Micromanipulation Techniques Research Articles

Establishment of CRISPR-Cas9 ribonucleoprotein mediated MSTN gene edited pregnancy in buffalo: Compare cells transfection and zygotes electroporation

Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.

Noble Dome: A Novel Air-Free Transfer System For Battery Research with Electron Microscopy

"Noble Dome: A Novel Air-Free Transfer System" introduces an innovative solution for loading air-sensitive samples into SEM/FIB instruments. Traditional loading methods expose samples to atmosphere during transfer, hindering investigations of reactive materials. The presented prototype, Noble Dome, directly attaches to the SEM/FIB, enabling sample transfer in an oxygen-free environment. The device's design and operational process are outlined, including airtight chambers, gas introduction, and modified venting systems. Experimental results demonstrate Noble Dome's effectiveness in preserving sample integrity. Compared to existing solutions, which often pose limitations, Noble Dome offers a practical approach for various research needs.Imaging and micromanipulation techniques such as Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB) can reveal critical information about the microstructure and composition of materials. To utilize these techniques, a sample must be loaded into the chamber of the instrument which is then pumped down to low vacuum conditions. In the process of removing a sample from its packaging and mounting it on the SEM/FIB stage, the sample is briefly exposed to atmosphere. This is problematic for researchers who are interested in investigating air sensitive samples or samples for which atmospheric contamination would detract from the investigation. This set of samples includes metals commonly used as battery materials such as lithium and sodium, which are highly reactive with elements in the air such as moisture or oxygen.We present a prototype for a glove box load lock that addresses this problem directly. This device attaches directly onto the side of a SEM/FIB and allows researchers to remove a sample from packaging and transfer it directly on the stage via an accessory port of the SEM/FIB in an oxygen-free environment and then pump the tool down to vacuum without any atmospheric exposure in the process, eliminating the use of an intermediary shuttle from a separate glove box. We call this device Noble Dome.In order to test Noble Dome’s ability to protect samples from atmosphere, we imaged and collected EDS spectra on the same location of a lithium metal sample in a pristine state, after sitting in Noble Dome for 30 minutes, and after sitting in atmosphere for 30 minutes.We note that there was very little change in the surface topography and oxygen counts between the sample in its pristine state and the sample after sitting in Noble Dome for 30 minutes. However, there is a marked increase in surface oxidation and topography after the sample sat in atmosphere for 30 minutes.Lithium-ion batteries are increasingly prolific in the landscape of modern technology. In order to improve the energy density, life cycle and safety of lithium-ion batteries, researchers require significant amounts of materials characterization pre-assembly, post-assembly and after several charge/discharge cycles. Electron microscopy is crucial for effective materials characterization, which necessitates a solution for loading samples into the vacuum chamber of the microscope without exposing these reactive samples to air.Other solutions currently on the market for loading air sensitive samples into a SEM/FIB involve manipulating the sample in a glove box which is separate from the instrument, loading the sample into an inert gas transfer shuttle which is closed by remote control, removing the shuttle from the glovebox, loading the shuttle into the SEM/FIB, and then opening the sample again via remote control. These solutions are not ideal because they're very expensive, have a variety of limitations on sample size, stage travel, and signal strength, and are high tech solutions prone to costly malfunction. These drawbacks highlight the demand for a more practical and efficient approach.The presented prototype, Noble Dome, addresses the challenge of loading air-sensitive samples into SEM/FIB instruments with a practical and innovative approach. The device eliminates the need for an intermediary glove box and inert gas transfer shuttle, allowing direct sample transfer onto the stage in an oxygen-free environment. The results demonstrate Noble Dome's efficacy in preserving sample integrity by minimizing atmospheric exposure, as evidenced by the minimal changes observed in surface topography and composition compared to samples exposed to atmosphere. Noble Dome enables research on reactive samples, used to pursue a carbon-free future. Figure 1

Bio-assembling and Bioprinting for Engineering Microvessels from the Bottom Up.

