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In this communication, local electrochemiluminescence (ECL) imaging is achieved using scanning electrochemical cell microscopy (SECCM) to create a localized region for the occurrence of ECL reaction. The system is demonstrated using a droplet at the micropipette that is contact with the prepared Ru-Silica@Au particles at indium tin oxide (ITO) slide. Tri-n-propylamine (TPrA) in the droplet reacts with ruthenium complex in a 10 μm-diameter-region to produce ECL emission, which is recorded by photo multiplier tube (PMT) underneath the ITO slide. By contacting each local region consecutively, the distribution of steady-state current and the related ECL emission at the ITO surface is imaged. The local ECL intensity show an approximately linear dependence with the current, but obvious deviation from the linear curve is observed at some regions. This result suggests that the ECL emission is not only determined by the transferred electron number in the electrochemical reaction, and also affected by local environments at ITO surface. As compared with the traditionally used ECL imaging in the bulk solution, this strategy avoids the cross-talking of ECL emission from nearby regions, and could offer a new way to unveil more molecular mechanism in the ECL reaction.

The deformability of leukocytes is relevant to a wide array of physiological and pathophysiological behaviors. The goal of this study is to provide a detailed, quantitative characterization of the mechanical properties of T cells and how those properties change with activation. We tested T cells and CD8+ cells isolated from peripheral blood samples of healthy donors either immediately (naïve population) or after 7 days of activation in vitro. Single-cell micropipette aspiration was used to test the mechanical properties. T cells exhibit the general characteristics of a highly viscous liquid drop with a cortical “surface” tension, Tcort. The time course of each cell entry into the micropipette was measured at two different aspiration pressures to test for shear thinning behavior. The data were analyzed in the framework of an approximate mechanical model of the cell deformation to determine the cortical tension, the cell volume, the magnitude of the initial cell entry, the characteristic viscosity μo, and the shear thinning coefficient, b. Activation generally caused increases in cellular resistance to deformation and a broadening of the distribution of cell properties. The cell volume increased substantially upon cell activation from ∼200 μm3 to ∼650 μm3. Naive and activated T cells had similar mean cortical tension (∼150 pN/μm). However, compared to naïve CD8+ cells, the cortical tension of activated CD8+ cells increased significantly to ∼250 pN/μm. Dynamic resistance of naive CD8+ T cells, as reflected in their characteristic viscosity, was ∼870 Pa and significantly increased to 1,180 Pa after in vitro activation. The magnitude of the instantaneous projection length as the cell enters the pipette (Linit) was more than doubled for activated vs. naive cells. All cell types exhibited shear thinning behavior with coefficients b in the range 0.5–0.65. Increased cell size, cortical tension, and characteristic viscosity all point to increased resistance of activated T cells to passage through the microvasculature, likely contributing to cell trapping. The increased initial elastic response of cells after activation was unexpected and could point to instability in the cell that might contribute to spontaneous cell motility.

Dendrimers are macromolecules with well-defined three-dimensional structures, sizes and surface charges. In this work, four generations of poly(amidoamine) (PAMAM) dendrimers were investigated at the micro-interface between two immiscible electrolyte solutions (μITIES) to understand their electrochemical responses as simple models of ionised macromolecules. Cyclic voltammetry (CV) across a range of aqueous phase pH revealed that all four generations (G0-G3) presented diffusion-controlled ion-transfer from aqueous to organic phase, while the reverse transfers from organic to aqueous phase varied with both pH and the dendrimer generation. The larger dendrimers (G2 and G3) show an adsorption behaviour at pH ≤ 3.5, but show a diffusional response at pH ≥ 6. On the other hand, the smaller dendrimers (G0 and G1) always show a diffusional response and are not impacted by the pH. This indicates that more highly charged dendrimers condense at the interface. The reverse scan of CVs showed that an increased applied potential was required to remove (desorb) these polycations from the interfaces in comparison to smaller, less charged species. Diffusion coefficients (D) were estimated, showing a decrease with increasing generation. Limits of detection for these dendrimers by CV at the μITIES were 0.4, 0.2, 0.7 and 0.5 μM for G0 to G3, respectively, while differential pulse voltammetry lowered the LODs (0.07, 0.05, 0.09 and 0.08 μM, respectively). These study shows that the μITIES provides a simple way to detect and evaluate the electrochemical behaviour of ionised macromolecules, providing a simple illustration of detection mechanism with diffusion or adsorption processes. [Display omitted] • Ionised PAMAM dendrimers are electroactive at liquid/liquid micro-interfaces. • Lower dendrimer generations show simple diffusion controlled behaviour. • Higher generations show adsorption behaviour on reverse CV sweeps. • Analytical sensitivity increases with generation number and decreasing pH. [ABSTRACT FROM AUTHOR]

