Characterising the neck motor system of the blowfly

Flying insects use visual, mechanosensory, and proprioceptive information to control their movements, both when on the ground and when airborne. Exploiting visual information for motor control is significantly simplified if the eyes remain aligned with the external horizon. In fast flying insects, head rotations relative to the body enable gaze stabilisation during highspeed manoeuvres or externally caused attitude changes due to turbulent air. Previous behavioural studies into gaze stabilisation suffered from the dynamic properties of the supplying sensor systems and those of the neck motor system being convolved. Specifically, stabilisation of the head in Dipteran flies responding to induced thorax roll involves feed forward information from the mechanosensory halteres, as well as feedback information from the visual systems. To fully understand the functional design of the blowfly gaze stabilisation system as a whole, the neck motor system needs to be investigated independently. Through X-ray micro-computed tomography (μCT), high resolution 3D data has become available, and using staining techniques developed in collaboration with the Natural History Museum London, detailed anatomical data can be extracted. This resulted in a full 3- dimensional anatomical representation of the 21 neck muscle pairs and neighbouring cuticula structures which comprise the blowfly neck motor system. Currently, on the work presented in my PhD thesis, μCT data are being used to infer function from structure by creating a biomechanical model of the neck motor system. This effort aims to determine the specific function of each muscle individually, and is likely to inform the design of artificial gaze stabilisation systems. Any such design would incorporate both sensory and motor systems as well as the control architecture converting sensor signals into motor commands under the given physical constraints of the system as a whole.Open Acces

Macro optical projection tomography for large scale 3D imaging of plant structures and gene activity

Optical projection tomography (OPT) is a well-established method for visualising gene activity in plants and animals. However, a limitation of conventional OPT is that the specimen upper size limit precludes its application to larger structures. To address this problem we constructed a macro version called Macro OPT (M-OPT). We apply M-OPT to 3D live imaging of gene activity in growing whole plants and to visualise structural morphology in large optically cleared plant and insect specimens up to 60 mm tall and 45 mm deep. We also show how M-OPT can be used to image gene expression domains in 3D within fixed tissue and to visualise gene activity in 3D in clones of growing young whole Arabidopsis plants. A further application of M-OPT is to visualise plant-insect interactions. Thus M-OPT provides an effective 3D imaging platform that allows the study of gene activity, internal plant structures and plant-insect interactions at a macroscopic scale

Evaluation of the Thorax of Manduca Sexta for Flapping Wing Micro Air Vehicle Applications

The tobacco hornworm hawkmoth (Manduca sexta) provides an excellent model from which to gather knowledge pertaining to the development of a Flapping Wing Micro Air Vehicle (FWMAV). One of the major challenges in design of a FWMAV is the energy demanding nature of low Reynolds number flapping flight. Therefore, an understanding of the power required by the flight muscles to actuate the wings is essential for the design of a FWMAV. The M.sexta wing/thorax mechanism was evaluated as a mechanical system in order to gain insight to the mechanical power required to produce the full natural wing stroke. A unique dynamic load device was designed and constructed to mechanically actuate the upstroke and downstroke of the M.sexta in order to achieve the full flapping motion. Additionally, the forces applied through the flight muscles were directly measured in order to attain the power requirements of the flight muscles simultaneously. The experiment yielded wing stroke amplitudes of + 60 and - 35, which is what is seen in nature during hovering. The DVM and DLM muscle groups were calculated to have a power density of 112 W/kg with the vehicle energy density being 2 W/kg. The power output requirement indicates the need for a lightweight and energy-dense power source/actuator combination for the development of FWMAVs

A Pipeline for Volume Electron Microscopy of the Caenorhabditis elegans Nervous System.

The "connectome," a comprehensive wiring diagram of synaptic connectivity, is achieved through volume electron microscopy (vEM) analysis of an entire nervous system and all associated non-neuronal tissues. White et al. (1986) pioneered the fully manual reconstruction of a connectome using Caenorhabditis elegans. Recent advances in vEM allow mapping new C. elegans connectomes with increased throughput, and reduced subjectivity. Current vEM studies aim to not only fill the remaining gaps in the original connectome, but also address fundamental questions including how the connectome changes during development, the nature of individuality, sexual dimorphism, and how genetic and environmental factors regulate connectivity. Here we describe our current vEM pipeline and projected improvements for the study of the C. elegans nervous system and beyond

Design, Manufacture, and Structural Dynamic Analysis of a Biomimetic Insect-Sized Wing for Micro Air Vehicles

