Vision guided automation for intra-cytoplasmic sperm injection

Biological cell injection is an effective technique in which a foreign material is directly introduced into the target cell. Intracytoplasmic Sperm Injection (ICSI) is a microinjection technique which is used for infertility treatment. In this technique, a single sperm cell is directly injected into an oocyte using micropipettes. The operations in this application are manually controlled by an embryologist and more importantly, this reduces the accuracy, repeatability, and consistency of the operation. Therefore, the full automation is a prerequisite for microinjection operations, particularly in ICSI application. This thesis focuses on enhancing the microinjection procedure by developing vision-guided processes prior to and during the operation. Initially, a vision-controlled technique was proposed to align the injection and holding pipettes in three orthogonal axes which is essential for successful microinjection. To conduct reliable injection, the vibrational displacement of the injection pipette’s tip needs to be evaluated and improved before the operations continue further. A novel vision-based sensor was developed to measure the displacement changes at the tip in three orthogonal axes. By employing the developed vision sensor, the effect of injection speed on vibrational displacement creation was analysed to determine the value of various injection parameters, such as force fluctuation, and penetration force on cell damages. An ultimate automation task is required in microinjection to position the randomly located biological cell within the Petri dish to the system’s field of view. The proposed technique fills a gap in the literature by proposing a real-time cell recognising and positioning system that can be employed with different types of biological cells at various maturation stages, as well as with different microscope types that are being used in microinjection applications

Size-tunable nanoneedle arrays for influencing stem cell morphology, gene expression and nuclear membrane curvature

High-aspect-ratio nanostructures have emerged as versatile platforms for intracellular sensing and biomolecule delivery. Here, we present a microfabrication approach in which a combination of reactive ion etching protocols was used to produce high-aspect-ratio, nondegradable silicon nanoneedle arrays with tip diameters that can be finely tuned between 20 and 700 nm. We used these arrays to guide the long-term culture of human mesenchymal stem cells (hMSCs). Notably, we used the nanoneedle tip diameter to control the morphology, nuclear size and F-actin alignment of interfaced hMSCs, and to regulate the expression of nuclear lamina genes, Yes-associated protein (YAP) target genes and focal adhesion genes. These topography-driven changes were attributed to signaling by Rho-family GTPase pathways, differences in the effective stiffness of the nanoneedle arrays and the degree of nuclear membrane impingement, with the latter clearly visualized using focused-ion beam scanning electron microscopy (FIB-SEM). Our approach to design high-aspect-ratio nanostructures will be broadly applicable to design biomaterials and biomedical devices used for long-term cell stimulation and monitoring

A Computational Study of The Electrical Response of Biological Cells with Realistic Three-Dimensional Morphologies

Title from PDF of title page, viewed September 21, 2022Dissertation advisor: Ahmed M. HassanVitaIncludes bibliogaphical references (pages 119-132)Dissertation (Ph.D)--Department of Computer Science and Electrical Engineering, Department of Physics and Astronomy. University of Missouri--Kansas City, 2021One of the unique features of a biological cell is the cell membrane that protects the cell interior by establishing a physical barrier between the cytoplasm and the extracellular matrix and governs the distribution of the cytoskeleton to control the three-dimensional (3D) morphology of a cell. From an electrical standpoint, the cell membrane represents an insulating layer with selective ion permeability, thereby administering an ionic imbalance between the conductive extracellular and intracellular fluids. Consequently, the electrical characterization of a biological cell mostly focuses on the specific electrical properties of the cell membrane and the means to modulate its semipermeable nature. Previously reported research studies had revealed many characteristics of the cell membrane. However, they did not explore the morphological feature to its fullest, especially in three dimensions. Motivated by this knowledge gap, this work explores the effect of the 3D variations in cell morphology on the electrical response of biological cells. The most diverse and accurate 3D cell database developed to date by the National Institute of Standards and Technology was incorporated in this study, and an extensive investigation of these cells’ electrical characteristics was manifested by computational means. The cell database has hundreds of morphologies that were reconstructed from stem cells grown in different environments. To quantify how cell morphology affects the electrostatic properties of these complex cells, a validation study was conducted to study the polarizability tensors of stem cell morphologies, using three independent computational techniques. To draw accurate conclusions, the polarizability tensors of more than 1000 stem cells were calculated and statistical analysis was conducted to identify which growth environment generates cells with similar electric properties. Next, we studied the induced transmembrane voltage (ITV) across the cell membrane when it is subjected to a static and dynamic external electrical stimulus. The ITV generated across a cell’s membrane plays a significant role in the process of electroporation since the membrane permeability increases when the ITV exceeds a certain threshold. By setting an arbitrary ITV electroporation threshold, the electroporated area for each stem cell morphology at different orientations was calculated and significant differences were shown in comparison to spherical cells of similar size. The significance of morphological variation is more prominent for dynamic frequency-dependent excitation. Using computational experiments, it has been observed that as the frequency of the excitation increases, the ITV decreases beyond a certain cutoff frequency that varies with cell morphology. While the ITV study is vital in low-frequency applications, the nonlinear membrane dynamics must be taken into consideration in case of high intensity ultra-short electrical stimulus, commonly used in supra-electroporation techniques to penetrate the internal organelles’ (i.e. nucleus, mitochondria, etc.) membrane. As a consecutive step of this research, a computational testbed was developed upon the stem cell geometries to investigate the supra-electroporation phenomenon in realistic cell shapes. The results obtained from this study suggest that supra-electroporation is highly dependent on the cell membrane irregularity, especially the location of the internal organelle with respect to any protrusion on the cell surface. The results from this observation can be utilized to engineer selective targeting of the desired cell with specific morphology.Analysis of different computational techniques for calculating the polarizability tensors of stem cells with realistic three-dimensional morphologies -- Induced transmembrane voltage in realistic three-dimensional morphologies of stem cells -- A computational study to investigate the effect of cell morphology in supra-electroporation using 2D cross-sections of realistic cell geometries -- 3D computational study of localized electroporation with realistic cell morphology -- Conclusion and future researc

