Study of Speed and Force in Biomanipulation

Ph.DDOCTOR OF PHILOSOPH

Three dimensional mechanical modelling and analysis of embryos microinjection.

This research has developed a novel 3D particle based cell model that is able to accurately estimate the membrane deformation and indentation force in bio-micromanipulation of an individual cell, especially embryos microinjection practice

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

Force Sensing and Control in Micromanipulation

Ph.DDOCTOR OF PHILOSOPH

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

Force-controlled Biomanipulation for Biological Cell Mechanics Studies

Ph.DDOCTOR OF PHILOSOPH

Deep learning image recognition enables efficient genome editing in zebrafish by automated injections

<div><p>One of the most popular techniques in zebrafish research is microinjection. This is a rapid and efficient way to genetically manipulate early developing embryos, and to introduce microbes, chemical compounds, nanoparticles or tracers at larval stages. Here we demonstrate the development of a machine learning software that allows for microinjection at a trained target site in zebrafish eggs at unprecedented speed. The software is based on the open-source deep-learning library Inception v3. In a first step, the software distinguishes wells containing embryos at one-cell stage from wells to be skipped with an accuracy of 93%. A second step was developed to pinpoint the injection site. Deep learning allows to predict this location on average within 42 μm to manually annotated sites. Using a Graphics Processing Unit (GPU), both steps together take less than 100 milliseconds. We first tested our system by injecting a morpholino into the middle of the yolk and found that the automated injection efficiency is as efficient as manual injection (~ 80%). Next, we tested both CRISPR/Cas9 and DNA construct injections into the zygote and obtained a comparable efficiency to that of an experienced experimentalist. Combined with a higher throughput, this results in a higher yield. Hence, the automated injection of CRISPR/Cas9 will allow high-throughput applications to knock out and knock in relevant genes to study their mechanisms or pathways of interest in diverse areas of biomedical research.</p></div

Cell adhesion and cell mechanics during zebrafish development

During vertebrate development, gastrulation leads to the formation of three distinct germlayers. In zebrafish a central process is the delamination and the ingression of single cells from a common ancestor tissue - that will lead to the formation of the germlayers. Several molecules have been identified to regulate this process but the precise cellular mechanisms are poorly understood. Differential adhesiveness, a concept first introduced by Steinberg over 40 years ago, has been proposed to represent a key phenomena by which single hypoblast cells separate from the epiblast to form the mesendoderm at later stages. In this work it is shown that differential adhesion among the germlayer progenitor cells alone cannot predict germlayer formation. It is a combination of several mechanical properties such as cell cortex tension, cell adhesion and membrane mechanical properties that influence the migratory behavior of the constituent cells

Generation of new transgenic zebrafish lines for studying neuronal circuits underlying behavior in zebrafish

A central goal of Neuroscience is to understand how the brain processes sensory stimuli and generates behavioral responses. To achieve this goal, it is crucial to monitor and manipulate the neuronal activity of single cells in real time, as well as to study the neuronal circuits and their connections throughout the whole-brain in a behaving animal. Thus, it is important to use a genetically tractable model organism with a relatively simple nervous system but with robust behavior. Zebrafish has become a promising model organism in the study of nervous system. The accessibility and optical transparency of embryos and larvae make possible the expression and visualization of genetically encoded fluorescent reporters, through transgenic techniques. In this work, recent genetically encoded fluorescent reporters (LSSmOrange, mScarlet and GCaMP6fEF05) were used for establishing new transgenic zebrafish lines, through the Tol2 transposon system. These lines will be used to study the communication between different populations of neurons and to record neural activity during behavioral responses. The generation of new genetic tools allied to the development of sophisticated imaging techniques has opened up the possibility of whole-brain imaging with single-cell resolution and high temporal precision. In the coming years, the challenge will be to combine the approaches developed and currently used in zebrafish to understand how behaviors are generated in higher vertebrates