Most current efforts to advance medical technology proceed along one of two tracks. The first is dedicated to the improvement of clinical tasks through the incremental refinement of medical instruments. The second comprises engineering endeavors to support basic science studies that often only remotely relate to human medicine. Here we survey emerging research approaches that aim to populate the sprawling frontier between these tracks. We focus on interdisciplinary single-live-cell techniques that have overcome limitations of traditional biological methods to successfully address vital questions about medically relevant cellular behavior. Most of the presented case studies are based on the controlled manipulation of nonadherent human immune cells using one or more micropipettes. The included studies have (i) examined one-on-one encounters of immune cells with real or model pathogens, (ii) assessed the physiological role of the expandable surface area of immune cells, and (iii) started to dissect the spatiotemporal organization of signaling processes within these cells. The unique aptitude of such single-live-cell studies to fill conspicuous gaps in our quantitative understanding of medically relevant cause-effect relationships provides a sound basis for new insights that will inform and drive future biomedical innovation.

Intro: Force measurements of the nucleus, the strongest organelle, have propelled the field of mechanobiology to understand the basic mechanical components of the nucleus and how these components properly support nuclear morphology and function. Micromanipulation force measurement provides separation of the relative roles of nuclear mechanical components chromatin and lamin A. Methods: To provide access to this technique, we have developed a universal micromanipulation apparatus for inverted microscopes. We outline how to engineer and utilize this apparatus through dual micromanipulators, fashion and calibrate micropipettes, and flow systems to isolate a nucleus and provide force vs. extensions measurements. This force measurement approach provides the unique ability to measure the separate contributions of chromatin at short extensions and lamin A strain stiffening at long extensions. We then investigated the apparatus' controllable and programmable micromanipulators through compression, isolation, and extension in conjunction with fluorescence to develop new assays for nuclear mechanobiology. Results: Using this methodology, we provide the first rebuilding of the micromanipulation setup outside of its lab of origin and recapitulate many key findings including spring constant of the nucleus and strain stiffening across many cell types. Furthermore, we have developed new micromanipulation-based techniques to compress nuclei inducing nuclear deformation and/or rupture, track nuclear shape post-isolation, and fluorescence imaging during micromanipulation force measurements. Conclusion: We provide the workflow to build and use a micromanipulation apparatus with any inverted microscope to perform nucleus isolation, force measurements, and various other biophysical techniques. [ABSTRACT FROM AUTHOR]

• Local ECL imaging is realized by the incorporation of SECCM and PMT modules. • Local ECL emission in a micro-sized droplet is recorded at the electrode surface. • Local ECL emission exhibits a high heterogeneity that might be caused by the different local environment at the electrode surface. In this communication, local electrochemiluminescence (ECL) imaging is achieved using scanning electrochemical cell microscopy (SECCM) to create a localized region for the occurrence of ECL reaction. The system is demonstrated using a droplet at the micropipette that is contact with the prepared Ru-Silica@Au particles at indium tin oxide (ITO) slide. Tri- n -propylamine (TPrA) in the droplet reacts with ruthenium complex in a 10 μm-diameter-region to produce ECL emission, which is recorded by photo multiplier tube (PMT) underneath the ITO slide. By contacting each local region consecutively, the distribution of steady-state current and the related ECL emission at the ITO surface is imaged. The local ECL intensity show an approximately linear dependence with the current, but obvious deviation from the linear curve is observed at some regions. This result suggests that the ECL emission is not only determined by the transferred electron number in the electrochemical reaction, and also affected by local environments at ITO surface. As compared with the traditionally used ECL imaging in the bulk solution, this strategy avoids the cross-talking of ECL emission from nearby regions, and could offer a new way to unveil more molecular mechanism in the ECL reaction. [ABSTRACT FROM AUTHOR]

The research hypothesis was that particle-size analysis and sand fractionation can be performed using small amounts of soil. The objectives were as follows: to calculate the errors resulting from the replacement of the reference method; to define the smallest mass to determine soil particle size; and to perform sand fractionation. The experiment was conducted with samples from the A and B horizons of an Argissolo Amarelo (Alfisol) in a completely randomized design, with four treatments: 2, 4, 6, and 20 g of Air-Dried Fine Earth (ADFE), and 10 replicates. In all cases, the F-test was used on the analysis of variance, and Dunnett's test was conducted for means comparison. The results with the reference method for clay, sand, and silt, in sequence, were as follows: 66 (±2), 867 (±6), and 67 (±5) g kg−1 for A horizon and 379 (±15), 557 (±17), and 64 (±5) g kg−1 for the B horizon. Among all treatments used to determine particle size, only the treatment with a soil mass of 6g does not statistically differ from the reference method (20 g) in the determination of sand, silt, and clay fractions. In terms of sand sorting, all treatments applied defined poorly sorted sands. [ABSTRACT FROM AUTHOR]

