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Micromachines May 2022Programmed mini-pumps play a significant role in various fields, such as chemistry, biology, and medicine, to transport a measured volume of liquid, especially in the...
Programmed mini-pumps play a significant role in various fields, such as chemistry, biology, and medicine, to transport a measured volume of liquid, especially in the current detection of COVID-19 with PCR. In view of the cost of the current automatic pipetting pump being higher, which is difficult to use in a regular lab, this paper designed and assembled a three-dimensional programmed mini-pump with the common parts and components, such as PLC controller, motor, microinjector, etc. With the weighting calibration before and after pipetting operation, the error of the pipette in 10 μL (0.2%), 2 μL (1.8%), and 1 μL (5.6%) can be obtained. Besides, the contrast test between three-dimensional programmed mini-pump and manual pipette was conducted with the ORF1ab and pGEM-3Zf (+) genes in qPCR. The results proved that the custom-made three-dimensional programmed mini-pump has a stronger reproducibility compared with manual pipette (ORF1ab: 24.06 ± 0.33 vs. 23.50 ± 0.58, = 0.1014; pGEM-3Zf (+): 11.83.06 ± 0.24 vs. 11.50 ± 0.34, = 0.8779). These results can lay the foundation for the functional, fast, and low-cost programmed mini-pump in PCR or other applications for trace measurements.
PubMed: 35630239
DOI: 10.3390/mi13050772 -
Methods in Molecular Biology (Clifton,... 2023Both patch amperometry (PA) and intracellular patch electrochemistry (IPE) take advantage of a recording configuration where an electrochemical detector-carbon fiber...
Both patch amperometry (PA) and intracellular patch electrochemistry (IPE) take advantage of a recording configuration where an electrochemical detector-carbon fiber electrode (CFE)-is housed inside a patch pipette. PA, which is employed in cell-attached or excised inside-out patch clamp configuration, offers high-resolution patch capacitance measurements with simultaneous amperometric detection of catecholamines released during exocytosis. The method provides precise information on single-vesicle size and quantal content, fusion pore conductance, and permeability of the pore for catecholamines. IPE, on the other hand, measures cytosolic catecholamines that diffuse into the patch pipette following membrane rupture to achieve the whole-cell configuration. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode, it provides more specific information on the nature of the detected molecules and may selectively quantify catecholamines, providing a direct approach to determine cytosolic concentrations of catecholaminergic transmitters and their metabolites. Here, we provide detailed instructions on setting up PA and IPE, performing experiments and analyzing the data.
Topics: Carbon Fiber; Catecholamines; Catechols; Chromaffin Cells; Electrochemistry; Exocytosis
PubMed: 36205899
DOI: 10.1007/978-1-0716-2671-9_17 -
Journal of Neuroscience Methods Jan 2021Whole-cell patch-clamp recording in vivo is the gold-standard method for measuring subthreshold electrophysiology from single cells during behavioural tasks, sensory...
BACKGROUND
Whole-cell patch-clamp recording in vivo is the gold-standard method for measuring subthreshold electrophysiology from single cells during behavioural tasks, sensory stimulations, and optogenetic manipulation. However, these recordings require a tight, gigaohm resistance, seal between a glass pipette electrode's aperture and a cell's membrane. These seals are difficult to form, especially in vivo, in part because of a strong dependence on the distance between the pipette aperture and cell membrane.
NEW METHOD
We elucidate and utilize this dependency to develop an autonomous method for placement and synchronization of pipette's tip aperture to the membrane of a nearby, moving neuron, which enables high-yield seal formation and subsequent recordings deep in the brain of the living mouse.
RESULTS
This synchronization procedure nearly doubles the reported gigaseal yield in the thalamus (>3 mm below the pial surface) from 26 % (n = 17/64) to 48 % (n = 32/66). Whole-cell recording yield improved from 10 % (n = 9/88) to 24 % (n = 18/76) when motion compensation was used during the gigaseal formation. As an example of its application, we utilized this system to investigate the role of the sensory environment and ventral posterior medial region (VPM) projection synchrony on intracellular dynamics in the barrel cortex.
COMPARISON WITH EXISTING METHOD(S)
Current methods of in vivo whole-cell patch clamping do not synchronize the position of the pipette to motion of the cell.
CONCLUSIONS
This method results in substantially greater subcortical whole-cell recording yield than previously reported and thus makes pan-brain whole-cell electrophysiology practical in the living mouse brain.
