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Methods in Molecular Biology (Clifton,... 2019A complete characterization of temperature -and voltage-activated TRP channel gating requires a precise determination of the absolute probability of opening in a wide...
A complete characterization of temperature -and voltage-activated TRP channel gating requires a precise determination of the absolute probability of opening in a wide range of voltages, temperatures, and agonist concentrations. We have achieved this in the case of the TRPM8 channel expressed in Xenopus laevis oocytes. Measurements covered an extensive range of probabilities and unprecedented applied voltages up to 500 mV. In this chapter, we describe animal care protocols of patch-clamp pipette preparation, temperature control methods, and analysis of ionic currents to obtain reliable absolute open channel probabilities.
Topics: Animals; Electrophysiology; Ion Channel Gating; Membrane Potentials; Oocytes; Probability; Temperature; Transient Receptor Potential Channels; Xenopus laevis
PubMed: 31028680
DOI: 10.1007/978-1-4939-9446-5_11 -
Electrophoresis Oct 2017Electrokinetically controlled microinjection is reported as an effective transport mechanism for microinjection into the wild-type strain of the widely studied model...
Electrokinetically controlled microinjection is reported as an effective transport mechanism for microinjection into the wild-type strain of the widely studied model microalga Chlamydomonas reinhardtii. A microinjection system using glass capillary pipettes was developed to capture and impale the motile cells. To apply an electric field and induce electrokinetic flow (e.g., electrophoresis and electroosmosis), an electrode was inserted directly into the solution inside the impaling injection pipette and another electrode was inserted into the external cell media. The viability of the impaled cells was confirmed for more than an hour under 0.01 V using the fluorescein diacetate/propidium iodide dual fluorescent dye based assay. The viability was also found to increase almost logarithmically with decreasing voltage and to depend strongly on the solution within the injection pipette. Successful electrokinetic microinjection into cells was confirmed by both an increase in cell volume under an applied voltage and electric field dependent delivery of fluorescent fluorescein molecules into an impaled cell. Our study offers novel opportunities for quantitative delivery of biomolecules into microalgae and advancing the research and development of these organisms as biosynthetic factories.
Topics: Computer Simulation; Electricity; Electroosmosis; Electrophoresis; Kinetics; Microalgae; Microfluidic Analytical Techniques; Microinjections
PubMed: 28375577
DOI: 10.1002/elps.201600548 -
Nano-micro Letters Dec 2021Additive manufacturing-also known as 3D printing-has attracted much attention in recent years as a powerful method for the simple and versatile fabrication of...
Additive manufacturing-also known as 3D printing-has attracted much attention in recent years as a powerful method for the simple and versatile fabrication of complicated three-dimensional structures. However, the current technology still exhibits a limitation in realizing the selective deposition and sorting of various materials contained in the same reservoir, which can contribute significantly to additive printing or manufacturing by enabling simultaneous sorting and deposition of different substances through a single nozzle. Here, we propose a dielectrophoresis (DEP)-based material-selective deposition and sorting technique using a pipette-based quartz tuning fork (QTF)-atomic force microscope (AFM) platform DEPQA and demonstrate multi-material sorting through a single nozzle in ambient conditions. We used Au and silica nanoparticles for sorting and obtained 95% accuracy for spatial separation, which confirmed the surface-enhanced Raman spectroscopy (SERS). To validate the scheme, we also performed a simulation for the system and found qualitative agreement with the experimental results. The method that combines DEP, pipette-based AFM, and SERS may widely expand the unique capabilities of 3D printing and nano-micro patterning for multi-material patterning, materials sorting, and diverse advanced applications.
PubMed: 34862935
DOI: 10.1007/s40820-021-00760-x -
Methods in Molecular Biology (Clifton,... 2016Many eukaryotic cells move in the direction of a chemical gradient. Several assays have been developed to measure this chemotactic response, but no complete mathematical...
Many eukaryotic cells move in the direction of a chemical gradient. Several assays have been developed to measure this chemotactic response, but no complete mathematical models of the spatial and temporal gradients are available to describe the fundamental principles of chemotaxis. Here we provide analytical solutions for the gradients formed by release of chemoattractant from a point source by passive diffusion or forced flow (micropipettes) and gradients formed by laminar diffusion in a Zigmond chamber. The results show that gradients delivered with a micropipette are formed nearly instantaneously, are very steep close to the pipette, and have a steepness that is strongly dependent on the distance from the pipette. In contrast, gradients in a Zigmond chamber are formed more slowly, are nearly independent of the distance from the source, and resemble the temporal and spatial properties of the natural cAMP wave that Dictyostelium cells experience during cell aggregation.
Topics: Algorithms; Cell Aggregation; Chemotactic Factors; Chemotaxis; Cyclic AMP; Dictyostelium; Diffusion; Models, Biological
PubMed: 27271915
DOI: 10.1007/978-1-4939-3480-5_26 -
Fertility and Sterility Nov 2023To present a novel trophectoderm biopsy method, independent of laser pulses, using innovatively designed micropipettes on blastocysts at different stages and show...