Mechanobiology is an emerging field at the interface of biology and mechanics, investigating the roles of mechanical forces within biomolecules, organelles, cells, and tissues. As a highlight, the recent advances of micropipette-based aspiration assays and dynamic force spectroscopies such as biomembrane force probe (BFP) provide unprecedented mechanobiological insights with excellent live-cell compatibility. In their classic applications, these assays measure force-dependent ligand–receptor-binding kinetics, protein conformational changes, and cellular mechanical properties such as cortical tension and stiffness. In recent years, when combined with advanced microscopies in high spatial and temporal resolutions, these biomechanical nanotools enable characterization of receptor-mediated cell mechanosensing and subsequent organelle behaviors at single-cellular and molecular level. In this review, we summarize the latest developments of these assays for live-cell mechanobiology studies. We also provide perspectives on their future upgrades with multimodal integration and high-throughput capability. [ABSTRACT FROM AUTHOR]

[Display omitted] Binding force between biomolecules has a crucial role in most biological processes. Receptor-ligand interactions transmit physical forces and signals simultaneously. Previously, we employed a robotic micropipette both in live cell and microbead adhesion studies to explore the adhesion force of biomolecules such as cell surface receptors including specific integrins on immune cells. Here we apply standard computational fluid dynamics simulations to reveal the detailed physical background of the flow generated by the micropipette when probing microbead adhesion on functionalized surfaces. Measuring the aspiration pressure needed to pick up the biotinylated 10 μm beads on avidin coated surfaces and converting it to a hydrodynamic lifting force on the basis of simulations, we found an unbinding force of 12 ± 2 nN, when targeting the beads manually; robotic targeting resulted in 9 ± 4 nN (mean ± SD). We measured and simulated the effect of the targeting offset, when the microbead was out of the axis (off-axis)of the micropipette. According to the simulations, the higher offset resulted in a higher lifting force acting on the bead. Considering this effect, we could readily correct the impact of the targeting offset to renormalize the experimental data. Horizontal force and torque also appeared in simulations in case of a targeting offset. Surprisingly, simulations show that the lifting force acting on the bead reaches a maximum at a flow rate of ~ 5 μl/s if the targeting offset is not very high (<5 μm). Further increasing the flow rate decreases the lifting force. We attribute this effect to the spherical geometry of the bead. We predict that higher flow rates cannot increase the hydrodynamic lifting force acting on the precisely targeted microbead, setting a fundamental force limit (16 nN in our setup) for manipulating microbeads with a micropipette perpendicular to the supporting surface. In order to extend the force range, we propose the offset targeting of microbeads. [ABSTRACT FROM AUTHOR]

Biological cells are contained by a fluid lipid bilayer (plasma membrane, PM) that allows for large deformations, often exceeding 50% of the apparent initial PM area. Isolated lipids self‐organize into membranes, but are prone to rupture at small (<2–4%) area strains, which limits progress for synthetic reconstitution of cellular features. Here, it is shown that by preserving PM structure and composition during isolation from cells, vesicles with cell‐like elasticity can be obtained. It is found that these plasma membrane vesicles store significant area in the form of nanotubes in their lumen. These act as lipid reservoirs and are recruited by mechanical tension applied to the outer vesicle membrane. Both in experiment and theory, it is shown that a "superelastic" response emerges from the interplay of lipid domains and membrane curvature. This finding allows for bottom‐up engineering of synthetic biomaterials that appear one magnitude softer and with threefold larger deformability than conventional lipid vesicles. These results open a path toward designing superelastic synthetic cells possessing the inherent mechanics of biological cells. [ABSTRACT FROM AUTHOR]

Abstract Biological cells are contained by a fluid lipid bilayer (plasma membrane, PM) that allows for large deformations, often exceeding 50% of the apparent initial PM area. Isolated lipids self‐organize into membranes, but are prone to rupture at small (

Accumulating evidence has shown transcranial low-intensity ultrasound can be potentially a non-invasive neural modulation tool to treat brain diseases. However, the underlying mechanism remains elusive and the majority of studies on animal models applying rather high-intensity ultrasound that cannot be safely used in humans. Here, we showed low-intensity ultrasound was able to activate neurons in the mouse brain and repeated ultrasound stimulation resulted in adult neurogenesis in specific brain regions. In vitro calcium imaging studies showed that a specific ultrasound stimulation mode, which combined with both ultrasound-induced pressure and acoustic streaming mechanotransduction, is required to activate cultured cortical neurons. ASIC1a and cytoskeletal proteins were involved in the low-intensity ultrasound-mediated mechanotransduction and cultured neuron activation, which was inhibited by ASIC1a blockade and cytoskeleton-modified agents. In contrast, the inhibition of mechanical-sensitive channels involved in bilayer-model mechanotransduction like Piezo or TRP proteins did not repress the ultrasound-mediated neuronal activation as efficiently. The ASIC1a-mediated ultrasound effects in mouse brain such as immediate response of ERK phosphorylation and DCX marked neurogenesis were statistically significantly compromised by ASIC1a gene deletion. Collated data suggest that ASIC1a is the molecular determinant involved in the mechano-signaling of low-intensity ultrasound that modulates neural activation in mouse brain.