Topics: Animals; Brain; Cell Membrane; Electrophysiological Phenomena; Mice; Neurons; Patch-Clamp Techniques
PubMed: 33242530
DOI: 10.1016/j.jneumeth.2020.109008 -
Frontiers in Neural Circuits 2020loose patch and breakthrough whole-cell recordings are useful tools for investigating the intrinsic and synaptic properties of neurons. However, the correlation among...
loose patch and breakthrough whole-cell recordings are useful tools for investigating the intrinsic and synaptic properties of neurons. However, the correlation among pipette resistance, seal condition, and recording time is not thoroughly clear. Presently, we investigated the recording time of different pipette resistances and seal conditions in loose patch and breakthrough whole-cell recordings. The recording time did not change with pipette resistance for loose patch recording (-loose) and first increased and then decreased as seal resistance for loose patch recording (-loose) increased. For a high probability of a recording time ≥30 min, the low and high cutoff values of -loose were 21.5 and 36 MΩ, respectively. For neurons with -loose values of 21.5-36 MΩ, the action potential (AP) amplitudes changed slightly 30 min after the seal. The recording time increased as seal resistance for whole-cell recording (-tight) increased and the zero-current membrane potential for breakthrough whole-cell recording (MP) decreased. For a high probability of a recording time ≥30 min, the cutoff values of -tight and MP were 2.35 GΩ and -53.5 mV, respectively. The area under the curve (AUC) of the MP receiver operating characteristic (ROC) curve was larger than that of the -tight ROC curve. For neurons with MP values ≤ -53.5 mV, the inhibitory or excitatory postsynaptic current amplitudes did not show significant changes 30 min after the seal. In neurons with -tight values ≥2.35 GΩ, the recording time gradually increased and then decreased as the pipette resistance for whole-cell recording (-tight) increased. For the high probability of a recording time ≥30 min, the low and high cutoff values of -tight were 6.15 and 6.45 MΩ, respectively. Together, we concluded that the optimal -loose range is 21.5-36 MΩ, the optimal -tight range is 6.15-6.45 MΩ, and the optimal -tight and MP values are ≥2.35 GΩ and ≤ -53.5 mV, respectively. Compared with -tight, the MP value can more accurately discriminate recording times ≥30 min and <30 min.
Topics: Acoustic Stimulation; Animals; Auditory Cortex; Excitatory Postsynaptic Potentials; Female; Inhibitory Postsynaptic Potentials; Membrane Potentials; Mice; Mice, Inbred C57BL; Patch-Clamp Techniques
PubMed: 32714153
DOI: 10.3389/fncir.2020.00034 -
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 -
Journal of Mass Spectrometry and... Nov 2023Pipettes are essential tools for biomedical and analytical laboratories, analogous to workstations for computer scientists. Variation in pipetting is a known unknown, as... (Review)
Review
Pipettes are essential tools for biomedical and analytical laboratories, analogous to workstations for computer scientists. Variation in pipetting is a known unknown, as it is generally accepted that variations exist, but thus far, there have been limited studies on the extent of these variations in practice. In this mini-review, we highlight how manual pipetting is a key technique in the laboratory, and, although simple, inaccuracy and imprecision exist. If variations are not adequately addressed, errors can be compounded and consequently compromise data quality. Determination of the accuracy and precision of manual pipetting is straightforward, and here we review two common approaches that use gravimetry and spectrophotometry as readouts. We also provide detailed protocols for determination of accuracy and precision using manual single and multi-channel pipettes. These simple-to-use methods can be used by any laboratory for competency training and regular checks. Having a common protocol for evaluation of variation will also enable cross-laboratory comparison and potentially facilitate establishment of a reference value of acceptable ranges for operator error. Such a value could be of relevance to the scientific community for benchmarking and assuring good laboratory practice.
PubMed: 37841753
DOI: 10.1016/j.jmsacl.2023.09.001 -
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 -
Frontiers in Cell and Developmental... 2021Utilizing microinjection to introduce biological molecules such as DNA, mRNA, siRNA, and proteins into the cell is well established to study oocyte maturation and early...