Blood vessels are essential in transporting nutrients, oxygen, metabolic wastes, and maintaining the homeostasis of the whole human body. Mass of engineered microvessels is required to deliver nutrients to the cells included in the constructed large three-dimensional (3D) functional tissues by diffusion. It is a formidable challenge to regenerate microvessels and build a microvascular network, mimicking the cellular viabilities and activities in the engineered organs with traditional or existing manufacturing techniques. Modular tissue engineering adopting the “bottom-up” approach builds one-dimensional (1D) or two-dimensional (2D) modular tissues in micro scale first and then uses these modules as building blocks to generate large tissues and organs with complex but indispensable microstructural features. Building the microvascular network utilizing this approach could be appropriate and adequate. In this review, we introduced existing methods using the “bottom-up” concept developed to fabricate microvessels including bio-assembling powered by different micromanipulation techniques and bioprinting utilizing varied solidification mechanisms. We compared and discussed the features of the artificial microvessels engineered by these two strategies from multiple aspects. Regarding the future development of engineering the microvessels from the bottom up, potential directions were also concluded.

Symmetry-group-protected microfluidics

Modern micromanipulation techniques typically involve trapping using electromagnetic, acoustic, or flow fields that produce stresses on the trapped particles thereby precluding stress-free manipulations. Here, we show that by employing polyhedral symmetries in a multichannel microfluidic design, we can separate the tasks of displacing and trapping a particle into two distinct sets of flow operations, each characterized and protected by their unique groups of symmetries. By combining only the displacing uniform flow modes to entrain and move targeted particles in arbitrary directions, we were able to realize symmetry-protected, stress-free micromanipulation in 3D. Furthermore, we engineered complex, microscale paths by programming and controlling the flow within each channel in real time, resulting in multiple particles simultaneously following desired paths in the absence of any supervision or feedback. Our work therefore provides a general symmetry-group-based framework for understanding and engineering microfluidics and a novel platform for 3D stress-free manipulations. Published by the American Physical Society 2024

Unravelling the Biophysical Properties of Chromatin Proteins and DNA Using Acoustic Force Spectroscopy.

Acoustic force spectroscopy (AFS) is a single-molecule micromanipulation technique that uses sound waves to exert force on surface-tethered DNA molecules in a microfluidic chamber. As large numbers of individual protein-DNA complexes are tracked in parallel, AFS provides insight into the individual properties of such complexes as well as their population averages. In this chapter, we describe in detail how to perform AFS experiments specifically on bare DNA, protein-DNA complexes, and how to extract their (effective) persistence length and contour length from force-extension relations.

Chromosomal analysis of single sperm cells from infertile couples with severe oligoteratozoospermia: A cross-sectional prospective study.

In this cross-sectional prospective study, advanced next-generation sequencing technology was used to compare the molecular karyotyping of individual human sperm cells in infertile couples with severe oligoteratozoospermia (i.e., low sperm count and motility) to those of infertile couples with normal semen. Fourteen infertile couples who were patients at Ramathibodi Hospital in Bangkok, Thailand, were recruited from January to November 2023, and they were categorized into two groups based on semen analysis results. The study group comprised couples with severe oligoteratozoospermia, whereas the control group exhibited normal semen. Individual sperm cells from the semen samples were isolated by the micromanipulation technique for subsequent whole-genome amplification and next-generation sequencing, where the primary outcome was the aneuploidy rate. Seventy individual sperm cells were isolated with a 90% success rate for amplification. The next-generation sequencing results showed that the aneuploidy rate was 25%-75%, with a mean of 48.28% in the study group. In contrast, the control group exhibited aneuploidy rates of 0-75%, with a mean of 15.15%. The difference between the two groups was statistically significant (odds ratio: 5.8, 95% confidence interval: 1.30-26.03). Sperm cells of the study group showed a threefold higher aneuploidy rate than those in the control group, even though the sperm cells were selected by micromanipulation for their normal morphology. Comprehensive counseling is recommended to address elevated aneuploidy rates that potentially surpass those of the general infertile population. Guidance on preimplantation genetic testing is also recommended to ensure the transfer of embryos with normal chromosomes.

Membrane Tubulation with a Biomembrane Force Probe.

