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PubMed2002

Determination of cellular strains by combined atomic force microscopy and finite element modeling.

Many organs adapt to their mechanical environment as a result of physiological change or disease. Cells are both the detectors and effectors of this process. Though many studies have been performed in vitro to investigate the mechanisms of detection and adaptation to mechanical strains, the cellular strains remain unknown and results from different stimulation techniques cannot be compared. By combining experimental determination of cell profiles and elasticities by atomic force microscopy with finite element modeling and computational fluid dynamics, we report the cellular strain distributions exerted by common whole-cell straining techniques and from micromanipulation techniques, hence enabling their comparison. Using data from our own analyses and experiments performed by others, we examine the threshold of activation for different signal transduction processes and the strain components that they may detect. We show that modulating cell elasticity, by increasing the F-actin content of the cytoskeleton, or cellular Poisson ratio are good strategies to resist fluid shear or hydrostatic pressure. We report that stray fluid flow in some substrate-stretch systems elicits significant cellular strains. In conclusion, this technique shows promise in furthering our understanding of the interplay among mechanical forces, strain detection, gene expression, and cellular adaptation in physiology and disease.

Actins↗
PubMed2002

Three-dimensional cellular deformation analysis with a two-photon magnetic manipulator workstation.

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within +/-10% over an area 500 x 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses may contain three-dimensional global deformations and show the suitability of this workstation to further studying cellular response to mechanical stresses.

3T3 Cells↗
PubMed2002

Stress response in Caenorhabditis elegans caused by optical tweezers: wavelength, power, and time dependence.

Optical tweezers have emerged as a powerful technique for micromanipulation of living cells. Although the technique often has been claimed to be nonintrusive, evidence has appeared that this is not always the case. This work presents evidence that near-infrared continuous-wave laser light from optical tweezers can produce stress in Caenorhabditis elegans. A transgenic strain of C. elegans, carrying an integrated heat-shock-responsive reporter gene, has been exposed to laser light under a variety of illumination conditions. It was found that gene expression was most often induced by light of 760 nm, and least by 810 nm. The stress response increased with laser power and irradiation time. At 810 nm, significant gene expression could be observed at 360 mW of illumination, which is more than one order of magnitude above that normally used in optical tweezers. In the 700-760-nm range, the results show that the stress response is caused by photochemical processes, whereas at 810 nm, it mainly has a photothermal origin. These results give further evidence that the 700-760-nm wavelength region is unsuitable for optical tweezers and suggest that work at 810 nm at normal laser powers does not cause stress at the cellular level.

Animals↗
PubMed1964

MECHANICAL PROPERTIES OF THE RED CELL MEMBRANE. I. MEMBRANE STIFFNESS AND INTRACELLULAR PRESSURE.

The technique of Mitchison and Swann (1954) was modified for determining the resistance to deformation, or "stiffness," of the red cell membrane and the pressure gradient across the cell wall. It requires a measure of the pressure needed to suck a portion of the cell into a micropipette. Stiffness of hypertonically crenated cells was less than that of biconcave discs or hypotonically swollen cells. Crenated cells showed zero pressure gradient and a stiffness, probably due to pure bending, equivalent to 0.007 +/- 0.001 (SE) dynes/cm. Normal and swollen cells showed a pressure gradient of 2.3 +/- 0.8 (SE) mm H(2)O and a stiffness, due to bending and tension in the membrane, equivalent to 0.019 +/- 0.002 (SE) dynes/cm. No difference in stiffness was found between the rim and the biconcavity of the cell or between biconcave discs and hypotonically swollen cells. Micromanipulation showed that the membrane can withstand large bending strains but limited tangential strains (stretching). These results have significant implications in any theory explaining the cell shape. For example, the data give no indication that the physical properties of the membrane are different at the rim from those of the biconcavities, and the existence of a positive pressure in the normal cell is established.

Biochemical Phenomena↗
PubMed1983

Free energy potential for aggregation of erythrocytes and phosphatidylcholine/phosphatidylserine vesicles in Dextran (36,500 MW) solutions and in plasma.