Reduction and nanoparticle nucleation of Cu2+ by decamethylferrocene was studied with cyclic and square‐wave voltammetry at a microscale liquid–liquid interface established at the tip of a micropipette. With square‐wave voltammetry, two reduction steps could be distinguished as two separate current waves. Comparing the experimental results of cyclic voltammetry with finite element method simulations, particle growth could be observed as an increasing limiting current. Furthermore, kinetic parameters could be estimated with square‐wave voltammetry simulations following Butler‐Volmer kinetics. [ABSTRACT FROM AUTHOR]

Micropipette is a quite simple, but an essential instrument used for the volume measurements and finds the applications in the field of chemistry, pharmacy, health care, biology, pathology, research and development, etc. The calibration laboratories must ensure that the results obtained using these instruments are accurate, and therefore, the calibration is an inevitable process. This report is on the micropipette calibration techniques with different methods considering various influencing factors those affect the measurement results. The measurements were done in controlled environmental conditions and taking all the necessary precautions. This report also presents a detailed comparison between the three distinct test methods used, i.e. "conventional method", "with evaporation loss correction" method and "without evaporation loss correction" method. This report summarizes how the change of techniques applied can affect the final volume of the micropipette and the associated uncertainty in the measurement. This study will contribute to the improvement of the whole process of micropipette calibration for performing the calibration in volume. Further such studies can also be conducted for glassware calibration to improve the overall upgradation of volume calibration. [ABSTRACT FROM AUTHOR]

Background: ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. Results: Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. Conclusions: We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes. [ABSTRACT FROM AUTHOR]

The nominal volume of a variable volume micropipette is the maximum volume of the range specified by the micropipette manufacturer. The purpose of the present study is to quantify the reproducibility standard uncertainty for non-nominal volume micropipette calibrations for variable volume micropipettes. An interlaboratory study was implemented for this purpose, using variable volume micropipettes with nominal volumes of 10 μL, 200 μL, and 1000 μL and setting the measurement volumes as 10 %, 50 %, and 100 % of the nominal volumes. In our previous paper (Accredit Qual Assur 19:377, 2014), we quantified the uncertainty due to reproducibility using only data obtained for nominal volume calibrations. From the results obtained, we found that the reproducibility uncertainties quantified for nominal volumes are too small to cover interlaboratory variations for non-nominal volume calibrations. For calibrations of 10 % and 50 % of the nominal volumes, the reproducibility uncertainties must be increased by the factors of 2.7 and 1.4, respectively, for micropipettes with nominal volumes of 10 μL to 1 000 μL, based on the results of this study. [ABSTRACT FROM AUTHOR]

The mechanic properties of cell membranes control many biological processes. The complexity of natural membranes is often dealt with by building synthetic vesicles (Giant Unilamellar Vesicles, GUVs), which can be thought as micron-sized minimal cells. Micropipette aspiration technique is the gold standard to characterize membrane mechanics, but it involves manual, long and tedious experiments. Microfluidics is perfectly suited to handle GUVs and permits in particular to conceive on-chip micropipettes for automated, systematic studies of membrane mechanical moduli. We developed a microfabrication process that enables obtaining the required 3-level channels including a micropipette in the intermediate level, with micrometric alignment, sufficiently low adhesion and roughness. We extended the theoretical analysis of micropipette, valid for cylindrical geometries that microfabrication does not allow, to the on-chip geometry, by considering the deformation of a vesicle in a square cross-section trap. We confirmed the validity of our approach thanks to systematic experiments performed on GUVs with well-characterized compositions: the obtained values of the membrane stretching modulus are in quantitative agreement with the literature. As a case study, we used our device to show that GUVs challenged with copolymer micelles, typically used for drug delivery, displayed a significantly decrease of the membrane stretching modulus, which could mediate internalization of these nanovectors. This study opens the path to systematic studies of the influence of physico-chemical environment on the mechanics of cell membranes.

Over the decades, nanochannels have been widely used for single molecule detection, smart sensors, and energy transfer and storage based on its unique ion transport properties. Although various ion transport phenomena of nanochannels have been reported, the discovery of new ion transport phenomena is still of great significance for understanding material transport of nanochannels and development of nanodevices with unique working capabilities. This article reports a novel nanochannel ion transport phenomenon - ion current rectification in combination with ion current saturation (ICR-S), which arised from a mesoporous titania conical microplug generated in situ in the glass micropipette tip cavity by space confinement evaporation. The ion current of forward voltage is greater than that of reverse voltage, and the saturation currents appear in both the forward and reverse voltages, the ratio of forward and reverse saturation current can reaches to 10. In addition, the influence of pH, ionic strength, and micropipette angle on ICR-S is also investigated. Image 1 • Ion current rectification with current saturation, ICR-S is found for the first time. • ICR-S occurs at mesoporous titania conical microplug (MTCP). • MTCP is generated in situ in glass micropipette tip by space confinement evaporation. • Forward voltage of ion current is greater than reverse voltage in absolute value. • Saturation currents appear in both of forward and reverse voltages with ratio of 10. [ABSTRACT FROM AUTHOR]