OBJECTIVE
To present a novel trophectoderm biopsy method, independent of laser pulses, using innovatively designed micropipettes on blastocysts at different stages and show variable characteristics.
DESIGN
A step-by-step demonstration of this method with narrated video.
SETTING
In vitro laboratory fertilization.
PATIENTS
Individuals whose embryos underwent preimplantation genetic testing.
INTERVENTIONS
Trophectoderm biopsy is accomplished using micropipettes that contain a set of innovative designs. Both biopsy and holding pipettes are characterized by sharp, flat opening ends. The holding pipette is designed with an inclined plane on the outer wall surface of its opening end. It is used to help the biopsy pipette make contact with the holding pipette with increased stability, preventing slipping during the detachment of the trophectoderm cells. There is a narrow structure inside the biopsy pipette, which is designed to trap released fragments and prevent sample loss. A trophectoderm biopsy for fully expanded blastocysts starts from artificial shrinkage, followed by zona pellucida drilling. Then, 5-10 trophectoderm cells are aspirated into a biopsy pipette. The blastocyst is released from the holding pipette, the edge of the opening end of the biopsy pipette is tightly pressed onto the inclined plane of the holding pipette, and the biopsy pipette is flicked directly without laser pulses or pulling off the trophectoderm cells. The aspirated trophectoderm cells are subsequently detached by mechanical friction between the edges of the biopsy and holding pipettes. Apart from drilling the zona pellucida for fully expanded blastocysts, the remaining steps do not require lasers. For hatching (including peanut-shaped and 8-shaped) and hatched blastocysts, a trophectoderm biopsy is accomplished by aspirating the cells without securing the blastocyst with a holding pipette, followed by detachment using the direct flicking method.
MAIN OUTCOME MEASURES
The biopsy time, sample loss rate, successful DNA amplification rate, and survival rate.
RESULTS
The innovatively designed micropipettes facilitate the successful detachment of trophectoderm cells through a single direct flicking procedure. This eliminates thermal damage caused by laser pulses, notably simplifying operational steps and shortening the biopsy time. Significant differences were noted between the direct flicking and conventional methods, wherein laser pulses and pulling of trophectoderm cells are prerequisites for cell detachment. When comparing the average biopsy time of fully expanded blastocyst (61 ± 8s vs. 104 ± 9s, P<.05), peanut-shaped hatching blastocyst (35 ± 6s vs. 113 ± 13s, P<.05), 8-shaped hatching blastocyst (32 ± 4s vs. 59 ± 6s, P<.05), and hatched blastocyst (34 ± 4s vs. 67 ± 8s, P<.05), the direct flicking method shows a significantly decreased biopsy time. The narrow structure inside the biopsy pipette effectively prevents sample loss, showing a significantly reduced sample loss rate (0%) compared with the conventional biopsy method (18%) for trainees. Moreover, a satisfactory survival rate (100%) and successful DNA amplification rate (99.5%) were achieved using the direct flicking method.
CONCLUSIONS
This innovative trophectoderm biopsy method, independent of laser pulses, has wide applicability and a satisfactory, stable performance. Moreover, the simplicity of the method makes it easy to master.
Topics: Humans; Biopsy; Blastocyst; DNA; Fertilization in Vitro; Lasers
PubMed: 37487821
DOI: 10.1016/j.fertnstert.2023.07.010 -
The Analyst Apr 2020Microfluidics has achieved integration of analytical processes in microspaces and realized miniaturized analyses in fields such as chemistry and biology. We have...
Microfluidics has achieved integration of analytical processes in microspaces and realized miniaturized analyses in fields such as chemistry and biology. We have proposed a general concept of integration and extended this concept to the 10-1000 nm scale exploring ultimate analytical performances (e.g. immunoassay of a single-protein molecule). However, a sampling method is still challenging for nanofluidics despite its importance in analytical chemistry. In this study, we developed a femtoliter (fL) sampling method for volume measurement and sample transport. Traditionally, sampling has been performed using a volumetric pipette and flask. In this research, a nanofluidic device consisting of a femtoliter volumetric pipette and flask was fabricated on glass substrates. Since gravity, which is exploited in bulk fluidic operations, becomes less dominant than surface effects on the nanometer scale, fluidic operation of the femtoliter sampling was designed utilizing surface tension and air pressure control. The working principle of an 11 fL volumetric pipette and a 50 fL flask, which were connected by a nanochannel, was verified. It was found that evaporation of the sample solution by air flow was a significant source of error because of the ultra-small volumes being processed. Thus, the evaporation issue was solved by suppressing the air flow. As a result, the volumetric measurement error was decreased to ±0.06 fL (CV 0.6%), which is sufficiently low for use in nanofluidic analytical applications. This study will present a fundamental technology for the development of novel analytical methods for femtoliter volume samples such as single molecule analyses.
PubMed: 32049074
DOI: 10.1039/c9an02258a -
Proceedings of the National Academy of... Jan 2023Gas vesicles used as contrast agents for noninvasive ultrasound imaging must be formulated to be stable, and their mechanical properties must be assessed. We report here...
