Nano handling and measurement of biological cells in culture

A thesis submitted to the University of Bedfordshire in partial fulfillment of the requirements for the degree of Doctor of PhilosophyThis thesis systematically investigates the nano handling and measurement techniques for biological cells in culture and studies the techniques to realize innovative and multi-functional applications in biomedicine. Among them, the technique based on AFM is able to visualize and quantify the dynamics of organic cells in culture on the nano scale. Especially, the cellular shear adhesion force on the various locations of biological cells was firstly accurately measured in the research of the cell-substrate interaction in terms of biophysical perspective. The innovative findings are conductive to study the cell-cell adhesion, the cell-matrix adhesion which is related to the cell morphology structure, function, deformation ability and adhesion of cells and better understand the cellular dynamic behaviors. Herein, a new liquid-AFM probe unit and an increment PID control algorithm were implemented suitable for scanning the cell samples under the air conditions and the liquid environments. The influence between the surface of sample and the probe, and the damage of probe during the sample scanning were reduced. The proposed system is useful for the nano handling and measurement of living cells. Besides, Besides, to overcome the limitations of liquid-AFMs, the multiple optical tweezers were developed to integrate with the liquid-AFM. The technique based on laser interference is able to characterize the optical trap stiffness and the escape velocity, especially to realize the capture and sorting of multiple cells by a polarization-controlled periodic laser interference. It can trap and move hundreds of cells without physical contact, and has broad application prospects in cytology. Herein, a new experimental method integrated with the positioning analysis in the Z direction was used to improve the fluid force method for the calibration and characterize the mechanical forces exerted on optical traps and living cells. Moreover, a sensitive and highly efficient polarization-controlled three-beam interference set-up was developed for the capture and sorting of multiple cells. By controlling the polarization angles of the beams, various intensity distributions and different sizes of dots were obtained. Subsequently, we have experimentally observed multiple optical tweezers and the sorting of cells with different polarization angles, which are in accordance with the theoretical analysis

X-ray Imaging Techniques to Quantify Spray Characteristics in the Near Field

Liquid sprays play a key role in many engineering processes, including, but not limited to, food processing, coating and painting, 3D printing, fire suppression, agricultural production, and combustion systems. Spray characteristics can easily be assessed in the mid- and far-field regions, well after liquid sheet breakup and droplet formation, using various optical and/or laser diagnostic techniques. The conditions in the near-field region influence mid- and far-field characteristics; however, near-field measurements are extremely challenging because the spray in this region is typically optically dense where optical and laser diagnostics are generally ineffective. This paper provides an overview of the various X-ray imaging techniques that can be used to characterize the near-field region of a spray. X-rays produced with tube sources as well as synchrotron sources will be discussed. Using tube-source X-rays, 2D radiographic videos are possible showing qualitative spray information. The 2D radiographs can also provide quantitative measurements of the optical depth (OD) in the near-field region. Tube sources can also provide X-ray computed tomography imaging that can produce time-average 3D density (mass distribution) maps of the spray. X-rays from synchrotron radiation provide a high-flux X-ray beam that can be used to provide high spatial and temporal resolution of the spray equivalent path length (EPL) as well as other characteristics, but it is more challenging to implement than using a common tube source. Various examples of these X-ray imaging techniques will be discussed

Review of optical breast imaging and spectroscopy

Diffuse optical imaging and spectroscopy of the female breast is an area of active research. We review the present status of this field and discuss the broad range of methodologies and applications. Starting with a brief overview on breast physiology, the remodeling of vasculature and extracellular matrix caused by solid tumors is highlighted that is relevant for contrast in optical imaging. Then, the various instrumental techniques and the related methods of data analysis and image generation are described and compared including multimodality instrumentation, fluorescence mammography, broadband spectroscopy, and diffuse correlation spectroscopy. We review the clinical results on functional properties of malignant and benign breast lesions compared to host tissue and discuss the various methods to improve contrast between healthy and diseased tissue, such as enhanced spectroscopic information, dynamic variations of functional properties, pharmacokinetics of extrinsic contrast agents, including the enhanced permeability and retention effect. We discuss research on monitoring neoadjuvant chemotherapy and on breast cancer risk assessment as potential clinical applications of optical breast imaging and spectroscopy. Moreover, we consider new experimental approaches, such as photoacoustic imaging and long-wavelength tissue spectroscopy

Quantitative Magnetic Resonance Imaging Methodology Development

Magnetic Resonance Imaging (MRI) is a non-invasive medical imaging modality that provides excellent soft tissue contrast and resolution. Objects with high magnetic susceptibility distort the magnetic field, leading to severe artifacts in conventional MRI. It is very challenging to image around metal implants. Novel strategies may exploit the field distortion for spatial encoding. The magnetic field map is required in the development of these methods. A robust field map can also be employed to quantify high susceptibility particles that play a major role in cell tracking studies and hyperthermic treatment of cancers. Pure phase encoding (PPE) techniques with short encoding times are largely immune to magnetic field inhomogeneity artifacts. Artifact-free MR images around titanium were acquired with PPE techniques, from which the magnetic field distribution was derived. The approach was extended to quantify iron microparticles and was compared with conventional MRI to demonstrate its superiority