Despite a 30% decline in mortality since 2000, malaria still affected 219 million subjects and caused 435,000 deaths in 2017. Red blood cells (RBC) host Plasmodium parasites that cause malaria, of which Plasmodium falciparum is the most pathogenic. The deformability of RBC is markedly modified by invasion and development of P. falciparum. Surface membrane area is potentially impacted by parasite entry and development, the cytoskeleton is modified by parasite proteins and cytosol viscosity is altered by parasite metabolism. RBC hosting mature parasites (second half of the asexual erythrocytic cycle) are abnormally stiff but the main reason for their absence from the circulation is their adherence to endothelial cells, mediated by parasite proteins exposed at the infected-RBC surface. By contrast, the circulation of non-adherent rings and gametocytes, depends predominantly on deformability. Altered deformability of rings and of uninfected-RBC altered by malaria infection is an important determinant of malaria pathogenesis. It also impacts the response to antimalarial therapy. Unlike conventional antimalarials that target mature stages, currently recommended first-line artemisinin derivatives and the emerging spiroindolones act on circulating rings. Methods to investigate the deformability of RBC are therefore critical to understand the clearance of infected- and uninfected-RBC in malaria. Herein, we review the main methods to assess the deformability of P. falciparum infected-RBC, and their contribution to the understanding of how P. falciparum infection causes disease, how the parasite is transmitted and how antimalarial drugs induce parasite clearance. [ABSTRACT FROM AUTHOR]

A small auto micropipette system is developed to improve the reliability and accuracy of the automatic enzyme immunoassay analyzer's microscale pipetting system. A sophisticated injection mechanism is designed by the means of dislocation parallel distribution of the screw and injector piston rod. It possesses the function of pipetting, taking and removing the pipette tips. In the control system, STM32 controller is used, controlling the single-axis S-type acceleration/deceleration algorithm and multi-threaded coordinated motion. The acceleration/deceleration curves are analyzed and optimized by using the method of segmentation; a minimum injection rate of 1 μL and a step rate of 0.05 μL are realized. The method of digital image processing is used to detect the amount of pipetting in micro-pipetting quantitatively. The liquid area is extracted by background contrast method, and the liquid volume in the tip is obtained by combining the geometric characteristics of the disposable tip, when the pipetting capacity is not qualified to carry out specific guidance on the pipetting system, and avoid the blocking needle, bubble and other abnormal pipetting phenomenon on the impact of pipetting accuracy. The experimental results show that the combination of the automatic sampling system and the image flow detection system can effectively improve the precision and reliability of the micropipetting system. Finally, the injection accuracy of the system at the test points with 10, 50 and 100 μL liquid volumes reaches 1.8%, 1.28% and 1.15% respectively. [ABSTRACT FROM AUTHOR]

Ion current rectification (ICR) is an electrodynamic phenomenon in electrolyte solution which is defined as the asymmetric potential-dependent ion flux through a confined environment, giving rise to asymmetric electrical current-voltage characteristics induced by the influence of an asymmetric electrical double layer structure. Since the discovery of the ICR phenomenon, the observation and application of ICR at nanoscale and microscale have been widely investigated experimentally and theoretically. Here, the recent progress of ICR from nanoscale to microscale is systematically reviewed. Nano/micropore structures of different materials, shapes and pore sizes are first discussed. Then, the factors influencing ICRs by thermodynamically or kinetically regulating the electrical double layer structure are introduced. Moreover, theoretical models are presented to explain the mechanism of ICRs. Based on the understanding of this phenomenon, the applications, especially in biosensors, are discussed. Finally, future developments of this area are briefly presented. This review covers the representative related literature published since 2010 and is intended to give a systematic introduction to this area. [ABSTRACT FROM AUTHOR]

The binding of integrin proteins to peptide sequences such as arginine-glycine-aspartic acid (RGD) is a crucial step in the adhesion process of mammalian cells. While these bonds can be examined between purified proteins and their ligands, live-cell assays are better suited to gain biologically relevant information. Here we apply a computer-controlled micropipette (CCMP) to measure the dissociation constant (Kd) of integrin-RGD-binding. Surface coatings with varying RGD densities were prepared, and the detachment of single cells from these surfaces was measured by applying a local flow inducing hydrodynamic lifting force on the targeted cells in discrete steps. The average behavior of the populations was then fit according to the chemical law of mass action. To verify the resulting value of Kd2d = (4503 ± 1673) 1/µm2, a resonant waveguide grating based biosensor was used, characterizing and fitting the adhesion kinetics of the cell populations. Both methods yielded a Kd within the same range. Furthermore, an analysis of subpopulations was presented, confirming the ability of CCMP to characterize cell adhesion both on single cell and whole population levels. The introduced methodologies offer convenient and automated routes to quantify the adhesivity of living cells before their further processing.