Tubulation is a common cellular process involving the formation of membrane tubes ranging from 50 nm to 1 µm in diameter. These tubes facilitate intercompartmental connections, material transport within cells and content exchange between cells. The high curvature of these tubes makes them specific targets for proteins that sense local geometry. In vitro, similar tubes have been created by pulling on the membranes of giant unilamellar vesicles. Optical tweezers and micromanipulation are typically used in these experiments, involving the manipulation of a GUV with a micropipette and a streptavidin-coated bead trapped in optical tweezers. The interaction forms streptavidin/biotin bonds, leading to tube formation. Here, we propose a cost-effective alternative using only micromanipulation techniques, replacing optical tweezers with a Biomembrane Force Probe (BFP). The BFP, employing a biotinylated erythrocyte as a nanospring, allows for the controlled measurement of forces ranging from 1 pN to 1 nN. The BFP has been widely used to study molecular interactions in cellular processes, extending beyond its original purpose. We outline the experimental setup, tube formation and characterization of tube dimensions and energetics, and discuss the advantages and limitations of this approach in studying membrane tubulation.

Review of Ultrasonic Particle Manipulation Techniques: Applications and Research Advances.

Ultrasonic particle manipulation technique is a non-contact label-free method for manipulating micro- and nano-scale particles using ultrasound, which has obvious advantages over traditional optical, magnetic, and electrical micro-manipulation techniques; it has gained extensive attention in micro-nano manipulation in recent years. This paper introduces the basic principles and manipulation methods of ultrasonic particle manipulation techniques, provides a detailed overview of the current mainstream acoustic field generation methods, and also highlights, in particular, the applicable scenarios for different numbers and arrangements of ultrasonic transducer devices. Ultrasonic transducer arrays have been used extensively in various particle manipulation applications, and many sound field reconstruction algorithms based on ultrasonic transducer arrays have been proposed one after another. In this paper, unlike most other previous reviews on ultrasonic particle manipulation, we analyze and summarize the current reconstruction algorithms for generating sound fields based on ultrasonic transducer arrays and compare these algorithms. Finally, we explore the applications of ultrasonic particle manipulation technology in engineering and biological fields and summarize and forecast the research progress of ultrasonic particle manipulation technology. We believe that this review will provide superior guidance for ultrasonic particle manipulation methods based on the study of micro and nano operations.

P-030 Clinical outcomes of microdissection testicular sperm extraction (micro TESE) and intracytoplasmic sperm injection (ICSI) in non-obstructive azoospermia (NOA) with the history of cryptorchidism

