Biomedical subjects

O N Mesquita

Publications and source records attributed to O N Mesquita.

9 recordsLinked to original sources

PubMed2006

Characterization of objective transmittance for optical tweezers.

We have measured the overall transmittance of a laser beam through an oil immersion objective as a function of the transverse size of the laser beam, using the dual-objective method. Our results show that the objective transmittance is not uniform and that its dependence on the radial beam's position can be modeled by a Gaussian function. This property affects the intensity distribution pattern in the sample region and should be taken into account in theoretical descriptions of optical tweezers. Moreover, one must consider this position dependence to determine the local laser power delivered at the sample region by the dual-objective method, especially when the beam overfills the objective's back entrance. If the transmittance is assumed to be uniform, the local power is overestimated.

Computer Simulation↗

PubMed2004

DNA-psoralen interaction: a single molecule experiment.

By attaching one end of a single lambda-DNA molecule to a microscope coverslip and the other end to a polystyrene microsphere trapped by an optical tweezers, we can study the entropic elasticity of the lambda-DNA by measuring force versus extension as we stretch the molecule. This powerful method permits single molecule studies. We are particularly interested in the effects of the photosensitive drug psoralen on the elasticity of the DNA molecule. We have illuminated the sample with different light sources, studying how the different wavelengths affect the psoralen-DNA linkage. To do this, we measure the persistence length of individual DNA-psoralen complexes.

Chemistry, Physical↗

PubMed2004

Defocusing microscopy.

Transparent objects (phase objects) are not visible in a standard brightfield optical microscope. In order to see such objects the most used technique is phase-contrast microscopy. In phase-contrast microscopy the contrast observed is proportional to the optical path difference introduced by the object. If the index of refraction is uniform, phase-contrast microscopy then yields a measure of the thickness profile of phase objects. We show that by slightly defocusing an optical microscope operating in brightfield, phase objects become visible. We modeled such an effect and show that the image contrast of a phase object is proportional to the amount of defocusing and proportional to the two-dimensional Laplacian of the optical path difference introduced by the object. For uniform index of refraction, defocusing microscopy then yields a measure of the curvature profile of phase objects. We extended our previous model for thin objects to thick objects. To check our theoretical model, we use as phase objects polystyrene spherical caps and compare their curvature radii obtained by defocusing microscopy (DM) to those obtained with atomic force microscopy (AFM). We also show that for thick curved phase objects one can reconstruct their thickness profiles from DM images. We illustrate the utility of defocusing microscopy in biological systems to study cell motility. In particular, we visualize and quantitatively measure real-time cytoskeleton curvature fluctuations of macrophages (a cell of the innate immune system). The study of such fluctuations might be important for a better understanding of the engulfment process of pathogens during phagocytosis.

Animals↗

PubMed2003

Measurements and modeling of water transport and osmoregulation in a single kidney cell using optical tweezers and videomicroscopy.

With an optical tweezer installed in our optical microscope we grab a single Madin Darby Canine kidney cell and keep it suspended in the medium without touching the glass substrate or other cells. Since the optically trapped cell remains with a closely round shape, we can directly measure its volume by using videomicroscopy with digital image analysis. We submit this cell to a hyperosmotic shock (up-shock) and video record the process: the cell initially shrinks due to osmotic efflux of water and after a while, due to regulatory volume increase (RVI), an osmoregulation response, it inflates again (water influx) until it reaches a new volume (the regulatory volume VR). In addition to considering standard osmotic water transport, we model RVI using a simple phenomenological model. We obtain an expression for cell volume variation as a function of time that fits very well with our experimental data, where two characteristic times appear naturally: one related to water transport and the other related to RVI. From the fit we obtain water permeability, osmolyte influx rate for RVI, and regulatory volume. With the addition of the hormone vasopressin, water permeability increases while the regulatory volume decreases until inhibition of RVI. In summary, we present a technique to measure directly volume changes of a single isolated kidney cell under osmotic shock and a phenomenological analysis of water transport that takes into account osmoregulation.

Adaptation, Physiological↗

PubMed2003

Cell surface fluctuations studied with defocusing microscopy.

Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional (2D) Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature (Laplacian of the local thickness) of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations (ruffles) on the surface of macrophages (cell of the innate immune system), and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the cell surface, increases the relaxation time of cytoskeleton fluctuations, and increases the phagocytosis time. Our results suggest that the methods developed in this work can be of utility to assess the importance of cytoskeleton motility in the dynamics of cellular processes such as phagocytosis exhibited by macrophages.

Actins↗

PubMed2002

Dynamic light scattering from an optically trapped microsphere.

Using a single microscope objective lens to optically trap, illuminate, and collect backscattered light of a dielectric microsphere, we measure the temporal-intensity-autocorrelation functions (ACFs), and intensity profiles to obtain the trap stiffness and friction coefficient of the bead. This is an interesting study of an harmonically bound Brownian particle, with nanometer resolution. We extend the work of Bar-Ziv et al. [Phys. Rev. Lett. 78, 154 (1997)] to more general situations allowing for the use of our simpler geometry in other applications. As examples, we present measurements of the parallel Stokes friction coefficient on the trapped bead as a function of its distance from a surface and the entropic force of a single lambda-DNA molecule.

Bacteriophage lambda↗

PubMed2001

Anomalous capillary length in cellular nematic-isotropic interfaces.

The long-standing puzzle of why capillary lengths measured in cellular nematic-isotropic interfaces are much longer than the value of 0.05 A predicted by Mullins-Sekerka theory has been solved. The resolution of the paradox is that in confined systems the substrate-nematic anchoring energy contributes to the capillary length which is greatly increased by selective adsorption of ions on the substrate.

Journal Article↗

PubMed1999

Experimental test of the Warren-Langer model in nematic-isotropic planar interfaces.

In a directional solidification apparatus, the recoil of the nonsteady planar nematic-isotropic interface of the liquid crystal 8CB doped with hexachloroethane was measured, for different pulling velocities. Results agree very well with the predictions of our two-sided extension of Warren and Langer's one-sided model [Phys. Rev. E 47, 2702 (1993)], therefore supporting the validity of their ansatz about the evolution of the dopant concentration field. From the comparison between experiment and theory we obtain values for the segregation and diffusion coefficients of hexachloroethane in 8CB comparable to those found in the literature and measured by other methods. Using the same procedure, we measured the value of the segregation coefficient of 8CB doped with water as a function of applied sinusoidal electric field perpendicular to the sample, along the homeotropic direction. The segregation coefficient increases with electric field. In addition, preliminary results on the cellular instability in this system show that the capillary length of the pattern also increases with electric field. To our knowledge, this is the first binary system with continuously tunable segregation coefficient and capillary length.

Journal Article↗

PubMed1998

Gramicidin channel kinetics under tension.

We have measured the effect of tension on dimerization kinetics of the channel-forming peptide gramicidin A. By aspirating large unilamellar vesicles into a micropipette electrode, we are able to simultaneously monitor membrane tension and electrical activity. We find that the dimer formation rate increases by a factor of 5 as tension ranges from 0 to 4 dyn/cm. The dimer lifetime also increases with tension. This behavior is well described by a phenomenological model of membrane elasticity in which tension modulates the mismatch in thickness between the gramicidin dimer and membrane.

Dimerization↗