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

拥有静水骨骼的陆地动物通过协调它们的软体如何压缩和变形来解决复杂的运动问题，以便与底物相互作用。以前的研究通过研究大型动物，如六色毛虫，在了解这些动物如何在各种底物上灵活地执行这一行为方面取得了进展。然而，由于没有足够的工具通过力的高时空分辨率从神经肌肉、行为和驱动的角度同时记录动物的行为，这一进展受到了限制。最近，发展了一种名为弹性谐振器应力干涉显微镜(ERISM)的技术，以便利用可变形弹性腔中的光干涉在细胞水平上对迁移力进行成像。这项技术在两个金镜之间使用弹性体，弹性体的长度决定了该腔内的共振光波长。当电池产生力时，空腔发生变形，从而缩短了总长度，从而移动了局部共振波长。因此，对于每个给定的腔长，基片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，这项技术还没有必要的时间分辨率来分辨超过0.5赫兹的区域，然而，这项技术的发展是为了利用更少的波长来允许在10赫兹时间范围内成像力-尽管这项技术仍然限于记录细胞力。该项目旨在开发ERISM，通过使用弹性模数更大的弹性体(20-60 kpa)来记录来自小动物的力，并将其与荧光显微镜相结合，以允许同时进行力测量和荧光活性测量。开发这项技术将使我们能够观察到软体动物产生的力量。一种这样的动物，黑腹果蝇幼虫，通过使用Gal-4 UAS系统，有着几乎无与伦比的遗传控制的历史。这种动物将允许我们通过遗传工具来研究肌肉的同时收缩，例如在每一块肌肉中表达荧光蛋白，或者通过在神经系统中直接表达基因编码的钙指示物(GECI)来观察神经肌肉活动。这种基因的易操纵性还允许我们在神经系统内的特定细胞中直接表达电离团--允许光基因控制动物运动行为的特定方面。这些幼虫也在一个有趣的规模内生长，在短短几天的时间里，从最大的细胞大小增长到最小的动物大小。这意味着他们必须使用同一机构来解决微观和宏观领域的问题。因此，这项研究旨在开发双波长ERRISM，以允许利用果蝇的完整遗传工具包，并将在理解软体动物的普通运动行为方面具有重要意义，但也可能为了解果蝇黑腹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的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.