The exceptional flying characteristics of airborne insects motivates the design of biomimetic wing structures that can exhibit a similar structural dynamic behavior. For this purpose, this investigation describes a method for both manufacturing a biomimetic insect-sized wing using the photolithography technique and analyzing its structural dynamic response. The geometry of a crane fly forewing (family Tipulidae) is acquired using a micro-computed tomography scanner. A computer-aided design model is generated from the measurements of the reconstructed scanned model of the insect wing to design the photomasks of the membrane and the venation network required for the photolithography procedure. A composite material wing is manufactured by patterning the venation network using photoresist SU-8 on a Kapton film for the assembling of the wing. A single material artificial wing is fabricated using the photoresist SU-8 for both the membrane and the network of veins. Experiments are conducted using a modal shaker and a digital image correlation (DIC) system to determine the natural frequencies and the mode shapes of the artificial wing from the fast Fourier transform of the displacement response of the wing. The experimental results are compared with those from a finite element (FE) model of the wing. A numerical simulation of the fluid-structure interaction is conducted by coupling the FE model of the artificial wing with a computational fluid dynamics model of the surrounding airflow. From these simulations, the deformation response and the coefficients of drag and lift of the artificial wing are predicted for different freestream velocities and angles of attack. Wind-tunnel experiments are conducted using the DIC system to determine the structural deformation response of the artificial wing under different freestream velocities and angles of attack. The vibration modes are dominated by a bending and torsional deformation response. The deformation along the span of the wing increases nonlinearly from the root of the wing to the tip of the wing with Reynolds number. The aerodynamic performance, defined as the ratio of the coefficient of lift to the coefficient of drag, of the artificial wing increases with Reynolds number and angle of attack up to the critical angle of attack

Design, Manufacture, and Structural Dynamic Analysis of a Biomimetic Insect-Sized Wing for Micro Air Vehicles

The exceptional flying characteristics of airborne insects motivates the design of biomimetic wing structures that can exhibit a similar structural dynamic behavior. For this purpose, this investigation describes a method for both manufacturing a biomimetic insect-sized wing using the photolithography technique and analyzing its structural dynamic response. The geometry of a crane fly forewing (family Tipulidae) is acquired using a micro-computed tomography scanner. A computer-aided design model is generated from the measurements of the reconstructed scanned model of the insect wing to design the photomasks of the membrane and the venation network required for the photolithography procedure. A composite material wing is manufactured by patterning the venation network using photoresist SU-8 on a Kapton film for the assembling of the wing. A single material artificial wing is fabricated using the photoresist SU-8 for both the membrane and the network of veins. Experiments are conducted using a modal shaker and a digital image correlation (DIC) system to determine the natural frequencies and the mode shapes of the artificial wing from the fast Fourier transform of the displacement response of the wing. The experimental results are compared with those from a finite element (FE) model of the wing. A numerical simulation of the fluid-structure interaction is conducted by coupling the FE model of the artificial wing with a computational fluid dynamics model of the surrounding airflow. From these simulations, the deformation response and the coefficients of drag and lift of the artificial wing are predicted for different freestream velocities and angles of attack. Wind-tunnel experiments are conducted using the DIC system to determine the structural deformation response of the artificial wing under different freestream velocities and angles of attack. The vibration modes are dominated by a bending and torsional deformation response. The deformation along the span of the wing increases nonlinearly from the root of the wing to the tip of the wing with Reynolds number. The aerodynamic performance, defined as the ratio of the coefficient of lift to the coefficient of drag, of the artificial wing increases with Reynolds number and angle of attack up to the critical angle of attack

