Development of high-speed imaging techniques for C. elegans nervous system studies

We report high-speed imaging techniques for C. elegans nervous systems studies. We introduce C. elegans, the main model organism in this dissertation, and neuroscientific and biomedical studies using C. elegans involving calcium imaging, nerve regeneration, and drug screening. We review technologies including confocal microscopy and microfluidic devices used in the neuroscientific and biomedical studies We discuss development of a high-speed laser scanning confocal microscope capable of flexible control of imaging conditions, fast imaging speed, and large field-of-view. We provides the design principles used in the development of the confocal microscope including the optical, electrical, and software implementation, and the details of the confocal microscope we built based on the design principles. We present the performance characterization of the confocal microscope, then a few sample images obtained with the confocal microscope. We present development of time-lapse volumetric confocal imaging of whole animal C. elegans Ca²⁺ dynamics. We provide the design of the time-lapse volumetric confocal imaging system including a microfluidic device to accommodate the whole animal within the field-of-view of the imaging system. We examine the feasibility of the volumetric confocal imaging of a whole animal, and demonstrate imaging of the whole animal C. elegans neurons’ response to NaCl within a 630 × 150 × 25 μm³ volume at 2 Hz rate. We report a high-throughput automated imaging platform for C. elegans nerve regeneration study. We describe the design of the automated imaging platform and the automation flow, and characterizes the performance of the platform. The imaging platform can obtain high-resolution 3D confocal images of 20 animals in 10 minutes. We show sample images of C. elegans anterior lateral microtubule nerve regeneration examples acquired via the automated imaging platform. We demonstrate a planar laser activated neuronal scanning platform (PLANS), a high-throughput animal examination system for drug screening. We explain the construction of PLANS involving the optics, the microfluidic device, and the electronics. The PLANS system can scan an animal in less than 5 ms with a spatial sampling resolution of 3 μm FWHM. We show sample scanning results of a Huntington’s disease model of C. elegans. We summarize the studies discussed in this dissertation, and suggest relevant future research to follow up on the studies.Electrical and Computer Engineerin

Optical Coherence Tomography guided Laser-Cochleostomy

Despite the high precision of laser, it remains challenging to control the laser-bone ablation without injuring the underlying critical structures. Providing an axial resolution on micrometre scale, OCT is a promising candidate for imaging microstructures beneath the bone surface and monitoring the ablation process. In this work, a bridge connecting these two technologies is established. A closed-loop control of laser-bone ablation under the monitoring with OCT has been successfully realised

Geometric model and calibration method for a solid-state LiDAR

This paper presents a novel calibration method for solid-state LiDAR devices based on a geometrical description of their scanning system, which has variable angular resolution. Determining this distortion across the entire Field-of-View of the system yields accurate and precise measurements which enable it to be combined with other sensors. On the one hand, the geometrical model is formulated using the well-known Snell’s law and the intrinsic optical assembly of the system, whereas on the other hand the proposed method describes the scanned scenario with an intuitive camera-like approach relating pixel locations with scanning directions. Simulations and experimental results show that the model fits with real devices and the calibration procedure accurately maps their variant resolution so undistorted representations of the observed scenario can be provided. Thus, the calibration method proposed during this work is applicable and valid for existing scanning systems improving their precision and accuracy in an order of magnitude.Peer ReviewedPostprint (published version

Development And Analysis Of Linear Resonant Scanner With Torsional Mechanism

Large size mirror scanners are needed in several scanning technologies such as, ultra short-throw projector, free-space optical communications and barcode scanner. Several researches on large size mirror in microelectromechanical systems (MEMS) scanner were conducted. For instance, research on micromachined polysilicon microscanners has been performed for barcode scanner. However, the curvature of the microscanners causes image distortion. Furthermore, high operation voltages of the MEMS scanner deter the usage of MEMS scanner in hand-held applications. In this research, a linear resonant scanner consisting of an electronically driven mechanically-resonant torsional spring-mirror system was developed for display applications. The scanner was designed according to the functional components such as compliant structure and actuator. The torsional spring which is the compliant structure was modeled with finite element analysis (FEA) and geometry studies were conducted. The optimized torsional spring with the lowest stress level was selected for the design. The actuator of air core coil (ACC) was used in the scanner; geometry study was used to maximize the magnetic forces of the ACC. The ACC of with minimum length, minimum inner radius and maximum outer radius was used. Besides, experimental analysis and FEA of the scanner showed that resonant frequency, angular displacement and stress level are affected by the magnet position on the suspended plate. After the scanner design, several characteristic studies were conducted. A nonlinear damping model is proven to be able to analyze and predict the free vibration response of the scanner based on experimental results

Development And Analysis Of Linear Resonant Scanner With Torsional Mechanism

Large size mirror scanners are needed in several scanning technologies such as, ultra short-throw projector, free-space optical communications and barcode scanner. Several researches on large size mirror in microelectromechanical systems (MEMS) scanner were conducted. For instance, research on micromachined polysilicon microscanners has been performed for barcode scanner. However, the curvature of the microscanners causes image distortion. Furthermore, high operation voltages of the MEMS scanner deter the usage of MEMS scanner in hand-held applications. In this research, a linear resonant scanner consisting of an electronically driven mechanically-resonant torsional spring-mirror system was developed for display applications. The scanner was designed according to the functional components such as compliant structure and actuator. The torsional spring which is the compliant structure was modeled with finite element analysis (FEA) and geometry studies were conducted. The optimized torsional spring with the lowest stress level was selected for the design. The actuator of air core coil (ACC) was used in the scanner; geometry study was used to maximize the magnetic forces of the ACC. The ACC of with minimum length, minimum inner radius and maximum outer radius was used. Besides, experimental analysis and FEA of the scanner showed that resonant frequency, angular displacement and stress level are affected by the magnet position on the suspended plate. After the scanner design, several characteristic studies were conducted. A nonlinear damping model is proven to be able to analyze and predict the free vibration response of the scanner based on experimental results. xxx The damping model is able to accommodate the frequency perturbation which happens when the scanner mounting is changed. The hysterical frequency response on the large scale torsional spring mechanism is first found in this research work. Furthermore, the relationship between scanning angle and driving frequency was employed for extra mechanical gain. The proposed resonant scanner extends the ability of the torsional mechanism scanner for large angular displacement of 87.1o with low voltage input of 5 V