The free energy potential (affinity) for aggregation of human red blood cells and lipid vesicles in Dextran solutions and blood plasma has been quantitated by measuring to what extent a vesicle is encapsulated by the red cell surface. The free energy reduction per unit area of contact formation (affinity) was computed from the observation of the fractional extent of encapsulation at equilibrium with the use of a relation based on the elastic compliance of the red cell membrane as it is deformed to adhere to the vesicle surface. Micromanipulation methods were used to select and transfer single lipid vesicles (2-3 X 10(-4) cm diameter) from a chamber that contained the vesicle suspension to a separate chamber on the microscope stage that contained red cells in an EDTA buffer with Dextran or whole plasma. The vesicle and a red cell were maneuvered into close proximity and contact allowed to take place without forcing the cells together. To evaluate the effects of surface charge density and steric interactions on aggregation, vesicles were made from mixtures of egg phosphatidylcholine (PC) and bovine phosphatidylserine (PS) over a range of mole ratios (PC/PS)from (1:0) to (1:1); the vesicles were formed by rehydration in buffer. The Dextran solutions were made with a sharp-cut fraction of 36,500 MW in a concentration range of 0-10% by weight in grams (wt/wt). It was found that the Dextran 36,500 MW fraction produced aggregation behavior for red cells and vesicles similar to red cell-red cell aggregation in Dextran 70,000-150,000 MW fractions, when the vesicle surface charge density was comparable with that of normal red cells (i.e., PC/PS ratio of -3:1). This result indicated that Dextran molecules penetrate between the carbohydrate groups on the red cell surface and that either steric interactions between cell surface carbohydrates are important or many of the charge groups on red cells are superficial. Electrostatic repulsion effects were apparent, as no aggregation in Dextran 36,500 MW occurred for PC/PS ratios <2.6:1; the level of affinity increased with the PC content at a specific Dextran concentration. Affinities were measured in the range of 0-2 x 10-2 ergs/cm2. When adherent red cell-vesicle pairs were transferred into a Dextran-free buffer, the pair did not spontaneously separate.They maintained adhesive contact until forcibly parted, after which they would not read here. This demonstrates that Dextran forms a "cross-bridge" between the membrane surfaces. Red cell-vesicle aggregation was also tested in whole plasma, which normally yields affinity values in the range of 2-4 x 10-3 ergs/cm2 for red cell-red cell aggregation.However, no red cell-vesicle aggregation occurred in plasma, even for pure PC vesicles. This result indicates that either the aggregating plasma proteins (primarily fibrinogen) do not bind sufficiently to the lecithin surface, or they are shielded from binding to the surface by the presence of other nonaggregating components (perhaps albumin).

Dextrans↗
PubMed1984

Free energy potential for aggregation of giant, neutral lipid bilayer vesicles by Van der Waals attraction.

Here, we report the first direct observation of Van der Waals' attraction between biomembrane capsules using measurements of the free energy reduction per unit area of membrane-membrane contact formation. In these studies, the membrane capsules were reconstituted neutral (egg phosphatidylcholine) lipid bilayers of giant (greater than 10(-3) cm diam) vesicles. Micromanipulation methods were used to select and maneuver two vesicles into proximity for contact; after adhesion was allowed to occur, the extent of contact formation was regulated through the vesicle membrane tensions that were controlled by micropipette suction. The free energy reduction per unit area of contact formation was proportional to the membrane tension multiplied by a simple function of the pipette and vesicle dimensions. The free energy potential for Van der Waals attraction between the neutral bilayers in 120 mM NaCl solutions was 1.5 X 10(-2) ergs/cm2. Also, when human serum albumin was added to the medium in the range of 0-1 mg/ml, the free energy potential for bilayer-bilayer adhesion was not affected. Using published values for equilibrium spacing between lipid bilayers in multilamellar lipid-water dispersions and the theoretical equation for van der Waals attraction between continuous dielectric layers, we calculated the value for the Hamaker coefficient of the Van der Waals attraction to be 5.8 X 10(-14) ergs.

Cell Aggregation↗
PubMed1987

Use of cell contour analysis to evaluate the affinity between macrophages and glutaraldehyde-treated erythrocytes.

Recently, several authors evaluated the affinity between lipid bilayers or erythrocyte membranes by analyzing the deformation of cells or vesicles they brought into close contact using micromanipulators. In the present report, we extend this approach in a study of the adhesive properties of rough nucleated cells. Rat peritoneal macrophages were made to bind human red cells modified with glutaraldehyde or glutaraldehyde and polylysine. Conjugates were examined with electron microscopy, and photomicrographs were digitized for quantification of cell surface roughness in and out of adhesion areas. Also, macrophages were subjected to micropipette aspiration to find a relationship between apparent surface tension and area increase. Assuming that this increase was a direct consequence of a smoothing of the cell surface on the submicrometer scale, the actual affinity between macrophages and erythrocytes was estimated. The obtained values ranged between 8.4 X 10(-5) and 18.2 X 10(-5) J/m2. It is concluded that cell surface roughness may be an important parameter of cell adhesion and perhaps deformation. This is made amenable to experimental study by the present approach.