Abstract Study question What is sperm retrieval rate (SRR) by micro TESE and the clinical outcomes using testicular sperm in couples with the history of cryptorchidism? Summary answer NOA couples with the history of cryptorchidism had a higher SRR by micro TESE but lower clinical outcomes by ICSI compared of unexplained NOA. What is known already Undescended testis (UT) which is exposed to a higher temperature compared with the scrotal temperature is associated with impairment of germ cell maturation, and progressive Leydig and Sertoli cell atrophy, and subsequent infertility in adulthood. There have been very few studies of ICSI with a focus on, or large enough numbers to examine, the specific outcomes associated with male factor infertility, however improvement in sperm retrieval techniques including micro TESE and micromanipulation techniques, such as ICSI, has led to excellent fertilization and pregnancy outcomes of treatment cycles. Study design, size, duration We performed a retrospective study based on two reproduction centers in Japan and evaluated 1521 azoospermic patients in our clinics between September 2013 and December 2022. We investigated SRR by micro TESE in these patients and therefore aimed to evaluate the prevalence and the significance of ICSI outcomes with embryonic development in NOA couples with the history of cryptorchidism. Participants/materials, setting, methods We evaluated SRR of micro TESE, two pronuclei (2PN) oocyte rates, blastocyst development, good-quality blastocyst (Grade 3BB and above on day 5 by the Gardner scoring), and clinical pregnancy rates per embryo transfer (ET) in 72 NOA cases with the history of UT, 953 cases of unexplained NOA, not including after orchidopexy, Klinefelter syndrome, cryptozoospermia, mumps orchitis, and 284 cases of obstructive azoospermia (OA). Statistical analysis was performed using unpaired t-tests and chi-squared tests. Main results and the role of chance SRR of first attempt micro TESE in UT (44/56=78.6%) was higher than unexplained NOA (160/748=21.4%) (p < 0.001). Spermatozoa were successfully retrieved in 13 of 22 (59.1%) UT group and 36 of 226 (15.9%) unexplained NOA who had previously undergone micro TESE with no sperm found. No correlation was found between serum FSH, LH, and T level with the success of sperm retrieval. Testicular volume and patient age at orchidopexy also did not affect the SRR for micro TESE. 2PN oocytes, blastocysts development, and good-quality blastocysts rates were 45.5%, 47.4%, and 18.9% in UT, 51.2%, 45.0%, and 20.9% in unexplained NOA, and 62.6%, 52.1%, and 23.7% in OA, respectively. Clinical pregnancy rates per ET were 29.3% in UT, 32.3% in unexplained NOA, and 41.6% in OA. Significant differences were only observed in 2PN oocytes between unexplained NOA and OA (p < 0.05) and clinical pregnancy between UT and unexplained NOA (p < 0.05), and OA and unexplained NOA (p < 0.01). Several UT patients showed a very small number of spermatozoa and even only immotile sperm of retrieved by micro TESE. Limitations, reasons for caution We included the patients only after a surgery of cryptorchidism, but not a delayed testicular descent without surgery. The cohort size of this study is not small, however, our screened population of azoospermic patients may be biased. Wider implications of the findings This study shows a high impact in micro TESE and ICSI outcomes with embryonic development for the NOA couples with the history of cryptorchidism. Our study emphasizes that history of cryptorchidism provides clinically valuable prognostic information to couples considering surgical sperm retrieval. Trial registration number not applicable

Viscoelastic Properties of Polymeric Microneedles Determined by Micromanipulation Measurements and Mathematical Modelling

Microneedles, including dissolvable ones made from biocompatible and biodegradable materials, have been widely studied and can potentially be used for transdermal drug delivery, disease diagnosis (sampling), skin care, etc. Characterizing their mechanical properties is essential, as being mechanically strong enough to pierce the skin barrier is one of the most fundamental and crucial requirements for them. The micromanipulation technique was based on compressing single microparticles between two flat surfaces to obtain force and displacement data simultaneously. Two mathematical models had already been developed to calculate the rupture stress and apparent Young's modulus, which can identify variations of these parameters in single microneedles within a microneedle patch. In this study, a new model has been developed to determine the viscoelasticity of single microneedles made of hyaluronic acid (HA) with a molecular weight of 300 kDa loaded with lidocaine by using the micromanipulation technique to gather experimental data. The modelling results from the micromanipulation measurements suggest that the microneedles were viscoelastic and their mechanical behaviour was strain-rate dependent, which implies that the penetration efficiency of viscoelastic microneedles can be improved by increasing their piercing speed into the skin.

A novel technique for large-fragment knock-in animal production without ex vivo handling of zygotes

CRISPR/Cas-based genome editing has dramatically improved genetic modification technology. In situ electroporation called genome editing via oviductal nucleic acid delivery (GONAD), which eliminates the need for ex vivo embryo handling, is technically the simplest method for gene transfer and can be performed in laboratories without developmental engineering expertise including micromanipulation techniques. However, the use of this method remains challenging in the case of large-fragment knock-in, such as gene expression cassettes. Adeno-associated viruses (AAV) act as donor DNA for homologous recombination in infected cells, including rodent embryos. In this study, we demonstrated simultaneous electroporation of AAV donors and CRISPR/Cas9 components into embryos to create knock-in animals, and successfully generated knock-in rats carrying a gene cassette with a length of 3.0 kb using a small number of animals and in situ electroporation. These findings indicate that this technique is an efficient high-throughput strategy for producing genetically modified rodents and may be applicable to other animal species.

Artificial Cells for Dissecting Exocytosis.