Abstract: Purpose: To evaluate and compare three tear sampling methods using two grading scales for administering the tear ferning test (TFT) to healthy dogs. Methods: In total, 90 dogs (180 eyes) were subjected to tear sampling using millimetered strips, reused after the Schirmer tear test (STT) (Schirmer group, SG). Then, the dogs were subdivided into three groups according to sampling approach: micropipette (MPG), microcapillary (MCG), and Schirmer sample 2 (S2G). The collected tears were dried on a clean microscope glass slide at room temperature and humidity. The ferning patterns were observed under a polarized light microscope and classified according to the Rolando and Masmali grading scales. Results: Although all three methods were feasible, the STT was easier to perform in clinical settings. Type I and Grade 1 were the most commonly observed (64.17% and 61.7%, respectively) regardless of collection method. There was no significant difference between the STT median values and the TFT classifications. Conclusions: The TFT is appropriate for dogs and can be performed using the three suggested sampling methods, with a higher frequency of Type I and Grade 1. Thus, it is possible to use both grading scales in the classification of tear ferning in dogs. [ABSTRACT FROM AUTHOR]

In this study, a facile strategy for fabricating a microfluidic flow cytometer using two glass micropipettes with different sizes and a 3D printed millifluidic aligner was presented. Particle confinement was achieved by hydrodynamic focusing using a single sample and sheath flow. Device performance was extracted using the forward and side-scattered optical signals obtained using fiber-coupled laser and photodetectors. The 3-D printing assisted glass capillary microfluidic device is ultra-low-cost, not labor-intensive and takes less than 10 min to fabricate. The present device offers a great alternative to conventional benchtop flow cytometers in terms of optofluidic configuration. [ABSTRACT FROM AUTHOR]

Scanning electrochemical microscopy (SECM) offers an alternative approach for precise local electrodeposition of micro- and nanometer structures driven by electrochemistry. The tip generation and substrate collection mode of SECM has been applied to deposit sub-micron palladium structures by using a Pd microelectrode. This was compared with a different approach based on scanning ion conductance microscopy (SICM). The latter was utilized also for the localized electrochemical deposition of Pd patterns using a pulled micropipette as a tip. The micropipette was filled with PdCl42− and biased versus a reference electrode placed in a NaCl solution. The application of a negative potential to the micropipette causes negatively charged ions, PdCl42−, to egress the pipette, which were electrochemically reduced on a conducting surface. The Pd patterns locally deposited by SECM and SICM were used for the local electroless deposition of Cu. Comparison between the two techniques shows that SICM is superior to SECM in terms of resolution and ease of tip preparation. [ABSTRACT FROM AUTHOR]

We report herein development of a novel glass micropipette thermal sensor fabricated in a cost-effective manner, which is capable of measuring steady thermal fluctuation at spatial resolution of ~2 µm with an accuracy of ±0.01 °C. We produced and tested various micrometer-sized sensors, ranging from 2 µm to 30 µm. The sensor comprises unleaded low-melting-point solder alloy (Sn-based) as a core metal inside a pulled borosilicate glass pipette and a thin film of nickel coating outside, creating a thermocouple junction at the tip. The sensor was calibrated using a thermally insulated calibration chamber, the temperature of which can be controlled with an accuracy of ±0.01 °C, and the thermoelectric power (Seebeck coefficient) of the sensor was recorded from 8.46 to 8.86 µV/°C. We have demonstrated the capability of measuring temperatures at a cellular level by inserting our temperature sensor into the membrane of a live retinal pigment epithelium cell subjected to a laser beam with a focal spot of 6 μm. We measured transient temperature profiles and the maximum temperatures were in the range of 38–55 ± 0.5 °C.

The key improvement of the corrosion resistance of metals requires understanding and resolving the electrochemical reactions involved at the local scale. Here, we present the use of the pumped-micropipette delivery/substrate collection (Pumped-MD/SC) mode of scanning electrochemical microscopy (SECM) to monitor the oxygen reduction reaction (ORR) during corrosion of an Aluminum (Al) / Copper (Cu) galvanic coupled electrode, a model system of copper-rich aluminum alloys. In this case, the micropipette scans the substrate electrode pumping out a controlled flow of air-saturated electrolyte solution, which allows, through convective transport, to achieve high mass-transfer rates under steady-state conditions at the substrate electrode. The most relevant parameters in pumped-MD/SC mode were studied, it was demonstrated that the ORR current is linearly dependent on the injection flow rate. In contrast, the maximum ORR current was reached regardless of the tip-substrate distance (d) within the range of L (d/ micropipette radius (a)) < 15, which represents a clear advantage in comparison with other modes of SECM. It was also proved that the ORR might take place on both Cu and Al electrodes depending on the applied potential. In addition, the pumped-MD/SC mode of SECM was successfully used for imaging large dimension surfaces with both high spatial and temporal resolution, as well as high imaging contrast thanks to the high mass-transfer rate of electroactive species achieved by forced convection. Moreover, relevant information to evaluate the effect of different corrosion inhibitors in solution such as Ce 2 (SO 4) 3 and Li 2 CO 3 on ORR was provided by individually addressing the substrate electrode in static pumped-MD/SC mode. It was demonstrated that only Ce 2 (SO 4) 3 played the role of cathodic inhibitor through the formation of a Ce3+ oxide/hydroxide precipitate on the substrate electrode during ORR, which was confirmed by SEM and EDX analysis. [Display omitted] [ABSTRACT FROM AUTHOR]