Aldehydes↗
PubMed1989

Theoretical and experimental studies on cross-bridge migration during cell disaggregation.

A micromanipulation method is used to determine the adhesive energy density (gamma) between pairs of cytotoxic T cells (F1) and their target cells (JY: HLA-A2-B7-DR4,W6). gamma is defined as the energy per unit area that must be supplied to reduce the region of contact between a conjugated cell pair. Our analysis of the data indicates that the force applied by the micropipette on the cell is not uniformly distributed throughout the contact region as we had previously assumed (Sung, K. L. P., L. A. Sung, M. Crimmins, S. J. Burakoff, and S. Chien. 1986. Science (Wash. DC). 234: 1405-1408), but acts only at the edges of the contact region. We show that gamma is not constant during peeling but increases with decreasing contact area of the conjugated cell pairs F1-JY, F1-F1, and JY-JY in contrast to the constancy of gamma for typical engineering adhesives. This finding supports the notion that the cross-linking protein molecules slide towards the conjugated area across the leading edge of the separation while remaining attached to both cells. Our mathematical analysis shows that the elastic energy stored in the cross-links by the membrane tensions balances the diffusive forces that act against cross-bridge migration. The binding affinity between F1-JY is found to be approximately 15-20 times larger than the corresponding affinity for F1-F1. The number of binding sites of F1 for attachment to JY is approximately the same for binding F1 to another F1 and vary between 10(5) and 10(6).

Animals↗
PubMed1990

Cell-cell conjugation. Transient analysis and experimental implications.

In the present study we investigate the transient conjugation of cell pairs by using a mathematical model. Macromolecules responsible for adhesion (bonds) are assumed to exist in two reversible states, attached and unattached, and exert a force elastic in nature only when they cross-link the two cell surfaces (attached state). Bonds form a link between the two cell surfaces only in the attached form. The unattached bridges are assumed laterally mobile in the plane of the cell membrane. Lateral mobility of attached bonds may be limited by structures on the undersurface of the cell membrane. Using this model we show that the bond density distribution between a cytotoxic T-cell (F-1) and a cancer cell (JY:HLA-A2-B7-DR4, W6) approaches equilibrium within 10 min, the incubation period used in experiments by Sung, K.L.P., L.A. Sung, M. Crimmins, S.J. Burakoff, and S. Chien (1986. Science [Wash. DC]. 234:1405-1408). If the diffusion coefficient of attached bonds is set equal to zero in the computations the model predictions indicate accumulation of bonds at the edge of conjugation. This prediction is consistent with present experimental data on lectin-induced red blood cell aggregation (Vayo, M., R. Skalak, P. Brunn, S. Usami, and S. Chien. 1987. Fed. Proc. 46:1043). It is concluded that significant features of micromanipulation data on specific adhesion can be explained by the diffusivity properties of bonds responsible for adhesion.

Cell Adhesion↗
PubMed1992

Spontaneous oscillation of tension and sarcomere length in skeletal myofibrils. Microscopic measurement and analysis.

We have devised a simple method for measuring tension development of single myofibrils by micromanipulation with a pair of glass micro-needles. The tension was estimated from the deflection of a flexible needle under an inverted phase-contrast microscope equipped with an image processor, so that the tension development is always accompanied by the shortening of the myofibril (auxotonic condition) in the present setup. The advantage of this method is that the measurement of tension (1/30 s for time resolution and about 0.05 micrograms for accuracy of tension measurement; 0.05 microns as a spatial resolution for displacement of the micro-needle) and the observation of sarcomere structure are possible at the same time, and the technique to hold myofibrils, even single myofibrils, is very simple. This method has been applied to study the tension development of glycerinated skeletal myofibrils under the condition where spontaneous oscillation of sarcomeres is induced, i.e., the coexistence of MgATP, MgADP and inorganic phosphate without free Ca2+. Under this condition, we found that the tension of myofibrils spontaneously oscillates accompanied by the oscillation of sarcomere length with a main period of a few seconds; the period was lengthened and shortened with stretch and release of myofibrils. A possible mechanism of the oscillation is discussed.

Animals↗
PubMed1995

Calcium response of helper T lymphocytes to antigen-presenting cells in a single-cell assay.