Utilizing microinjection to introduce biological molecules such as DNA, mRNA, siRNA, and proteins into the cell is well established to study oocyte maturation and early embryo development . However, microinjection is an empirical technology. The cellular survival after microinjection is mainly dependent on the operator, and an experienced operator should be trained for a long time, from several months to years. Optimizing the microinjection to be highly efficient and quickly learned should be helpful for new operators and some newly established laboratories. Here, we combined the tip pipette and piezo-assisted micromanipulator to microinject the oocyte and early embryos at different stages of mouse. The results showed that the survival rate after microinjection was more than 85% for cumulus-oocyte complex, germinal vesicle oocyte, two-cell, and four-cell embryos, and close to 100% for MII oocyte and zygotes. The high-rate survival of microinjection can save many experimental samples. Thus, it should be helpful in studying some rare animal models such as aging and conditional gene knockout mice. Furthermore, our protocol is much easier to learn for new operators, who can usually master the method proficiently after several training times. Therefore, we would like to publicly share this experience, which will help some novices master microinjection skillfully and save many laboratory animals.
PubMed: 34540848
DOI: 10.3389/fcell.2021.735971 -
Sensors (Basel, Switzerland) Sep 2023A patch clamp is the "gold standard" method for studying ion-channel biophysics and pharmacology. Due to the complexity of the operation and the heavy reliance on...
A patch clamp is the "gold standard" method for studying ion-channel biophysics and pharmacology. Due to the complexity of the operation and the heavy reliance on experimenter experience, more and more researchers are focusing on patch-clamp automation. The existing automated patch-clamp system focuses on the process of completing the experiment; the detection method in each step is relatively simple, and the robustness of the complex brain film environment is lacking, which will increase the detection error in the microscopic environment, affecting the success rate of the automated patch clamp. To address these problems, we propose a method that is suitable for the contact between pipette tips and neuronal cells in automated patch-clamp systems. It mainly includes two key steps: precise positioning of pipettes and contact judgment. First, to obtain the precise coordinates of the tip of the pipette, we use the Mixture of Gaussian (MOG) algorithm for motion detection to focus on the tip area under the microscope. We use the object detection model to eliminate the encirclement frame of the pipette tip to reduce the influence of different shaped tips, and then use the sweeping line algorithm to accurately locate the pipette tip. We also use the object detection model to obtain a three-dimensional bounding frame of neuronal cells. When the microscope focuses on the maximum plane of the cell, which is the height in the middle of the enclosing frame, we detect the focus of the tip of the pipette to determine whether the contact between the tip and the cell is successful, because the cell and the pipette will be at the same height at this time. We propose a multitasking network CU-net that can judge the focus of pipette tips in complex contexts. Finally, we design an automated contact sensing process in combination with resistance constraints and apply it to our automated patch-clamp system. The experimental results show that our method can increase the success rate of pipette contact with cells in patch-clamp experiments.
Topics: Robotic Surgical Procedures; Robotics; Brain; Automation; Neurons
PubMed: 37836974
DOI: 10.3390/s23198144 -
Precision Engineering Oct 2016Many experimental biological techniques utilize hollow glass needles called micropipettes to perform fluid extraction, cell manipulation, and electrophysiological...
Many experimental biological techniques utilize hollow glass needles called micropipettes to perform fluid extraction, cell manipulation, and electrophysiological recordings For electrophysiological recordings, micropipettes are typically fabricated immediately before use using a "pipette puller", which uses open-loop control to heat a hollow glass capillary while applying a tensile load. Variability between manufactured micropipettes requires a highly trained operator to qualitatively inspect each micropipette; typically this is achieved by viewing the pipette under 40-100x magnification in order to ensure that the tip has the correct shape (e.g., outer diameter, cone angle, taper length). Since laboratories may use hundreds of micropipettes per week, significant time demands are associated with micropipette inspection. Here, we have automated the measurement of micropipette tip outer diameter and cone angle using optical microscopy. The process features repeatable constraint of the micropipette, quickly and automatically moving the micropipette to bring its tip into the field of view, focusing on the tip, and computing tip outer diameter and cone angle measurements from the acquired images by applying a series of image processing algorithms. As implemented on a custom automated microscope, these methods achieved, with 95% confidence, ±0.38 µm repeatability in outer diameter measurement and ±5.45° repeatability in cone angle measurement, comparable to a trained human operator. Accuracy was evaluated by comparing optical pipette measurements with measurements obtained using scanning electron microscopy (SEM); optical outer diameter measurements differed from SEM by 0.35 ± 0.36 µm and optical cone angle measurements differed from SEM by -0.23 ± 2.32°. The algorithms we developed are adaptable to most commercial automated microscopes and provide a skill-free route to rapid, quantitative measurement of pipette tip geometry with high resolution, accuracy, and repeatability. Further, these methods are an important step toward a closed-loop, fully-automated micropipette fabrication system.
PubMed: 27672230
DOI: 10.1016/j.precisioneng.2016.04.003