Single molecule tracking with light sheet microscopy

The work presented here concentrates on light sheet based fluorescence microscopy (LSFM) and its application to single molecule tracking. In LSFM the sample is illuminated perpendicular to the detection axis with a thin light sheet. In this manner a simple optical sectioning microscope is created, because only the focal plane of the detection optics is illuminated and no out-of-focus fluorescence is generated. This results in an enhancement of the signal-to-noise-ratio and combined with the high acquisition speed of a video microscopy a powerful tool is created to study single molecule dynamics on a millisecond timescale, A completely new setup was designed and constructed, that combines light sheet illumination technique with single molecule detection ability. Theoretical calculations and quantitative measurements of the illumination light sheet thickness (2-3 µm thick) and the microscope point spread function were performed. A direct comparison of LSFM and epi-illumination of model samples with intrinsic background fluorescence illustrated the clear contrast improvement of LSFM for thick samples. Single molecule detection is limited by the number of photons emitted by a single fluorophore per observation time. So, the ability to track single molecules is dependent on molecule speed, background, detection sensitivity and frame rate. The imaging speed with the concomitant high signal-to-noise ratio that could be realized within the setup was unprecedented until then. It permitted the observation of single protein trajectories in aqueous solution with a diffusion coefficient greater than 100 µm²/s. The in vivo imaging of single molecules in thick biological samples was demonstrated in living salivary gland cell nuclei of Chironomus tentans larvae. These cell nuclei afford exceptional possibilities for the study of RNA mobility, but provide a microscopic challenge with a diameter of 50-75 µm and up 200 µm deep within the sample. To image the intranuclear mobility of individual messenger RNA particles, they were indirectly labeled via the fluorescently labeled RNA binding protein hrp36. Thus it was possible to identify at least three different diffusion modes of the mRNA particles and indirectly measure the nuclear viscosity. A high flexibility and easy adaptation of the optical sectioning thickness is required to visualize biological samples of various sizes. Often, however, the sheet geometry is fixed, whereas it would be advantageous to adjust the sheet geometry to specimens of different dimensions. Therefore, an afocal cylindrical zoom lens system comprising only 5 lenses and a total system length of less than 160 mm was developed. Two movable optical elements were directly coupled, so that the zoom factor could be adjusted from 1x to 6.3x by a single motor. Polytene chromosomes of salivary gland cell nuclei of C.tentans larvae were imaged in vivo to demonstrate the advantages in image contrast by imaging with different light sheet dimensions. The light sheet microscope introduced in this thesis proofed its suitability for in vivo single molecule imaging deep within a biological sample. It has the potential to reveal new dynamic single molecule interactions in vivo and enables new studies and experiments of intracellular processes

Manufacturing and Evaluation of a Biologically Inspired Engineered MAV Wing Compared to the Manduca Sexta Wing Under Simulated Flapping Conditions

In recent years, researchers have expressed a vested interest in the concepts surrounding flapping wing micro air vehicles (FWMAVs) that are capable of both range and complex maneuvering. Most research in this arena has found itself concentrated on topics such as flapping dynamics and the associated fluid-structure interactions inherent in the motion, however there still remains myriad questions concerning the structural qualities intrinsic to the wings themselves. Using nature as the template for design, FWMAV wings were constructed using carbon fiber and Kapton and tested under simplified flapping conditions by analyzing frozen\u27 digital images of the deformed wing by methods of photogrammetry. This flapping motion was achieved via the design and construction of a flapper that emulates several of the kinematic features that can be seen in naturally occurring flyers. The response to this motion was then compared to the inspiring specimen\u27s wings, the North American Hawkmoth (Manduca Sexta), under the same flapping conditions in order to identify some of the key features that nature has deemed necessary for successful flight

Systems analysis of gaze stabilization behaviour and imaging of motor systems in the blowfly calliphora

A fast and powerful gaze stabilization reflex supports self-motion es- timation and flight control in flies. Changes in body posture are conveyed by a variety of sensory modalities and compensated for by fast and accurate head movements. This thesis aims to further our understanding of the behavioural basis of compensatory head move- ments, and presents a first foray into the in vivo imaging of the motor systems that actuate these control reflexes. Major sensors that contribute to gaze stabilisation are the visual ocelli and compound eyes on the head, and the mechanosensory halteres on the thorax. The integration of visual feedback and mechanosensory feedforward control gives rise to a two-degree-of-freedom controller, a design which is extensively used in engineering applications. I per- formed a linear systems analysis of compensatory head roll in response to forced thorax oscillations in the fly. The feedforward pathway ex- hibited a high bandwidth and constant gain and reduced the response delay of the reflex. Large stability margins in the feedback pathway supplied by the compound eyes guaranteed stable behaviour in the face of response variability. The occlusion of the ocelli did not change the gain of the feedback pathway, but significantly reduced the la- tency. I investigated the use of iodine-enhanced computed x-ray microto- mography (microCT) to perform fast three-dimensional imaging of the neck and flight motor systems. Virtual dissections of major func- tional units illustrate the possibilities and limitations of microCT. To observe the configuration of motor systems in behaving flies I per- formed gated microtomography using hard x-rays at the TOMCAT beamline of the Swiss Light Source, a third generation synchrotron. While 3D tomograms of the neck motor system proved elusive, this thesis presents the first in vivo tomograms of the flight motor and wing hinge during tethered flight.Open Acces