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

High Performance Optical Computed Tomography for Accurate Three-Dimensional Radiation Dosimetry

Optical computed tomography (CT) imaging of radiochromic gel dosimeters is a method for truly three-dimensional radiation dosimetry. Although optical CT dosimetry is not widely used currently due to previous concerns with speed and accuracy, the complexity of modern radiotherapy is increasing the need for a true 3D dosimeter. This thesis reports technical improvements that bring the performance of optical CT to a clinically useful level. New scanner designs and improved scanning and reconstruction techniques are described. First, we designed and implemented a new light source for a cone-beam optical CT system which reduced the scatter to primary contribution in CT projection images of gel dosimeters from approximately 25% to approximately 4%. This design, which has been commercially implemented, enables accurate and fast dosimetry. Second, we designed and constructed a new, single-ray, single-detector parallel-beam optical CT scanner. This system was able to very accurately image both absorbing and scattering objects in large volumes (15 cm diameter), agreeing within ∼1% with independent measurements. It has become a reference standard for evaluation of optical CT geometries and dosimeter formulations. Third, we implemented and characterized an iterative reconstruction algorithm for optical CT imaging of gel dosimeters. This improved image quality in optical CT by suppressing the effects of noise and artifacts by a factor of up to 5. Fourth, we applied a fiducial-based ray path measurement scheme, combined with an iterative reconstruction algorithm, to enable optical CT reconstruction in the case of refractive index mismatch between different media in the scanner’s imaged volume. This improved the practicality of optical CT, as time-consuming mixing of liquids can be avoided. Finally, we applied the new laser scanner to the difficult dosimetry task of small-field measurement. We were able to obtain beam profiles and depth dose curves for 4 fields (3x3 cm2 and below) using one 15 cm diameter dosimeter, within 2 hours. Our gel dosimetry depth-dose curves agreed within ∼1.5% with Monte Carlo simulations. In conclusion, the developments reported here have brought optical CT dosimetry to a clinically useful level. Our techniques will be used to assist future research in gel dosimetry and radiotherapy treatment techniques

Evaluation of the region-specific risks of accidental radioactive releases from the European Spallation Source

The European Spallation Source (ESS) is a neutron research facility under construction in southern Sweden. The facility will produce a wide range ofradionuclides that could be released into the environment. Some radionuclides are of particular concern such as the rare earth gadolinium-148. In this article, the local environment was investigated in terms of food production and rare earth element concentration in soil. The collected data will later be used to model thetransfer of radioactive contaminations from the ESS

Multi-scale imaging of porous media and flow simulation at the pore scale

In the last decade, the fundamental understanding of pore-scale flow in porous media has been undergoing a revolution through the recent development of new pore-scale imaging techniques, reconstruction of three-dimensional pore space images, and advances in the computational methods for solving complex fluid flow equations directly or indirectly on the reconstructed three-dimensional pore space images. Important applications include hydrocarbon recovery from - and CO2 storage in - reservoir rock formations. Of particular importance is the consideration of carbonate reservoirs, as our understanding of carbonates with respect to geometry and fluid flow processes is still very limited in comparison with sandstone reservoirs. This thesis consists of work mainly performed within the Qatar Carbonates and Carbon Storage Research Centre (QCCSRC) project, focusing on development of three dimensional imaging techniques for accurately characterizing and predicting flow/transport properties in both complex benchmark carbonate and sandstone rock samples. Firstly, the thesis presents advances in the application of Confocal Laser Scanning Microscopy (CLSM), including the improvement of existing sample preparation techniques and a step-by step guide for imaging heterogeneous rock samples exhibiting sub-micron resolution pores. A novel method has been developed combining CLSM with sequential grinding and polishing to obtain deep 3D pore-scale images. This overcomes a traditional limitation of CLSM, where the depth information in a single slice is limited by attenuation of the laser light. Other features of this new method include a wide field of view at high resolution to arbitrary depth; fewer grinding steps than conventional serial sectioning using 2D microscopy; the image quality does not degrade with sample size, as e.g. in micro-computed tomography (micro- CT) imaging. Secondly, it presents two fundamental issues – Representative Element of Volume (REV) and scale dependency which are addressed with qualitative and quantitative solutions for rocks increasing in heterogeneity from beadpacks to sandpacks to sandstone to carbonate rocks. The REV is predicted using the mathematical concept of the Convex Hull, CH, and the Lorenz coefficient, LC, to investigate the relation between two macroscopic properties simultaneously, in this case porosity and absolute permeability. The effect of voxel resolution is then studied on the segmented macro-pore phase (macro-porosity) and intermediate phase (micro-porosity) and the fluid flow properties of the connected macro-pore space using lattice-Boltzmann (LB) and pore network (PN) modelling methods. A numerical coarsening (up-scaling) algorithm have also been applied to reduce the computational power and time required to accurately predict the flow properties using the LB and PN methods. Finally, a quantitative methodology has been developed to predict petrophysical properties, including porosity and absolute permeability for X-ray medical computed tomography (CT) carbonate core images of length 120 meters using image based analysis. The porosity is calculated using a simple segmentation based on intensity grey values and the absolute permeability using the Kozeny-Carman equation. The calculated petrophysical properties were validated with the experimental plug data.Open Acces