• Micropipette users commonly experience hand and upper body pain. • Systematic study of micropipette design features can improve micropipette ergonomics. • MRI-based finite element analysis of simulated micropipetting evaluated joint contact mechanics. • Micropipette handle diameter and presence of finger hook were examined. • Contact mechanics measures decreased with increasing handle diameter. Micropipette users commonly experience problems in the hand and upper limbs. Mechanical factors are thought to contribute to osteoarthritis (OA) initiation and progression in the basilar thumb joint. Finite element analysis can be used to examine the effects of micropipette design on contact mechanics measures within the basilar thumb joint. This pilot study examined the effect of micropipette handle diameter (12 mm, 25 mm, and 40 mm) and the presence of a finger rest on contact area, contact force, and peak contact pressure in the basilar thumb joint. All contact mechanics measures decreased with increasing handle diameter with significant differences between the 12 mm diameter and the 40 mm diameter handles (contact area down about 30 mm2, contact force down about 15 N, and peak pressure down about 1 MPa). Decreasing contact mechanics measures with increasing diameter matched our expectations that contact measures would decrease with a more open grip. Contact mechanics measures were higher (p < 0.05) with a finger rest for contact area and force. We expected peak contact pressure and contact area to decrease with the presence of a finger rest. The unexpected outcome may have been due to non-randomized testing order and fatigue during testing. [ABSTRACT FROM AUTHOR]

The deformability of leukocytes is relevant to a wide array of physiological and pathophysiological behaviors. The goal of this study is to provide a detailed, quantitative characterization of the mechanical properties of T cells and how those properties change with activation. We tested T cells and CD8 + cells isolated from peripheral blood samples of healthy donors either immediately (naïve population) or after 7 days of activation in vitro . Single-cell micropipette aspiration was used to test the mechanical properties. T cells exhibit the general characteristics of a highly viscous liquid drop with a cortical "surface" tension, T . The time course of each cell entry into the micropipette was measured at two different aspiration pressures to test for shear thinning behavior. The data were analyzed in the framework of an approximate mechanical model of the cell deformation to determine the cortical tension, the cell volume, the magnitude of the initial cell entry, the characteristic viscosity cort . The time course of each cell entry into the micropipette was measured at two different aspiration pressures to test for shear thinning behavior. The data were analyzed in the framework of an approximate mechanical model of the cell deformation to determine the cortical tension, the cell volume, the magnitude of the initial cell entry, the characteristic viscosity μ o , and the shear thinning coefficient, b . Activation generally caused increases in cellular resistance to deformation and a broadening of the distribution of cell properties. The cell volume increased substantially upon cell activation from ∼200 μm 3 to ∼650 μm 3 . Naive and activated T cells had similar mean cortical tension (∼150 pN/μm). However, compared to naïve CD8 + cells, the cortical tension of activated CD8 + cells increased significantly to ∼250 pN/μm. Dynamic resistance of naive CD8 + T cells, as reflected in their characteristic viscosity, was ∼870 Pa and significantly increased to 1,180 Pa after in vitro activation. The magnitude of the instantaneous projection length as the cell enters the pipette ( L init ) was more than doubled for activated vs. naive cells. All cell types exhibited shear thinning behavior with coefficients b in the range 0.5-0.65. Increased cell size, cortical tension, and characteristic viscosity all point to increased resistance of activated T cells to passage through the microvasculature, likely contributing to cell trapping. The increased initial elastic response of cells after activation was unexpected and could point to instability in the cell that might contribute to spontaneous cell motility., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Waugh, Lomakina, Amitrano and Kim.)

