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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]

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]

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]

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]

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]

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 quality of diagnostic tests conducted by the Animal Quarantine Service (AQS) is critical to the safety of national livestock production. In procedures for tests such as polymerase chain reaction and enzyme-linked immunosorbent assay, measurement of tiny quantities of samples or reagents is quite common. Therefore, many efforts have been made to maintain the quality of micropipette operations, including routine calibration of equipment and training programs for operators. In this study, we developed a pilot training program to analyze the effects of training on pipetting accuracy, i.e., trueness and precision. The program involved a self-instruction A4-sized leaflet describing how to improve pipetting technique. Thirty-three staff members from seven AQS laboratories participated in the program. Each participant repeated sampling of 300 μL of distilled water 10 times before and after reading the leaflet, and the effects of training on the trueness and precision of the pipetting results were analyzed. To determine pipetting trueness, we examined the variation of the delivered volume from the target volume (300 μL) using the proportional odds mixture model. Our data demonstrated that participation in the training program and the level of experience with pipetting significantly improved trueness. Analysis of standard deviations of the ten samplings, as an indicator of precision, also showed that participation in the training program improves pipetting precision. Thus, our data supported the implementation of such simple, quick training sessions to improve pipetting trueness and precision in diagnostic laboratories. [ABSTRACT FROM AUTHOR]

The gravimetric method specified by ISO 8655-6 is a standard method for calibrating piston pipettes (micropipettes). The quality of the calibration can be assessed by uncertainty evaluation, the procedure for which is described in ISO/Technical Report (TR) 20461. However, the existence of 'method uncertainty' due to ambiguity in the calibration operation, which is not described in the TR, has been found in various experiments, such as the interlaboratory comparison of CCM.FF-K4.2.2011. In this report, the 'method standard uncertainty' is quantified as exp(−4.51 + 0.36 ln( V/μL) + 0.05{ln( V/μL)}) μL for a nominal volume, V, in the range from 2 μL to 10000 μL. Furthermore, the reported values in an interlaboratory comparison are confirmed to be consistent using the quantified method standard uncertainty. [ABSTRACT FROM AUTHOR]

The micropipette technique has been widely used to determine the mechanical response of living cells. Cell is a complex structure and has highly nonlinear behavior when aspirated by the micropipette. Different models exist in the literature to determine the physical properties of the cells from the micropipette aspiration. In this work, we model the cell's response during micropipette aspiration using continuum nonlinear membrane theory. The peripheral surface of the cell is modeled as a nonlinear membrane. The membrane is homogenous, isotropic, and of hyper-elastic material. The cytoplasm of the cell is modeled as an incompressible fluid. Our results show good agreement with the experiments reported in the literature. [ABSTRACT FROM AUTHOR]

Understanding mechanotransduction pathways leading to thrombosis will require progressive steps, including determination of the mechanical behavior of the platelet membrane in response to applied loads. The platelet membrane deformation capacity, as quantified by membrane progression into a borosilicate glass micropipette of defined internal diameter, was probed in murine platelets using a controlled range of negative pressure (0-7 cm HO). Based on our observations that the platelet portion outside the micropipette was mostly spherical and that the platelet volume did not change upon aspiration, a novel continuum mechanics-based model of the platelet micropipette aspiration experiment was created, and a new hyperelastic isotropic material model including membrane residual tension was proposed for the platelet membrane. Murine platelet membranes maintained an average linear deformation behavior: L/ R = 146,100 p × R + 19.923, where L is the platelet length aspirated in the micropipette (m), R is micropipette radius (m) and p is the aspiration pressure (Pa). The theoretical model was used to generate material constants for the murine platelet membrane that allowed for an accurate simulation of the micropipette aspiration experiments. From published results, another set of material constants was established for the human platelet membrane. Limited cases of platelet lysis upon aspiration were analyzed using the theoretical model to determine preliminary membrane tension strength values. [ABSTRACT FROM AUTHOR]