The fusion of vesicles and exocytosis release of neurotransmitters into the extracellular space for detection and chemical signal decoding by neighboring cells is the key process in neuronal communication. It is important to understand what regulates exocytosis because the amount of neurotransmitters released into the synaptic cleft has a direct impact on brain function such as cognition learning and memory as well as on brain malfunctions. Much success in molecular biology can be credited for the existence of simplified model systems. Therefore, for gaining deeper insights into the details of exocytosis and what controls vesicle-mediated neurotransmission, functional artificial cells for exocytosis have been developed that can be used for studying various biophysical aspects and roles of molecules affecting exocytosis, which is difficult to study in living cells. Here, we describe the design and fabrication of specific artificial cell models and how chemical measurements at these cells can be implemented for probing dynamics of the exocytosis fusion pore and its effect on the regulation of neurochemical release. We introduce bottom-up synthetic methods for constructing model cells using protein-free giant unilamellar vesicles (GUV) as starting material, which allows further tuning of molecular complexity in a manner that is not possible in living cells and therefore can be used for dissecting the role of essential molecular components affecting the exocytosis process. The experimental setup uses microscopy video recording, micromanipulation and microelectroinjection techniques, and amperometry detection to study neurotransmitter release from these cells mimicking exocytosis.

A Simple and Efficient Method for Generating KO Rats Using In Vitro Fertilized Oocytes.

The development of ZFN, TALEN, and CRISPR/Cas9 systems has simplified the process of generating knockout (KO) and knock-in (KI) rats in addition to mice. However, in rats, an efficient genome editing technique that uses in vitro fertilized oocytes has not been established. Recently, we reported the stable generation of offspring from five standard strains of rats by superovulation and in vitro fertilization (IVF). Furthermore, genome-edited rats can be easily generated by electroporation. First, juvenile female rats are administered LHRH (luteinizing hormone-releasing hormone) to synchronize the estrous cycle and then AIS (Automatic Identification System) with PMSG (pregnant mare serum gonadotropin) before hCG (human chorionic gonadotropin) for superovulation. Sperm collected from a sexually mature male rat the following morning is then pre-cultured. Cumulus cell-oocyte complexes (COCs) are collected from female rats under anesthesia, and COCs are induced into a medium containing concentration-adjusted sperm. Thereafter, oocytes with two pronucleus are selected as fertilized oocytes. Next, fertilized oocytes are transferred into a glass chamber containing CRISPR ribonucleoprotein (RNP) complexes formed from gRNA and Cas9 protein. After electroporation, fertilized oocytes are then immediately transferred to culture medium. The next day, embryos are transferred into the oviduct of pseudopregnant female rats. Using the above method, offspring can be obtained 22days after the day of embryo transfer. In this paper, we outline a method allowing simple and efficient generation of genetically modified rats without the need for technically difficult micromanipulation techniques.

Advancements in the future of automating micromanipulation techniques in the IVF laboratory using deep convolutional neural networks.

To determine if deep learning artificial intelligence algorithms can be used to accurately identify key morphologic landmarks on oocytes and cleavage stage embryo images for micromanipulation procedures such as intracytoplasmic sperm injection (ICSI) or assisted hatching (AH). Two convolutional neural network (CNN) models were trained, validated, and tested over three replicates to identify key morphologic landmarks used to guide embryologists when performing micromanipulation procedures. The first model (CNN-ICSI) was trained (n = 13,992), validated (n = 1920), and tested (n = 3900) to identify the optimal location for ICSI through polar body identification. The second model (CNN-AH) was trained (n = 13,908), validated (n = 1908), and tested (n = 3888) to identify the optimal location for AH on the zona pellucida that maximizes distance from healthy blastomeres. The CNN-ICSI model accurately identified the polar body and corresponding optimal ICSI location with 98.9% accuracy (95% CI 98.5-99.2%) with a receiver operator characteristic (ROC) with micro and macro area under the curves (AUC) of 1. The CNN-AH model accurately identified the optimal AH location with 99.41% accuracy (95% CI 99.11-99.62%) with a ROC with micro and macro AUCs of 1. Deep CNN models demonstrate powerful potential in accurately identifying key landmarks on oocytes and cleavage stage embryos for micromanipulation. These findings are novel, essential stepping stones in the automation of micromanipulation procedures.

