Image-Guided Robot-Assisted Techniques with Applications in Minimally Invasive Therapy and Cell Biology

There are several situations where tasks can be performed better robotically rather than manually. Among these are situations (a) where high accuracy and robustness are required, (b) where difficult or hazardous working conditions exist, and (c) where very large or very small motions or forces are involved. Recent advances in technology have resulted in smaller size robots with higher accuracy and reliability. As a result, robotics is fi nding more and more applications in Biomedical Engineering. Medical Robotics and Cell Micro-Manipulation are two of these applications involving interaction with delicate living organs at very di fferent scales.Availability of a wide range of imaging modalities from ultrasound and X-ray fluoroscopy to high magni cation optical microscopes, makes it possible to use imaging as a powerful means to guide and control robot manipulators. This thesis includes three parts focusing on three applications of Image-Guided Robotics in biomedical engineering, including: Vascular Catheterization: a robotic system was developed to insert a catheter through the vasculature and guide it to a desired point via visual servoing. The system provides shared control with the operator to perform a task semi-automatically or through master-slave control. The system provides control of a catheter tip with high accuracy while reducing X-ray exposure to the clinicians and providing a more ergonomic situation for the cardiologists. Cardiac Catheterization: a master-slave robotic system was developed to perform accurate control of a steerable catheter to touch and ablate faulty regions on the inner walls of a beating heart in order to treat arrhythmia. The system facilitates touching and making contact with a target point in a beating heart chamber through master-slave control with coordinated visual feedback. Live Neuron Micro-Manipulation: a microscope image-guided robotic system was developed to provide shared control over multiple micro-manipulators to touch cell membranes in order to perform patch clamp electrophysiology. Image-guided robot-assisted techniques with master-slave control were implemented for each case to provide shared control between a human operator and a robot. The results show increased accuracy and reduced operation time in all three cases

Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology

Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed “blind,” meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum

Spatio-temporal modulation of light for stimulating and recording neuronal activity

The spectacular facets of light have made light ubiquitous in all fields of science. Light's interaction with matter allows for accurate manipulation of atomic and molecular structures that enabled fundamental breakthroughs in physics, chemistry, and biomedical research. The transfer of light's energy on molecules and genetically expressed proteins can be used to stimulate cells and emulate cellular processes such as synaptic inputs spatially distributed along the neuron's dendritic tree. Here, we show basic neuronal functions derived via numerical modelling and describe how we can use light to emulate these functions in order to provide a systematic study of the neuron's response. We focus on cortical pyramidal neurons and use the NEURON simulation environment to analyze how spatio-temporal stimulation patterns along various dendritic locations sets the neuron to fire an output. We then show an equivalent response from experiments via complex spatial light patterns for stimulating across different regions along the dendritic tree. Furthermore, we use the same spatial light patterns to simultaneously visualize neuronal responses via functional calcium imaging predicted via the same neuron model. Visualizing dendritic responses from back-propagating action potentials can provide new insights to some important features of dendritic computation.This work has been supported by partly by the Australian Research Council Discovery Project (DP140101555) and the National Health and Medical Research Council Project Grant (PG1105944)

Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

Astrocyte effects on hippocampal synaptogenesis in culture and near-field microscopy

This dissertation presents work in two parts. First, a presentation of effects of astrocytes on synapse formation of hippocampal neurons. The second describes a new near-field microscope for biological samples.;The formation of chemical synapses is a poorly understood process that is likely to critically rely on the presence of molecular cues that arise from presynaptic and postsynaptic neurons as well as associated glial cells. Rat hippocarnpal cultures were established in astrocyte-deplete and enriched conditions to ask whether the presence of astrocytes stimulated formation of chemical synapses. Co-culture with astrocytes selectively augmented formation of excitatory synapses. Immunostaining demonstrated that astrocytes stimulate export of synaptic proteins to the neurites, and the nerve terminal, which on ultrastructural examination were shown to contain increased number of synaptic vesicles within the presynaptic terminal. Also, local contact with astrocytes led to a selective augmentation of the magnitude of the N-type calcium current. The N-type calcium current is known to be associated with newly formed synapses. We propose that local interactions between the process of an astrocyte and developing pre- and postsynaptic terminals will augment calcium influx and thus synaptic transmission to place this tripartite structure at a competitive advantage over neighboring developing synapses that are devoid of interactions with astrocytes.;Sub-diffraction optical resolution achieved using near-field optical microscopy has the potential for new approaches and insights into sub-cellular function and molecular dynamics. Despite this potential it has been difficult to apply near-field microscopy to biology. Sample thickness causes the optical information to be comprised of a composite signal containing both near- and far-field fluorescence. To overcome this issue we have developed an approach in which a near-field optical fiber is translated toward the cell. Increased fluorescence intensity during z-translation contains two components; far-field fluorescence and combined near- and far-field fluorescence. Fitting a regression curve to the far-field intensity as the illumination aperture approaches the cell; it is possible to isolate near-field fluorescent signals. We demonstrate ability to resolve actin filaments in fixed glial cells. Comparison of composite signals with extracted near-field fluorescence demonstrates this approach significantly increases ability to detect sub-cellular structures at sub-diffraction resolution

