Ros-Drill Automation: Visual Feedback Control And Rotational Motion Tracking

ICSI (intra-cytoplasmic sperm injection) has attracted research interest from both biological and engineering groups. The technology is constantly evolving to perform this procedure with precision and speed. One such development is the contribution of this thesis. We focus on a relatively recent procedure called Ros-Drill© (rotationally oscillating drill), of which the early versions have already been effectively utilized for the mice. In the first part, we present a procedure to automate a critical part of the operation: initiation of the rotational oscillation, Visual feedback is used to track the pipette tip. Predetermined species-specific penetration depth is successfully utilized to initiate the rotational oscillation command. Penetration-depth-based decisions concur with a curvature-based approach. In the second part for the automation we improve the performance of the rotational motion tracking. Ros-Drill© is an inexpensive set-up, which creates high-frequency rotational oscillations at the tip of an injection pipette tracking a harmonic motion profile. These rotational oscillations enable the pipette to drill into cell membranes with minimum biological damage. Such a motion control procedure presents no particular difficulty when it uses sufficiently precise motion sensors. However, size, costs and accessibility of technology on hardware components may severely constrain the sensory capabilities. Then the trajectory tracking is adversely affected. In this thesis we handle such a practical case, and present hardware and software improvements using a commonly available microcontroller and extremely low-resolution position measurements. Biological tests are performed and it is confirmed that the mechanical structure plays a crucial role for success

The Role of MEMS in In-Vitro-Fertilization

The assisted reproduction has been considered a viable solution for the infertility of humankind for more than four decades. In-Vitro-Fertilization (IVF) is one of the most successful assisted reproduction techniques, where the reproductive cell of the female partner is fertilized outside of her body. Initially, the IVF process has been conducted manually by an experienced embryologist. However, even with a highly experienced individual, the operation had extremely lower success rates due to the limited control in environmental conditions and the requirement of precise movements. Therefore, to address this technological deficit, the feasibility of the mechatronics devices for IVF procedures has been investigated. Among the different mechatronics concepts, micro-electromechanical system (MEMS) technologies have been gradually attracted to the IVF process and improved its capabilities. The purpose of this paper is to present a brief overview of the role of MEMS technologies in IVF. The article classifies the MEMS technologies in IVF based on their application in order to emphasize its contribution. In addition, the article extensively discusses the state-of-the-art mechatronic techniques utilized in Intracytoplasmic Sperm Injection (ICSI), one of the most popular techniques used in IVF. This review article expects to become extremely beneficial for the engineering researchers new to this field who seek critical information on IVF in simple terms with highlights on the possible advancements and challenges that may emerge in the future

Dynactin-dependent cortical dynein and spherical spindle shape correlate temporally with meiotic spindle rotation in Caenorhabditis elegans.

Oocyte meiotic spindles orient with one pole juxtaposed to the cortex to facilitate extrusion of chromosomes into polar bodies. In Caenorhabditis elegans, these acentriolar spindles initially orient parallel to the cortex and then rotate to the perpendicular orientation. To understand the mechanism of spindle rotation, we characterized events that correlated temporally with rotation, including shortening of the spindle in the pole-to pole axis, which resulted in a nearly spherical spindle at rotation. By analyzing large spindles of polyploid C. elegans and a related nematode species, we found that spindle rotation initiated at a defined spherical shape rather than at a defined spindle length. In addition, dynein accumulated on the cortex just before rotation, and microtubules grew from the spindle with plus ends outward during rotation. Dynactin depletion prevented accumulation of dynein on the cortex and prevented spindle rotation independently of effects on spindle shape. These results support a cortical pulling model in which spindle shape might facilitate rotation because a sphere can rotate without deforming the adjacent elastic cytoplasm. We also present evidence that activation of spindle rotation is promoted by dephosphorylation of the basic domain of p150 dynactin

A Rotating Spiral Micromotor for Noninvasive Zygote Transfer

Embryo transfer (ET) is a decisive step in the in vitro fertilization process. In most cases, the embryo is transferred to the uterus after several days of in vitro culture. Although studies have identified the beneficial effects of ET on proper embryo development in the earlier stages, this strategy is compromised by the necessity to transfer early embryos (zygotes) back to the fallopian tube instead of the uterus, which requires a more invasive, laparoscopic procedure, termed zygote intrafallopian transfer (ZIFT). Magnetic micromotors offer the possibility to mitigate such surgical interventions, as they have the potential to transport and deliver cellular cargo such as zygotes through the uterus and fallopian tube noninvasively, actuated by an externally applied rotating magnetic field. This study presents the capture, transport, and release of bovine and murine zygotes using two types of magnetic micropropellers, helix and spiral. Although helices represent an established micromotor architecture, spirals surpass them in terms of motion performance and with their ability to reliably capture and secure the cargo during both motion and transfer between different environments. Herein, this is demonstrated with murine oocytes/zygotes as the cargo; this is the first step toward the application of noninvasive, magnetic micromotor‐assisted ZIFT

Sperm-Driven Micromotors Moving in Oviduct Fluid and Viscoelastic Media

Biohybrid micromotors propelled by motile cells are fascinating entities for autonomous biomedical operations on the microscale. Their operation under physiological conditions, including highly viscous environments, is an essential prerequisite to be translated to in vivo settings. In this work, a sperm-driven microswimmer, referred to as a spermbot, is demonstrated to operate in oviduct fluid in vitro. The viscoelastic properties of bovine oviduct fluid (BOF), one of the fluids that sperm cells encounter on their way to the oocyte, are first characterized using passive microrheology. This allows to design an artificial oviduct fluid to match the rheological properties of oviduct fluid for further experiments. Sperm motion is analyzed and it is confirmed that kinetic parameters match in real and artificial oviduct fluids, respectively. It is demonstrated that sperm cells can efficiently couple to magnetic microtubes and propel them forward in media of different viscosities and in BOF. The flagellar beat pattern of coupled as well as of free sperm cells is investigated, revealing an alteration on the regular flagellar beat, presenting an on–off behavior caused by the additional load of the microtube. Finally, a new microcap design is proposed to improve the overall performance of the spermbot in complex biofluids. © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinhei

Custom-Designed Biohybrid Micromotor for Potential Disease Treatment

Micromotors are recognized as promising candidates for untethered micromanipulation and targeted cargo transport. Their future application is, however, hindered by the low efficiency of drug encapsulation and their poor adaptability in physiological conditions. To address these challenges, one potential solution is to incorporate micromotors with biological materials as the combination of functional biological entities and smart artificial parts represents a manipulable and biologically friendly approach. This dissertation focuses on the development of custom-designed micromotors combined with sperm and their potential applications on targeted diseases treatment. By means of 2D and 3D lithography methods, microstructures with complex configurations can be fabricated for specific demands. Bovine and human sperm are both for the first time explored as drug carriers thanks to their high encapsulation efficiency of hydrophilic drugs, their powerful self-propulsion and their improved drug-uptake relying on the somatic-cell fusion ability. The hybrid micromotors containing drug loaded sperm and constructed artificial enhancements can be self-propelled by the sperm flagella and remotely guided and released to the target at high precision by employing weak external magnetic fields. As a result, micromotors based on both bovine and human sperm show significant anticancer effect. The application here can be further broadened to other biological environments, in particular to the blood stream, showing the potential on the treatment of blood diseases like blood clotting. Finally, to enhance the treatment efficiency, in particular to control sperm number and drug dose, three strategies are demonstrated to transport swarms of sperm. This research paves the way for the precision medicine based on engineered sperm-based micromotors

Doctor of Philosophy

dissertationMicrofluidic technology has the unique potential to separate sperm from unwanted debris while improving the effectiveness of assisted reproductive technologies (ART). Limitations of current clinical protocols regarding separation of sperm from other cells and cellular debris can lead to low sperm recovery when the sample contains low concentrations of mostly low motility sperm and a high concentration of unwanted cells or cellular debris, such as occurs with surgical testis dissection samples from nonobstructive azoospermia (NOA) patients who have undergone microsurgical testicular sperm extraction (mTESE), and semen samples from leukospermia patients (high white blood cell (WBC) semen). Over the years, most microfluidic sperm separation approaches have relied on sperm motility for separation with added features through which only highly motile sperm can pass. Thus, these techniques can separate only progressive motile sperm from semen samples, but they lose a significant number of sperm cells including viable nonprogressive motile and nonmotile sperm. This dissertation demonstrates label-free separation of sperm from challenging sperm samples using inertial microfluidics. The approach does not require any externally applied forces except the movement of the fluid sample through the instrument. In this way, it is possible to recover not only any motile sperm, but also viable less-motile and nonmotile sperm with high recovery rates. The results show the usefulness of inertial microfluidics to significantly reduce the concentrations of unwanted cells/cellular debris (Red blood cells/White blood cells) significantly by flow focusing of debris within a spiral channel flow. The majority (∼80%) of sperm cells collect to the designated outlet and ∼98% of debris goes to the waste outlet. The estimated sample process time is more rapid (∼5minutes) and autonomous than conventional methods which may take between ∼1 hour (semen purification) and 10 ∼18 hours (manual mTESE sample search process). The flow focusing results of sperm and blood cells included that sharp flow focusing of RBC and WBC, but not of sperm cell where sharp flow focusing didn't appear. The successful flow focusing of RBC and WBC imply that the spherical model did accurately predict the behavior of RBCs and WBCs, but the lack of definitive focusing of sperm cells imply that the modeling of sperm cells wasn't accurate. This partial success of sperm modeling was caused by a lack of understanding of sperm behavior in the curved channel. This dissertation presents an improved model of sperm cell behavior in curved channels based on both 2D COMSOL® simulations and experimental studies. The results show promising evidence that the proposed method should able to generate more precise sperm separation for mTESE samples. Lastly this dissertation also performed viability, toxicity, and recovery tests on the proposed sperm separation method for biocompatibility verification. These tests should provide initial validation of clinical usefulness

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Department of Biomedical EngineeringThe optical imaging has a critical role in biomedical research to analyze functional and morphological variation of an organ, tissue and even a single cell of animal models. Since the optical imaging modality has features of indirect access, volumetric analysis and high resolution, it has been used for biomedical analysis. Especially, as a low coherence interferometric imaging technique, optical coherence tomography (OCT) has been applied in scientific and medical fields from few decades ago. Since OCT can provide endogenous contrast of biological tissue using the infrared light source, it has high potential to be applied in practical medical diagnosis. However, it is hard to acquire uneven or thick sample due to the limited imaging window and penetration depth. To overcome those limitations, lots of optical, mathematical and chemical solutions comes within a decade such as adaptive optics, full-range method and tissue clearing. Despite the existence of suggested solutions, practical application of OCT is limitation due to the cost of time and effort. Here, we present practical methods to enhance acquirable endogenous information of sample through versatile scanning optical coherence tomography(VS-OCT). Conventional OCT utilizes dual-axis based flat focal plane scanning method providing limited depth information of curved samples. Thus, we developed advanced OCT, called VS-OCT, which can fully optimize imaging window by changing focal plane to dual plane and cylindrical plane. The VS-OCT is demonstrated for 1) quantification of engineered skin, 2) monitoring of tadpole development, 3) screening phenotype of zebrafish and 4) quantification of spinal cord injury (SCI) of mouse.ope