Gas vesicles used as contrast agents for noninvasive ultrasound imaging must be formulated to be stable, and their mechanical properties must be assessed. We report here the formation of perfluoro--butane microbubbles coated with surface-active proteins that are produced by filamentous fungi (hydrophobin HFBI from ). Using pendant drop and pipette aspiration techniques, we show that these giant gas vesicles behave like glassy polymersomes, and we discover novel gas extraction regimes. We develop a model to analyze the micropipette aspiration of these compressible gas vesicles and compare them to incompressible liquid-filled vesicles. We introduce a sealing parameter to characterize the leakage of gas under aspiration through the pores of the protein coating. Utilizing this model, we can determine the elastic dilatation modulus, surface viscosity, and porosity of the membrane. These results demonstrate the engineering potential of protein-coated bubbles for echogenic and therapeutic applications and extend the use of the pipette aspiration technique to compressible and porous systems.
Topics: Porosity
PubMed: 36649434
DOI: 10.1073/pnas.2211509120 -
Archives of Gynecology and Obstetrics Jun 2020In this prospective study, endometrial biopsy by pipette was compared with hysteroscopy with curettage in patients with an abnormal uterine bleeding (ABU)...
PURPOSE
In this prospective study, endometrial biopsy by pipette was compared with hysteroscopy with curettage in patients with an abnormal uterine bleeding (ABU) (hypermenorrhea, intermediate bleeding, continuous bleeding, postmenopausal bleeding) and patients with a sonographically abnormal endometrium.
METHODS
176 patients were included. The pipette samples were taken during the usual planned procedure under general anaesthesia. Thereafter, the planned hysteroscopy with curettage was completed. The study was performed as a double-blind study. The obtained histologies (of pipelle and curettage) were sent separately to the same pathologist. The pipelle material was encoded by a specific number without any patient data.
RESULTS
In 97% of the cases using the biopsy with pipette were obtained an adequate sample. The biopsy with pipette had a sensitivity and specificity of 100% in the diagnosis of endometrial carcinoma and atypical hyperplasia. Pipette showed a significantly lower accuracy with a sensitivity of 28% in the diagnosis of endometrial hyperplasia without atypia. Pipette showed the lowest sensitivity for polyps, myomas and atrophic endometrium.
CONCLUSION
The study shows that pipette sampling is a safe, accurate, low-cost ambulatory procedure with high sensitivity for the detection of atypical endometrial hyperplasia and endometrial carcinoma. In the case of sonographically definable findings (polyp, myoma), hysteroscopy with curettage is preferred.
Topics: Curettage; Double-Blind Method; Endometrial Hyperplasia; Female; Humans; Hysteroscopy; Prospective Studies
PubMed: 32350598
DOI: 10.1007/s00404-020-05551-0 -
ENeuro 2021Patch clamp electrophysiology is a common technique used in neuroscience to understand individual neuron behavior, allowing one to record current and voltage changes...
Patch clamp electrophysiology is a common technique used in neuroscience to understand individual neuron behavior, allowing one to record current and voltage changes with superior spatiotemporal resolution compared with most electrophysiology methods. While patch clamp experiments produce high fidelity electrophysiology data, the technique is onerous and labor intensive. Despite the emergence of patch clamp systems that automate key stages in the typical patch clamp procedure, full automation remains elusive. Patch clamp pipettes can miss the target cell during automated experiments because of positioning errors in the robotic manipulators, which can easily exceed the diameter of a neuron. Further, when patching in acute brain slices, the inherent light scattering from non-uniform brain tissue can complicate pipette tip identification. We present a convolutional neural network (CNN), based on ResNet101, to identify and correct pipette positioning errors before each patch clamp attempt, thereby preventing the deleterious effects of and accumulation of positioning errors. This deep-learning-based pipette detection method enabled superior localization of the pipette within 0.62 ± 0.58 μm, resulting in improved cell detection success rate and whole-cell patch clamp success rates by 71% and 59%, respectively, compared with the state-of-the-art cross-correlation method. Furthermore, this technique reduced the average time for pipette correction by 81%. This technique enables real-time correction of pipette position during patch clamp experiments with similar accuracy and quality of recording to manual patch clamp, making notable progress toward full human-out-of-the-loop automation for patch clamp electrophysiology.
Topics: Automation; Electrophysiological Phenomena; Humans; Machine Learning; Neurons; Patch-Clamp Techniques
PubMed: 34312222
DOI: 10.1523/ENEURO.0051-21.2021 -
Small (Weinheim An Der Bergstrasse,... Jan 2016An integrated method for blood plasma separation is presented by combining a pneumatic device, which is referred to as a "smart pipette," and a hydrophoretic...
An integrated method for blood plasma separation is presented by combining a pneumatic device, which is referred to as a "smart pipette," and a hydrophoretic microchannel as a microfluidic pipette tip for whole-blood sample preparation. This method enables hemolysis-free, high-purity plasma separation through smart pipetting of whole blood, potentially providing the means for rapid, inexpensive blood sample preparation for point-of-care testing.
Topics: Animals; Dogs; Erythrocytes; Microfluidics; Plasma; Pressure
PubMed: 26568206
DOI: 10.1002/smll.201502719