Developing Instrumentation for Multi-parametric Investigation of Mechanisms of Mechanosensitivity in Ion Channels

Mechanosensitive (MS) channels are implicated in pathologies of the renal and pulmonary systems. Abnormal activity in MS channel reduces cell viability causing a variety of pathologies. MS channels are also responsible for sensation of pain and hearing. Despite the vital importance of MS channels, very little is known about the gating mechanisms of these channels. Attempts to study the mechanisms are severely limited by the lack of suitable instrumentation. A better understanding of the structure-function interaction of MS channels is necessary to find pharmacological leads for the pathologies. Activation data based on indirect activation of MS channels using hypo- or hyper-osmotic solutions or viscous drag is confounded by factors like membrane stretch and cytoskeletal stress. Traditional patch clamp does not allow direct access to the cell by other probes. While a planar patch clamp chip may allow for such access, most of the existing planar patch clamp chips are focused on high throughput screening for pharmaceutical targets and have designs that limit multi-parametric studies. We present here instrumentation that combines atomic force microscopy with cellular electrophysiology based on planar patch clamp approach. The instrumentation allows multi-parametric studies on single cells and provides unique insights into mechanisms of activation of not just MS channels, but ion channels in general by combining cellular electrophysiology, optical microscopy and atomic force microscopy. Using HaCaT cells as our model system we have obtained functional maps of distribution MS channels across cell surface. The maps reveal that the distribution of MS channels on HaCaT cells is highly non-uniform and that the channels are present in small clusters instead of dispersed as single entities. Our results using direct mechanical stimulation of single cells reveal that threshold stress level is required in order to activate MS channels and that the stress has a limited spatial range. Investigation of kinetics of the electrical response to direct mechanical stimulation reveals that the MS channels respond to the mechanical signal after a small time lag, which we attribute to the conformational changes necessary while the channel is being gated. We hope that the insights gained from studying the mechanosensitive channels of HaCaT cells will also advance the understanding of MS channels in general. Apart from opening new avenues in MS channel research, the instrumentation can also be useful in studying the dynamics and gating of ligand gated channels by appropriately tagging the AFM cantilever. With further improvements in the speed of AFM imaging, it will also be possible to observe the gating of channels in real time at molecular scale by imaging the channel on the cell while the channel is being gated

Development of multifunctional nano-probes for neuroscience research

The contribution of nanotechnology to the field of Neuroscience is increasing exponentially. In order to understand the relationship of structure to function at the cellular level, and to decipher the mysteries of nervous system, development of new tools to manipulate and measure cellular function at a local level is necessary. It is a continuing challenge to develop easily fabricated, multipurpose nano-probes which are able to target neural nanostructures for the local manipulation and measurement of functional responses. This thesis is focused on the fabrication, characterisation and implementation of a nano-pipette on a Scanning Ion Conductance Microscopy (SICM). The nano-pipette mounted on a SICM set-up acts as a proximity sensor for non-contact imaging of cellular features. SICM platform to accommodate electrochemical experiments is discussed. In particular, the development of a novel electrochemical probe, fabricated by pyrolytic decomposition of carbon within a quartz nano-pipette is discussed. This method is simple and carbon nano-electrodes of variable size can be fabricated in a single step. The nano-pipette‘s distance controlled feedback system was exploited for local delivery of chemicals to neuronal structures. Experimental and theoretical data are compared in order to calculate the concentration of molecules at the tip of the nano-pipette as a function of the driving force (voltage or pressure) and distance. The quantitative delivery of molecules from a 100 nm nano-pipette is demonstrated. In particular capsaicin-filled nano-pipette is used to trigger capsaicin-sensitive TRPV1 receptors in sensory neurons and transfected cells. Finally some preliminary results for the future development and potential application of nano-pipettes are shown. The nano-pipette is easily fabricated and is shown to be multi-functional. It provides an invaluable tool in the investigation of the nano-physiology of neurons. The SICM multipoint delivery competence can contribute to the various endeavours in drug discovery and to the yield of in vitro pharmacological assays.Open Acces