Optogenetic Interrogation and Manipulation of Vascular Blood Flow in Cortex

Understanding blood flow regulatory mechanisms that correlate the regional blood flow with the level of local neuronal activity in brain is an ongoing research. Discerning different aspects of this coupling is of substantial importance in interpretation of functional imaging results, such as functional magnetic resonance imaging (fMRI), that rely on hemodynamic recordings to detect and image brain neuronal activity. Moreover, this understanding can provide insight into blood flow disorders under different pathophysiological conditions and possible treatments for such disorders. The blood regulatory mechanisms can be studied at two different; however, complementary levels: at the cellular level or at the vascular level. To fully understand the regulatory mechanisms in brain, it is essential to discern details of the coupling mechanism in each level. While, the cellular pathways of the coupling mechanism has been studied extensively in the past few decades, our understanding of the vascular response to brain activity is fairly basic. The main objective of this dissertation is to develop proper methods and instrumentation to interrogate regional cortical vasodynamics in response to local brain stimulation. For this purpose we offer the design of a custom-made OCT scanner and the necessary lens mechanisms to integrate the OCT system, fluorescence imaging, and optogenetic stimulation technologies in a single system. The design uses off-the-shelf components for a cost-effective design. The modular design of the device allows scientists to modify it in accordance with their research needs. With this multi-modal system we are able to monitor blood flow, blood velocity, and lumen diameter of pial vessels, simultaneously. Additionally, the system design provides the possibility of generating arbitrary spatial stimulation light pattern on brain. These abilities enables researchers to capture more diverse datasets and, eventually, obtain a more comprehensive picture of the vasodynamics in the brain. Along with the device we also proposed new biological experiments that are tailored to investigate the spatio-temporal properties of the vascular response to optical neurostimulation of the excitatory neurons. We demonstrate the ability of the proposed methods to investigate the effect of length and amplitude of stimulation on the temporal pattern of response in the blood flow, blood velocity, and diameter of the pial vessels. Moreover, we offer systemic approaches to investigate the spatial characteristics of the response in a vascular network. In these methods we apply arbitrary spatial patterns of optical stimulation to the cortex of transgenic mice and monitor the attributes of surrounding vessels. With this flexibility we were able to image the brain region that is influenced by a pial artery. After characterizing the spatio-temporal properties of the vascular blood flow response to optical neuro-modulation, we demonstrate the design and application of an optogenetic-based closed-loop controller mechanism in the brain. This controller, uses a proportional–integral–derivative (PID) compensator to engineer temporal optogenetic stimulation light pulses and maintain the flow of blood at various user defined levels in a set of selected arteries. Upon tuning the gain values of the PID controller we obtained a near to critically-damped response in the blood flow of selected arterial vessels

Development and applications of high speed and hyperspectral nonlinear microscopy

Nonlinear microscopy refers to a range of laser scanning microscopy techniques that are based on nonlinear optical processes such as two-photon excited fluorescence and second harmonic generation. Nonlinear microscopy techniques are powerful because they enable the visualization of highly scattering biological samples with subcellular resolution. This capability is especially valuable for in vivo and live tissue imaging since it can provide both structural and functional information about tissues in their native environment. With the use of a range of exogenous dyes and intrinsic contrast, in vivo nonlinear microscopy can be used to characterize and measure dynamic processes of tissues in their normal environment. These advances have been particularly relevant in neuroscience, where truly understanding the function of the brain requires that its neural and vascular networks be observed while undisturbed. Despite these advantages, in vivo nonlinear microscopy still faces several major challenges. First, observing dynamics that occur in large areas over short time scales, such as neuronal signaling and blood flow, is challenging because nonlinear microscopy generally requires scanning to create an image. This limits the study of dynamic behavior to either a single plane or to a small subset of regions within a volume. Second, applications that rely on the use of exogenous dyes can be limited by the need to stain tissues before imaging, the availability of dyes, and specificity that can be achieved. Usually considered a nuisance, endogenous tissue contrast from autofluorescence or structures exhibiting second harmonic generation can produce stunning images for visualizing subcellular morphology. Imaging endogenous contrast can also provide valuable information about the chemical makeup and metabolic state of the tissue. Few methods have been developed to carefully and quantitatively examine endogenous fluorescence in living tissues. In this thesis, these two challenges in nonlinear microscopy are addressed. The development of a novel hyperspectral two-photon microscopy method to acquire spectroscopic data from tissues and increase the information available from endogenous contrast is presented. This system was applied to visualize and identify sources of endogenous contrast in gastrointestinal tissues, providing robust references for the assessment of normal and diseased tissues. Secondly, three methods for high speed volumetric imaging using laser scanning nonlinear microscopy were developed to address the need for improved high-speed imaging in living tissues. A spectrally-encoded high-speed imaging method that can provide simultaneous imaging of multiple regions of the living brain in parallel is presented and used to study spontaneous changes in vascular tone in the brain. This technique is then extended for use with second harmonic generation microscopy, which has the potential to greatly increase the degree of multiplexing. Finally, a complete system design capable of volumetric scan rates >1Hz is shown, offering improved performance and versatility to image brain activity