Using the method of gene transfection with liposome, we obtained the mouse erythroleukemia cell line MEL-TF19, which stably carries TFAR19, a novel apoptosis-related gene. The expression of TFAR19 was detected by Western blot. Growth curve and flow cytometry analysis showed that after being transfected with TFAR19 gene, the growth of MEL-TF19 is suppressed and its apoptosis is accelerated because of the serum deprivation. Our biorheological study indicated that in the apoptotic process, compared with MEL cells, MEL-TF19 cells exhibit larger osmotic fragility, lower cell surface charge density, increased elastic modulus K 1 which is inversely proportional to cells’ maximal deformation ability, obviously diminished surface viscosity μ, with elastic modulus K 2having no distinct changes. The above results provided some bases for recognizing the function of TFAR19 completely from the viewpoint of biorheology. [ABSTRACT FROM AUTHOR]

Selectin/ligand interactions initiate the multistep adhesion and signaling cascades in the recruitment of leukocytes from circulation to inflamed tissues and may also play a role in tumor metastasis. Kinetic properties of these interactions are essential determinants governing blood-borne cells' tethering to and rolling on the vessel wall. Extending our recently developed micropipette method, we have measured the kinetic rates of E-selectin/ligand interactions. Red cells coated with an E-selectin construct were allowed to bind HL-60 or Colo-205 cells bearing carbohydrate ligands. Specific adhesions were observed to occur at isolated points, the frequency of which followed a Poisson distribution. These point attachments were formed at the same rate with both the HL-60 and Colo-205 cells (0.14 ± 0.04 and 0.13 ± 0.03 µ m2s-1 per unit density of E-selectin, respectively) but dissociated from the former at a rate twice as fast as did from the latter (0.92 ± 0.23 and 0.44 ± 0.10s-1, respectively). The reverse rates agree well with those measured by the flow chamber. The forward rates are orders of magnitude higher than those of Fcγ receptors interacting with IgG measured under similar conditions, consistent with the rapid kinetics requirement for the function of E-selectin/ligand binding, which is to capture leukocytes on endothelial surfaces from flow. © 2001 Biomedical Engineering Society. PAC2001: 8717-d, 8719Tt [ABSTRACT FROM AUTHOR]

This paper describes an electronic device for the measurement of the diameter of the tip opening of micropipettes. The device measures the threshold pressure required to produce bubbles from the micropipette submerged in a liquid and displays the tip inner diameter in micrometres. We demonstrate that this technique can be used effectively in a range of liquids of different density, surface tension and viscosity and that the thickness of the glass from which the micropipettes are made does not influence the measurements. [ABSTRACT FROM AUTHOR]

The ampullary wall of the posterior semicircular canal was isolated from the frog, cut and the cupula exposed in frog Ringer's solution. The cupula was stimulated by depression with a fine glass micropipette. Three points on the cupula were selected for depression: point A, the lowest point on the midline of the cupula surface on the utricular side; point B, the uppermost on the midline of the same surface; and point C, in the middle of the lateral surface of the cupula. Depression at points A and B was toward the canal, whereas depression at point C was toward the centre of the cupula. The amount of depression was controlled by a micromanipulator. At point A, the minimum depression facilitating the posterior canal action potential was 1 μ; at point B, a greater amount of depression was needed to produce the action potential. Even a large amount of depression at point C resulted in only a small action potential, possibly because the direction of the sensory cell polarity is along the long axis of the canal. [ABSTRACT FROM AUTHOR]

We have determined the effect of hydrostatic edema on total and segmental vascular resistances in the rat lung. Lungs of 12 adult rats, body weight 515±42 g, were isolated and perfused with blood. To investigate the role of vasoactivity on edema effects, we studied two groups of lungs; group I (n=6) were untreated and group II (n=6) were treated with papaverine hydrochloride to paralyze the vasculature. Initially blood flow was adjusted to keep pulmonary artery pressure ∼15 cmHO, left atrial and airway pressures being 8 and 7 cmHO, respectively, and then kept unchanged thereafter (18±3 ml/kg/min). Hydrostatic edema was induced by raising venous pressure and pulmonary artery pressure measured continuously. In 4 lungs from each group, during baseline and after the development of severe edema, we partitioned the pulmonary circulation into arteries, microvessels, and veins by measuring pressures in 20-50 µm diameter subpleural arterioles and venules with the micropipette-Servonull method. Baseline total vascular resistance was similar in the two groups. Interstitial and early alveolar edema did not affect pulmonary vascular pressures. With severe edema (W/D ratio: 17±2), pressures in pulmonary artery and arterioles increased significantly in both groups; venular pressures did not change. Total resistance increased by 250% in group I and by 224% in group II lungs. Arterial resistance increased 3-5-fold in both groups, as did microvascular resistance. Venous resistances also increased in both groups, although to a lesser extent. The increase in total and segmental vascular resistances was not significantly different in the two groups of lungs. We conclude that in isolated rat lungs only severe edema results in an increase in total vascular resistance, mainly due to an increase in arterial and microvascular resistances, with a smaller increase in venous resistance. This appears to be a mechanical effect of edema on the vasculature and not a result of active vasomotion. [ABSTRACT FROM AUTHOR]

