。这种遗传易感性也允许我们直接在神经系统内的特定细胞中表达离子载体——允许光遗传学控制动物运动行为的特定方面。这些幼虫的生长规模也很有趣，在短短几天的时间里，它们从最大的细胞长到最小的动物细胞。这意味着他们必须用同一个身体来解决微观和宏观领域的问题。因此，本研究旨在开发双波长ERISM，以允许利用黑颊果蝇的完整遗传工具包，这对于理解软体动物的普通运动行为非常重要，但也可能为黑颊果蝇疾病模型的行为表型提供见解-允许对帕金森模型中发现的受损神经肌肉驱动进行动态研究。

Terrestrial animals with hydrostatic skeletons solve complex motor problems by orchestrating how their soft bodies compress and deform in order to interact with the substrate. Previous studies have made progress in understanding how these animals perform this action flexibly over a wide variety of substrates by researching large animals such as Manduca sexta caterpillars. However, progress has been limited by the insufficient tools to simultaneously record the animal's behaviour from a neuromuscular, behavioural and actuation through forces perspective with high spatiotemporal resolution. Recently, a technique, elastic resonator stress interference microscopy (ERISM), was developed in order to allow for the imaging of migratory forces on a cellular level using the interference of light in a deformable elastic cavity. This technique uses an elastomer between two gold mirrors, the length of which determines the resonant light wavelengths within this cavity. As cells produce forces the cavity deforms, thereby decreasing the overall length, thereby shifting the local resonant wavelengths. Thus, for each given cavity length, the pushing and pulling forces on the substrate can be determined by examining this shift in local resonance. Until recently, this technique did not have the requisite time resolution for resolving beyond the 0.5 Hz region, however, this technique was developed to make use of fewer wavelengths in order to allow for imaging of forces within the 10 Hz timeframe - although this technique is still limited to recording cellular forces. This project aims to develop ERISM to allow for recording forces from small animals by making use of an elastomer with a greater elasticity modulus (20-60 kPa) and to combine it with epifluorescence microscopy to allow for simultaneous force measurement and fluorescence activity measurement. Developing this technique will allow us to observe the forces produced by soft bodied animals. One such animal, Drosophila melanogaster larvae, has a history of almost unrivalled genetic control through use of the Gal-4 UAS system. This animal will allow us to study the simultaneous contraction of muscles via genetic tools such as expressing fluorescent proteins in each of its muscles or by expressing genetically encoded calcium indicators (GECIs) directly in its nervous system to observe neuromuscular activity. This genetic tractability also allows us to express ionophores directly in the specific cells within the nervous system - allowing for optogenetic control of specific aspects of the animal's locomotor behaviour. These larvae also grow within an interesting scale, growing from the size of the largest of cells to the size of the smallest of animals over the course of mere days. This means they have to solve both micro and macro domain problems using the same body. Thus, this study aims to develop two-wavelength ERISM to allow for the utilisation of the full genetic toolkit of Drosophila melanogaster and will be important in understanding both ordinary locomotor behaviour of soft-bodied animals, but also may offer insights into the behavioural phenotypes of Drosophila melanogaster disease models - allowing for a dynamic study of impaired neuromuscular actuation found in Parkinson's models.