Study on the Manipulation Strategy of Metallic Microstructures Based on Electrochemical-Assisted Method

Microcomponent manipulation (MCM) technology plays a decisive role in assembling complex systems at the micro- and nanoscale. However, the existing micromanipulation methods are difficult to widely apply in the manufacturing of microelectromechanical systems (MEMSs) due to the limited manipulation space and complex application objects, and the manipulation efficiency is relatively low, which makes it difficult to industrialize these micromanipulating systems. To solve the above problems, this paper proposes an efficient metal MCM strategy based on the electrochemical method. To verify the feasibility and repeatability of the strategy, the finite element model (FEM) incorporating the hydrodynamic and electrochemical theories is used to calculate the local stress distribution of the contact position during the dynamic pick-up process. Based on the simulation results, we defined the relationship between the parameters, such as the optimal manipulating position and angle for picking, transferring and releasing. The failure behaviors of pick-up are built to realize the efficient three-dimensional manipulation of microcopper wire of 300 μm. By establishing a theoretical model and experimental verification, it was concluded that the middle point was the best manipulating position when picking up the microcopper wire, the most efficient picking angle was between 45 and 60 degrees for the pipette, and the average time was 480 s in three sets of picking-release manipulation experiments. This paper provides an achievable idea for different types of micro-object manipulations and promotes the rapid application of micromanipulation techniques in MEMSs.

Microplastic-Free Microcapsules to Encapsulate Health-Promoting Limonene Oil.

Fast-moving consumer goods (FMCG) industry has long included many appealing essential oils in products to meet consumers' needs. Among all, the demand for limonene (LM) has recently surged due to its broad-spectrum health benefits, with applications in cosmetic, detergent, and food products. However, LM is extremely volatile, hence has often been encapsulated for a longer shelf-life. To date, mostly non-biodegradable synthetic polymers have been exploited to fabricate the microcapsule shells, and the resulting microcapsules contribute to the accumulation of microplastic in the environment. So far, information on LM-entrapping microcapsules with a natural microplastic-free shell and their mechanism of formation is limited, and there is lack of an in-depth characterisation of their mechanical and adhesive properties, which are crucial for understanding their potential performance at end-use applications. The present research aims towards developing safe microcapsules with a core of LM fabricated via complex coacervation (CC) using gum Arabic (GA) and fungally sourced chitosan (fCh) as shell precursors. The encapsulation efficiency (EE) for LM was quantified by gas chromatography (GC) separation method. The morphology of microcapsules was investigated via bright-field optical microscopy and scanning electron microscopy, and their mechanical properties were characterised using a micromanipulation technique. Moreover, the adhesive properties of the resulting microcapsules were studied via a bespoke microfluidic device fitted with a polyethylene-terephthalate (PET) substrate and operating at increasingly hydrodynamic shear stress (HSS). Spherical core-shell microcapsules (EE ~45%) with a mean size of 38 ± 2 μm and a relatively smooth surface were obtained. Their mean rupture force and nominal rupture stress were 0.9 ± 0.1 mN and 2.1 ± 0.2 MPa, respectively, which are comparable to those of other microcapsules with synthetic shells, e.g., urea- and melamine-formaldehyde. It was also found that the fCh-GA complexed shell provided promising adhesive properties onto PET films, leading to a microcapsule retention of ~85% and ~60% at low (≤50 mPa) and high shear stress (0.9 Pa), respectively. Interestingly, these values are similar to the adhesion data available in literature for microplastic-based microcapsules, such as melamine-formaldehyde (50-90%). Overall, these findings suggest that microplastics-free microcapsules with a core of oil have been successfully fabricated, and can offer a potential for more sustainable, consumer- and environmentally friendly applications in FMCGs.