Spectrin forms aggregates in solution when incubated at relatively high concentrations (several millimolar) of divalent cations. According to the evidence of electron microscopy, aggregates of globular appearance and rather uniform size are cooperatively formed from spectrin dimers, no intermediate structures being seen. Inter-dimer chemical cross-linking of spectrin in intact red cell membranes is enhanced if magnesium ions at a concentration of 0.5 mM or more are present. On the other hand, the elimination of magnesium from the interior of intact cells causes no significant change in shear elastic modulus, measured by micropipette assays, nor is there any dependence of membrane filtration rate on intracellular free magnesium concentration in the range 0-1 mM. Magnesium-depleted cells are, however, converted into echinocytes within a short period, in which control cells, exposed to ionophore and external magnesium ions, remain completely discoid. Magnesium-depleted cells also undergo structural changes on heating below the temperature at which vesiculation sets in. These reveal themselves by the transformation of the cells to a unique characteristic shape, by grossly reduced filtrability, and by extensive agglutination of the cells when treated with a bifunctional reagent. Magnesium ions thus regulate the stability, but not to any measurable extent the gross elasticity, of the red cell membrane. [ABSTRACT FROM AUTHOR]

Desensitization of ACh-receptors occurs when ACh-micropipettes with resistances smaller than 100 MΩ are used, even if an applied 'braking' current is set to an optimum. High sensitivity with almost no desensitization can be obtained with pipettes of higher resistances, but the current passing capability is normally reduced and a high time constant makes ACh-pulses of 1 msec duration a problem. These drawbacks can be reduced by using a potent electronic constant current source which simultaneously neutralizes the pipette's capacity. The resistance range from 120 to 200 MΩ seems to be optimal for microapplication of ACh, especially in exploring regions of low sensitivity. Rather uniform pipettes in this range can be obtained by applying a cooling air jet during the pull of the pipettes. [ABSTRACT FROM AUTHOR]

Isolation of single cells is a fundamental technique for single-cell analyses. High resolution of controlled volume is required to isolate the target cell from a cell group with tiny medium of single-cell level. Furthermore, the speed of isolation is also an important requirement to capture a motile cell. To achieve cell isolation with high resolution at high speed, we present a high-speed picoliter pipette as an isolation device, which is driven by a piezoelectric actuator. We evaluated performances of the developed picoliter pipette. The results showed a high-resolution and high-speed response of 0.42 pL and 3.9 ms, respectively. In addition, we applied the picoliter pipette to isolate the Euglena gracilis (E. gracilis) cell which was a motile cell. Utilizing the fast response of developed picoliter pipette, we succeeded in picking up a swimming single E. gracilis cell. After the picking up, we isolated the picked cells into other culture dish individually. We evaluated viability of the isolated E. gracilis cells, and 99.1 % (total: 110 cells) of the cells showed motility and division ability. From these results, we achieved the high-speed picoliter pipetting for single-cell isolation. [ABSTRACT FROM AUTHOR]

A novel micropipette holder has been designed to overcome the problem of drift enountered when experimenting on small preparations using patchclamp techniques. In order to increase the stiffness and thermal stability of the system, the holder is designed such that the micropipette is clamped to a rigid quartz rod, and isolated from stresses arising in the connectors. The pressure line, made from stiff tubing, and electrical leads are firmly attached to the table and micromanipulator. This design was able to hold the micropipette tip stable to with 100 nm for more than 1 h, even with changes of 26.7 kPa (200 mmHg) in the pressure line. [ABSTRACT FROM AUTHOR]