Characterizing the diffusional behavior and trafficking pathways of Kv2.1 using single particle tracking in live cells

2013 Spring.Includes bibliographical references.Studying the diffusion pattern of membrane components yields valuable information regarding membrane structure, organization, and dynamics. Single particle tracking serves as an excellent tool to probe these events. We are investigating of the dynamics of the voltage gated potassium channel, Kv2.1. Kv2.1 uniquely localizes to stable, micro-domains on the cell surface where it plays a non-conducting role. The work reported here examines the diffusion pattern of Kv2.1 and determines alternate functional roles of surface clusters by investigating recycling pathways using single particle tracking in live cells. The movement of Kv2.1 on the cell surface is found to be best modeled by the combination of a stationary and non-stationary process, namely a continuous time random walk in a fractal geometry. Kv2.1 surface structures are shown to be specialized platforms involved in trafficking of Kv channels to and from the cell surface in hippocampal neurons and transfected HEK cells. Both Kv2.1 and Kv1.4, a non-clustering membrane protein, are inserted and retrieved from the plasma membrane at the perimeter of Kv2.1 clusters. From the distribution of cluster sizes, using a Fokker-Planck formalism, we find there is no evidence of a feedback mechanism controlling Kv2.1 domain size on the cell surface. Interestingly, the sizes of Kv2.1 clusters are rather governed by fluctuations in the endocytic and exocytic machinery. Lastly, we pinpoint the mechanism responsible for inducing Kv2.1 non-ergodic dynamics: the capture of Kv2.1 into growing clathrin-coated pits via transient binding to pit proteins

Novel miniaturised and highly versatile biomechatronic platforms for the characterisation of melanoma cancer cells

There has been an increasing demand to acquire highly sensitive devices that are able to detect and characterize cancer at a single cell level. Despite the moderate progress in this field, the majority of approaches failed to reach cell characterization with optimal sensitivity and specificity. Accordingly, in this study highly sensitive, miniaturized-biomechatronic platforms have been modeled, designed, optimized, microfabricated, and characterized, which can be used to detect and differentiate various stages of melanoma cancer cells. The melanoma cell has been chosen as a legitimate cancer model, where electrophysiological and analytical expression of cell-membrane potential have been derived, and cellular contractile force has been obtained through a correlation with micromechanical deflections of a miniaturized cantilever beam. The main objectives of this study are in fourfold: (1) to quantify cell-membrane potential, (2) correlate cellular biophysics to respective contractile force of a cell in association with various stages of the melanoma disease, (3) examine the morphology of each stage of melanoma, and (4) arrive at a relation that would interrelate stage of the disease, cellular contractile force, and cellular electrophysiology based on conducted in vitro experimental findings. Various well-characterized melanoma cancer cell lines, with varying degrees of genetic complexities have been utilized. In this study, two-miniaturized-versatile-biomechatronic platforms have been developed to extract the electrophysiology of cells, and cellular mechanics (mechanobiology). The former platform consists of a microfluidic module, and stimulating and recording array of electrodes patterned on a glass substrate, forming multi-electrode arrays (MEAs), whereas the latter system consists of a microcantilever-based biosensor with an embedded Wheatstone bridge, and a microfluidic module. Furthermore, in support of this work main objectives, dedicated microelectronics together with customized software have been attained to functionalize, and empower the two-biomechatronic platforms. The bio-mechatronic system performance has been tested throughout a sufficient number of in vitro experiments.Open Acces