We developed a dynamic, single-cell assay involving alternating differential interference contrast and fluorescence microscopy, together with digital imaging, for both viewing the physical interaction of live helper T lymphocytes (Th cells) with antigen-presenting cells (APCs) and monitoring the increases in the intracellular free calcium concentration of the Th cell, an early event in Th cell activation. We obtained Th-APC conjugates by allowing the Th cells to migrate toward and interact with APCs that either settled nearby or had been micromanipulated in close proximity to the Th cells. Th cell motility played an important role in initiating Th-APC contacts but not in determining the Th cell calcium response. We found that the intracellular calcium responses of individual Th cells are heterogeneous and an all-or-none phenomenon, independent of antigen concentration. However, the fraction of Th-APC conjugates involving responding Th cells is an increasing function of the antigen concentration. Finally, we measured some characteristics of the developing Th-APC contact area. We used all of these data together with previously developed mathematical models to estimate that only 1 to 20 major histocompatibility class II-antigen complexes are required in the initial Th-APC contact area to elicit a Th cell calcium response.

Analysis of Variance↗
PubMed1995

Evidence for localized cell heating induced by infrared optical tweezers.

The confinement of liposomes and Chinese hamster ovary (CHO) cells by infrared (IR) optical tweezers is shown to result in sample heating and temperature increases by several degrees centigrade, as measured by a noninvasive, spatially resolved fluorescence detection technique. For micron-sized spherical liposome vesicles having bilayer membranes composed of the phospholipid 1,2-diacyl-pentadecanoyl-glycero-phosphocholine (15-OPC), a temperature rise of approximately 1.45 +/- 0.15 degrees C/100 mW is observed when the vesicles are held stationary with a 1.064 microns optical tweezers having a power density of approximately 10(7) W/cm2 and a focused spot size of approximately 0.8 micron. The increase in sample temperature is found to scale linearly with applied optical power in the 40 to 250 mW range. Under the same trapping conditions, CHO cells exhibit an average temperature rise of nearly 1.15 +/- 0.25 degrees C/100 mW. The extent of cell heating induced by infrared tweezers confinement can be described by a heat conduction model that accounts for the absorption of infrared (IR) laser radiation in the aqueous cell core and membrane regions, respectively. The observed results are relevant to the assessment of the noninvasive nature of infrared trapping beams in micromanipulation applications and cell physiological studies.

Animals↗
PubMed1997

Extension of torsionally stressed DNA by external force.

Metropolis Monte Carlo simulation was used to study the elasticity of torsionally stressed double-helical DNA. Equilibrium distributions of DNA conformations for different values of linking deficit, external force, and ionic conditions were simulated using the discrete wormlike chain model. Ionic conditions were specified in terms of DNA effective diameter, i.e., hard-core radius of the model chain. The simulations show that entropic elasticity of the double helix depends on how much it is twisted. For low amounts of twisting (less than about one turn per twist persistence length) the force versus extension is nearly the same as in the completely torsionally relaxed case. For more twisting than this, the molecule starts to supercoil, and there is an increase in the force needed to realize a given extension. For sufficiently large amounts of twist, the entire chain is plectonemically supercoiled at low extensions; a finite force must be applied to obtain any extension at all in this regime. The simulation results agree well with the results of recent micromanipulation experiments.

Computer Simulation↗
PubMed1998

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique.

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level.

Adsorption↗
PubMed1998

Behavior of supercoiled DNA.

We study DNA supercoiling in a quantitative fashion by micromanipulating single linear DNA molecules with a magnetic field gradient. By anchoring one end of the DNA to multiple sites on a magnetic bead and the other end to multiple sites on a glass surface, we were able to exert torsional control on the DNA. A rotating magnetic field was used to induce rotation of the magnetic bead, and reversibly over- and underwind the molecule. The magnetic field was also used to increase or decrease the stretching force exerted by the magnetic bead on the DNA. The molecule's degree of supercoiling could therefore be quantitatively controlled and monitored, and tethered-particle motion analysis allowed us to measure the stretching force acting on the DNA. Experimental results indicate that this is a very powerful technique for measuring forces at the picoscale. We studied the effect of stretching forces ranging from 0.01 pN to 100 pN on supercoiled DNA (-0.1 < sigma < 0.2) in a variety of ionic conditions. Other effects, such as stretching-relaxing hysteresis and the braiding of two DNA molecules, are discussed.

Biophysics↗
PubMed2000

Sperm chromosome analysis and outcome of IVF in patients with non-mosaic Klinefelter's syndrome.