Development of experimental setups for the characterization of the mechanoelectrical coupling of cells in vitro

The field of mechanobiology emerged from the many evidences that mechanical forces acting on cells have a central role in their development and physiology. Cells, in fact, convert such forces into biochemical activities and gene expression in a process referred as mechanotransduction. In vitro models that mimic cell environment also from the mechanical point of view represent therefore a key tool for modelling cell behaviour and would find many applications, e.g. in drug development and tissue engineering. In this work I introduce novel tools for the study of mechanotransduction. In particular, I present a system for the evaluation of the complex response of electrically active cells, such as neurons and cardiomyocytes. This system integrates atomic force microscopy, extracellular electrophysiological recording, and optical microscopy in order to investigate cell activity in response to mechanical stimuli. I also present cell scaffolds for the in vitro study of cancer. Obtained results, although preliminary, show the potential of the proposed systems and methods to develop accurate in vitro models for mechanobiology studies

Creation of Defined Single Cell Resolution Neuronal Circuits on Microelectrode Arrays

The way cell-cell organization of neuronal networks influences activity and facilitates function is not well understood. Microelectrode arrays (MEAs) and advancing cell patterning technologies have enabled access to and control of in vitro neuronal networks spawning much new research in neuroscience and neuroengineering. We propose that small, simple networks of neurons with defined circuitry may serve as valuable research models where every connection can be analyzed, controlled and manipulated. Towards the goal of creating such neuronal networks we have applied microfabricated elastomeric membranes, surface modification and our unique laser cell patterning system to create defined neuronal circuits with single-cell precision on MEAs. Definition of synaptic connectivity was imposed by the 3D physical constraints of polydimethylsiloxane elastomeric membranes. The membranes had 20μm clear-through holes and 2-3μm deep channels which when applied to the surface of the MEA formed microwells to confine neurons to electrodes connected via shallow tunnels to direct neurite outgrowth. Tapering and turning of channels was used to influence neurite polarity. Biocompatibility of the membranes was increased by vacuum baking, oligomer extraction, and autoclaving. Membranes were bound to the MEA by oxygen plasma treatment and heated pressure. The MEA/membrane surface was treated with oxygen plasma, poly-D-lysine and laminin to improve neuron attachment, survival and neurite outgrowth. Prior to cell patterning the outer edge of culture area was seeded with 5x105 cells per cm and incubated for 2 days. Single embryonic day 7 chick forebrain neurons were then patterned into the microwells and onto the electrodes using our laser cell patterning system. Patterned neurons successfully attached to and were confined to the electrodes. Neurites extended through the interconnecting channels and connected with adjacent neurons. These results demonstrate that neuronal circuits can be created with clearly defined circuitry and a one-to-one neuron-electrode ratio. The techniques and processes described here may be used in future research to create defined neuronal circuits to model in vivo circuits and study neuronal network processing

Remote Access and Computerized User Control of Robotic Micromanipulators

Nano- and micromanipulators are critical research tools in numerous fields including micro-manufacturing and disease study. Despite their importance, nano- and micromanipulation systems remain inaccessible to many groups due to price and lack of portability. An intuitive and remotely accessible manipulation system helps mitigate this access problem. Previously, optimal control hardware for single-probe manipulation and the effect of latency on user performance were not well understood. Remote access demands full computerization; graphical user interfaces with networking capabilities were developed to fulfill this requirement and allow the use of numerous hardware controllers. Virtual environments were created to simulate the use of a manipulator with full parametric control and measurement capabilities. Users completed simulated tasks with each device and were surveyed about their perceptions. User performance with a commercial manipulator controller was exceeded by performance with both a computer mouse and pen tablet. Latency was imposed within the virtual environment to study it’s effects and establish guidelines as to which latency ranges are acceptable for long-range remote manipulation. User performance began to degrade noticeably at 100 ms and severely at 400 ms and performance with the mouse degraded the least as latency increased. A computer vision system for analyzing carbon nanotube arrays was developed so the computation time could be compared to acceptable system latency. The system characterizes the arrays to a high degree of accuracy and most of the measurement types of obtainable fast enough for real-time analysis