The electrochemical behaviors of ten derivatives of ketone as the organic phases for the establishment of liquid/liquid interfaces were investigated by cyclic voltammetry and micropipettes. The ionic species limiting the potential window were determined, and relationships between the structure of solvents and the potential window were evaluated. For heptanones and nonanones, the limits of the potential window were affected by the places of carbonyl and the branched structures in carbon chains. We have found that the water/2,6-dimethyl-4-heptanone interface has the widest potential window among these ten ketone derivatives. Simple ion transfers of TPAs + , TBA + , BF 4 − , IO 4 − and ClO 4 − were observed at the water/2,6-dimethyl-4-heptanone interface with BTPPATPBCl and LiCl as the respective organic and aqueous supporting electrolytes, and their thermodynamic data were calculated. [ABSTRACT FROM AUTHOR]

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 40–100× 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. [ABSTRACT FROM AUTHOR]

We present herein localized galvanostatic and potentiodynamic measurements on lithium iron phosphate (LFP) particles, using the combination of a scanning micropipette contact method (SMCM) and scanning electron microscopy (SEM). The proposed technique allows small substrate areas (∼10 μm in diameter) decorated with LFP particles to be probed within a lithium electrolyte solution in organic carbonate solvents (0.1 M LiClO 4 in propylene carbonate in the present study). SEM images of the scanned area allow the correlation of each electrochemical response to the number, and volume of the probed particles. Under favorable conditions, single particles are found within the measurement areas, thus enabling the determination of single particle properties in an anaerobic environment without the additional complications arising from the presence of binders and electronically conductive fillers. The ability to analyze a material with complementary experimental techniques at the single particle level should create new opportunities for fundamental studies and for the quality control of granular materials. [ABSTRACT FROM AUTHOR]

Dynamic evolution of liquid/liquid/solid contact line at micrometer length scale, corresponding to the Bond number Bo∼0, was investigated in a system with tuneable interfacial properties. Viscous oil was spontaneously displaced by water on a micron size spherical glass surface. By controlling wettability of the substrate and chemistry of surrounding aqueous solution, the displacement dynamics of three phase contact lines over a wide range of capillary numbers were studied. The rates of spontaneous oil displacement showed a strong dependence on fluid viscosity and interfacial properties of the system. The observed wetting dynamics were modeled using hydrodynamic and molecular-kinetic theory. Energy dissipation in the immediate neighborhood of the contact line was shown to determine the wetting dynamics of our micron scale experiments, even when capillary numbers were well above 10 −4 in which hydrodynamic flow regime could be usually expected for conventional macroscopic systems. [ABSTRACT FROM AUTHOR]

We describe a new kind of polymer monolithic column for polymer monolith microextraction (PMME) of carbamate insecticide (methomyl, carbofuran and pirimicarb) residues from water samples. The monolith was prepared inside the tip of a micropipette via thermally initiated free-radical polymerization of methacrylic acid as the monomer, ethylene glycol dimethacrylate as the cross-linker, and graphene oxide (GO) dispersed in the mixed porogens (1-propanol and 1,4-butanediol). The resulting monolith was characterized by scanning electron microscopy and IR spectrometry. The effects of sample flow rate, sample volume, sample pH, desorption solvent and flow rate on PMME were optimized. Compared to a monolith without GO, the new monolith displays higher enrichment capacity, and is reusable and stable. Following extraction, the three carbamates were quantified by HPLC. The limits of detection (for S/N = 3) for the three carbamates are in the range of 0.3-0.7 μg∙L, and the intra- and inter-day precisions are <5.7 %. The new monolith was successfully applied for the determination of the insecticides in various (spiked) environmental water samples, with recoveries in the range from 78.9 to 103.4 %. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

In this work, we established a convenient while reproduceable method for stabilizer-free silver nanoparticle (AgNP)-coated micropipettes by the combination of magnetron sputtering and surface coupling agent. The clear surfaces of the AgNPs are beneficial for absorbing biological or functional molecules on their surfaces. By optimizing the operating parameters, such as sputtering current and sputtering time, the tip of micropipettes coated with AgNPs exhibits excellent surface-enhanced Raman scattering (SERS) performance. Finally, the Raman spectra of a single A549 lung adenocarcinoma cell are successfully acquired by these advanced SERS-active micropipettes. [ABSTRACT FROM AUTHOR]

The preservative methylchloroisothiazolinone/methylisothiazolinone (MCI/MI) is a well-known sensitiser and present in most baseline series since at least 20 years. The proportions of MCI/MI are 3:1. MI alone has been used as a preservative in occupational and household products, and cosmetics since less than 10 years. MCI/ MI tested at 100 ppm fails to detect a significant percentage of contact-allergic reactions to MI. Our aim was to investigate whether a separate test preparation with MI picks up additional cases of contact allergy to MI not detected with MCI/MI 200 ppm. MI was inserted into the baseline series of the Malmö clinic in 2003 starting at 475 ppm, then 900 ppm, then 1,000 ppm, 1,500 ppm and finally 2,000 ppm. In 5,881 consecutively tested dermatitis patients the contact allergy rate for MI varied between 0.5 and 6.5%, with a marked increase in recent years. The contact allergy rate to MI 2,000 ppm alone, not traced by MCI/MI 200 ppm, varied between 0 and 1.9%. In conclusion, due to the increase of contact allergy to MI not traced by MCI/MI 200 ppm, MI in water at 2,000 ppm should be tested in a baseline series. Independent of patch test technique a dose of 60 μg/cm2 should not be exceeded to avoid adverse reactions and particularly patch test sensitisation [ABSTRACT FROM AUTHOR]