OBJECTIVE: The aim of the study was to determine the potential risk for fetal chromosomal anomalies in non-mosaic Klinefelter's syndrome patients undergoing IVF and intracytoplasmic sperm injection. DESIGN: Individually collected spermatozoa were isolated from wet testicular tissue preparations and fixed on glass slides using micromanipulation. Their nuclei were analyzed for chromosomes X, Y, and 18 by fluorescent in situ hybridization. SETTING: Assisted reproductive technology program. PATIENT(S): Consenting patients with non-mosaic Klinefelter's syndrome undergoing testicular biopsy and IVF (fresh specimens) or following such treatment (cryopreserved specimens). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The rates of numerical chromosome abnormalities for chromosomes X, Y, and 18 among spare testicular sperm and the pregnancy outcome following treatment. RESULT(S): Testicular sperm were found in 8 of 20 patients. Four couples became pregnant following embryo replacement. Sperm chromosomes were analyzed in five patients. One hundred and five sperm of 112 analyzed (93.7%) were normal with X to Y ratio of 50:55 (NS) respectively. Among the 112 sperm tested, seven (6.3%) demonstrated chromosomal abnormalities, of which five were related to the sex chromosomes and two to chromosome 18. One set of triplets, one set of twins, and two singletons (four males and three females) with normal karyotypes were born. CONCLUSION(S): Most of the testicular sperm retrieved from Klinefelter's syndrome patients demonstrates a normal pattern of sex chromosome segregation. Therefore, the risk of transmitting numerical sex chromosome abnormalities is relatively low and probably comparable with the rates found in other severe male factor infertility patient groups.

Chromosome Aberrations↗
PubMed2000

Assisted reproductive technology in the United States: 1997 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry.

OBJECTIVE: To summarize the procedures and outcomes of assisted reproductive technology (ART) initiated in the United States in 1997. DESIGN: Data were collected electronically by using Society for Assisted Reproductive Technology Clinical Outcome Reporting System software and were submitted to the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. PARTICIPANT(S): 335 programs submitted data on procedures performed in 1997. Data were collated after November 1998 so that the outcome of all pregnancies established would be known. MAIN OUTCOME MEASURE(S): Incidence of clinical pregnancy, ectopic pregnancy, abortion, stillbirth, delivery, and structural and functional abnormalities. RESULT(S): Programs reported initiating 73,069 cycles of ART treatment. Of these, 51,344 cycles involved IVF (with and without micromanipulation), with a delivery rate per retrieval of 27.9%; 1,943 were cycles of GIFT, with a delivery rate per retrieval of 30.0%; and 1,104 were cycles of zygote intrafallopian transfer, with a delivery rate per retrieval of 28.0%. The following additional ART procedures were also initiated: 4,616 donor oocyte cycles, with a delivery rate per transfer of 40.0%; 10,181 frozen embryo transfer procedures, with a delivery rate per transfer of 18.8%; 1,584 frozen embryo transfers using donated oocytes, with a delivery rate per transfer of 22.2%; and 600 cycles using a host uterus, with a delivery rate per transfer of 34.6%. Furthermore, 1,173 cycles were reported as combinations or more than one treatment type, 40 cycles as research, 258 as embryo banking, and 226 as other (unclassified) cycle types. As a result of all procedures, 17,311 deliveries resulting in 25,059 babies were reported. CONCLUSION(S): In 1997, more programs reported ART treatment and the number of reported cycles increased significantly (10.9%) compared with 1996. In comparable cycle types, the overall success rate (deliveries per retrieval) increased by 1.8%, which represents an increase of 6.9% compared with the success rate for 1996.

Abortion, Spontaneous↗
PubMed2001

Sperm extraction at orchiectomy for testis cancer.

OBJECTIVE: To evaluate the value of aspirating sperm from the vas and epididymis at orchiectomy in azoospermic patients. DESIGN: Retrospective clinical study. SETTING: Tertiary care academic hospital. PATIENT(S): Three patients with known azoospermia who presented with testicular masses suspected to be cancerous. INTERVENTION(S): At orchiectomy, immediately after ligation of the spermatic cord, the contents of the epididymis and vas deferens were extracted into preserving media. MAIN OUTCOME MEASURE(S): Fertility rate. RESULT(S): Sperm retrieval was successful in all three patients. The mean total sperm count was 2.3 x 10(6)/mL with 20% motility. Intracytoplasmic injection of sperm harvested by using this method was successful in two couples, one of which delivered a healthy infant. CONCLUSION(S): Sperm can be aspirated from the vas deferens and epididymis at orchiectomy for preservation. In azoospermic patients, this procedure may salvage enough sperm for successful use in micromanipulation techniques. It may be worthwhile to perform sperm aspiration during orchiectomy for testis cancer in any patient with known or suspected infertility.

Cryopreservation↗