CLINICAL OUTCOMES OF MICRODISSECTION TESTICULAR SPERM EXTRACTION (MICRO TESE) AND INTRACYTOPLASMIC SPERM INJECTION (ICSI) IN NON-OBSTRUCTIVE AZOOSPERMIA (NOA) WITH THE HISTORY OF CRYPTORCHIDISM

Undescended testis (UT) is associated with impairment of germ cell maturation and subsequent infertility in adulthood. The undescended testis is exposed to a higher temperature compared with the scrotal temperature and there is progressive Leydig and Sertoli cell atrophy. There have been very few studies of ICSI with a focus on, or large enough numbers to examine, the specific outcomes associated with male factor infertility. Improvement in sperm retrieval techniques including micro TESE and micromanipulation techniques, such as ICSI, has led to excellent fertilization and pregnancy outcomes of treatment cycles.

Y-STR mixture deconvolution by single-cell analysis.

Since Y-STR typing only amplifies male Y chromosomal DNA, it can simplify the interpretation of some DNA mixtures that contain female DNA. However, if there are multiple male contributors, mixed Y-STR DNA profiles will often be obtained. Y-STR mixture analysis cases are particularly challenging though as, currently, there are no validated probabilistic genotyping (PG) software solutions commercially available to aid in their interpretation. One approach to fully deconvoluting these challenging mixtures into their individual donors is to conduct single-cell genotyping by isolating individual cells from a mixture prior to conducting DNA typing. In this work, a physical micromanipulation technique involving a tungsten needle and direct PCR with decreased reaction volume and increased cycle number was applied to equimolar 2- and 3-person buccal cell male DNA mixtures and a mock touch DNA case scenario involving the consecutive firing of a handgun by two males. A consensus DNA profiling approach was then utilized to obtain YFiler™ Plus Y-STR haplotypes. Buccal cells were used to optimize and test the direct single-cell subsampling approach, and 2-3 person male buccal cell mixtures were fully deconvoluted into their individual donor Y-STR haplotypes. Single-cell (or agglomerated cell clump) subsampling from the gun's trigger recovered single-source Y-STR profiles from both individuals who fired the gun, the owner, and the other unrelated male. Only the non-owner's DNA was found in the cells recovered from the handle. In summary, direct single-cell subsampling as described represents a potential simple way to analyze and interpret Y-STR mixtures.

Egg microinjections – method experimental viral infections in brook trout Salvelinus fontinalis (Mitchill, 1815) and rainbow brook trout Salvelinus fontinalis ♂ × Oncorhynchus mykiss ♀

Abstract Microinjection is a micromanipulation technique that has been used in various fields and in a variety of species, including teleost fish, for over a hundred years. Microinjection is mainly used in genetic, molecular, and toxicological research. The paper describes an experimental study of a microinjection technique for the administration of Infectious Pancreatic Necrosis Virus (IPNV) in fertilized eggs of brook trout, Salvelinus fontinalis (Mitchill), and a rainbow trout Oncorhynchus mykiss (Walbaum) hybrid, Salvelinus fontinalis ♂ × Oncorhynchus mykiss ♀. The oocytes and embryos of these salmonids have complex structures that require modifying the method commonly used in zebrafish, Danio rerio (Hamilton), research. A modular apparatus and technique for injecting brook trout and rainbow brook trout fish embryos with IPNV are described. The modified microinjection method can be used successfully to administer to salmonid eggs infectious substances that simulate the vertical transmission of pathogens.

Dynamic manipulation of WGM lasing by tailoring the coupling strength.

Miniaturized lasing with dynamic manipulation is critical to the performance of compact and versatile photonic devices. However, it is still a challenge to manipulate the whispering gallery mode lasing modes dynamically. Here, we design the quasi-three-dimensional coupled cavity by a micromanipulation technique. The coupled cavity consists of two intersection polymer microfibers. The mode selection mechanism is demonstrated experimentally and theoretically in the coupled microfiber cavity. Dynamic manipulation from multiple modes to single-mode lasing is achieved by controlling the coupling strength, which can be quantitatively controlled by changing the coupling angle or the coupling distance. Our work provides a flexible alternative for the lasing mode modulation in the on-chip photonic integration.