Characterization of Ambra1 heterozygous mice as genetic mouse model of female-specific autism

Autism is known as a heritable neurodevelopmental disorder, diagnosed prior to the age of three years in humans based on three major domains: (1) impairment in social interaction (2) communication deficits (3) restricted interests and repetitive behaviors. Since it is a very heterogeneous disorder with various causes and different combinations of phenotypes, it is also called autism spectrum disorder (ASD). Monogenic heritable forms of ASD enable us to develop genetic mouse models of autism in order to obtain mechanistic insight in this disorder. Ambra1 is a positive regulator of Beclin1, a major player in the formation of autophagosomes during the process of autophagy. While Ambra1 null mutation leads to embryonic lethality, we could show that Ambra1 heterozygous mice (Ambra1+/-) display autism-like behavior only in females. Purpose of this thesis was therefore to characterize this mouse line further. It turned out that communication deficits, measured by ultrasound vocalization, start in the neonatal stage of females, while physical or neurological development is normal in Ambra1+/-. Female Ambra1 mutants had a stronger reduction in Ambra1 expression than male mutants, which gives first hints of the female-specific autism-like behavior in this mouse line. Mild enlargement of whole brain and hippocampus was detected in both Ambra1+/- males and females, with no change of ventricle size. Since β-galactosidase, used as reporter expressed under the Ambra1 promoter, was found only in neuronal cells, I focused on understanding the neural mechanism of its phenotype. Short-term and long-term synaptic plasticity in the hippocampus was normal for males and females of both genotypes. However, the power of gamma oscillations (γ-power), indicative of change in the balance of excitation and inhibition, was age-dependently altered in Ambra1+/- females only. However, this difference was not detected in male. Moreover, increased susceptibility to seizures, a known comorbid condition of ASD was restricted to females, suggesting an association between autism-like behavior, gamma oscillation and seizure propensity in female Ambra1+/- mice. Next, I approached the neuronal substrate of these three phenotypes by morphological analysis of hippocampal pyramidal neurons, such as dendritic arborization and synapse number. A genotype-associated difference of dendritic arborization was detected in neither males nor females. The quantification of spines or synapses and cellular electrophysiology are still on-going. First signals point to an imbalance between excitation and inhibition as a cause of the female autism-like behavior in Ambra1+/- mice

Characterization of Ambra1 heterozygous mice as genetic mouse model of female-specific autism

Autism is known as a heritable neurodevelopmental disorder, diagnosed prior to the age of three years in humans based on three major domains: (1) impairment in social interaction (2) communication deficits (3) restricted interests and repetitive behaviors. Since it is a very heterogeneous disorder with various causes and different combinations of phenotypes, it is also called autism spectrum disorder (ASD). Monogenic heritable forms of ASD enable us to develop genetic mouse models of autism in order to obtain mechanistic insight in this disorder. Ambra1 is a positive regulator of Beclin1, a major player in the formation of autophagosomes during the process of autophagy. While Ambra1 null mutation leads to embryonic lethality, we could show that Ambra1 heterozygous mice (Ambra1+/-) display autism-like behavior only in females. Purpose of this thesis was therefore to characterize this mouse line further. It turned out that communication deficits, measured by ultrasound vocalization, start in the neonatal stage of females, while physical or neurological development is normal in Ambra1+/-. Female Ambra1 mutants had a stronger reduction in Ambra1 expression than male mutants, which gives first hints of the female-specific autism-like behavior in this mouse line. Mild enlargement of whole brain and hippocampus was detected in both Ambra1+/- males and females, with no change of ventricle size. Since β-galactosidase, used as reporter expressed under the Ambra1 promoter, was found only in neuronal cells, I focused on understanding the neural mechanism of its phenotype. Short-term and long-term synaptic plasticity in the hippocampus was normal for males and females of both genotypes. However, the power of gamma oscillations (γ-power), indicative of change in the balance of excitation and inhibition, was age-dependently altered in Ambra1+/- females only. However, this difference was not detected in male. Moreover, increased susceptibility to seizures, a known comorbid condition of ASD was restricted to females, suggesting an association between autism-like behavior, gamma oscillation and seizure propensity in female Ambra1+/- mice. Next, I approached the neuronal substrate of these three phenotypes by morphological analysis of hippocampal pyramidal neurons, such as dendritic arborization and synapse number. A genotype-associated difference of dendritic arborization was detected in neither males nor females. The quantification of spines or synapses and cellular electrophysiology are still on-going. First signals point to an imbalance between excitation and inhibition as a cause of the female autism-like behavior in Ambra1+/- mice