32 results on '"Mesquita, O. N."'
Search Results
2. Transition on the entropic elasticity of DNA induced by intercalating molecules.
- Author
-
Rocha, M. S., Ferreira, M. C., and Mesquita, O. N.
- Subjects
DNA ,NUCLEIC acids ,MOLECULES ,GENES ,MATHEMATICAL physics ,STRENGTH of materials - Abstract
We use optical tweezers to perform stretching experiments on DNA molecules when interacting with the drugs daunomycin and ethidium bromide, which intercalate the DNA molecule. Our results show that the persistence length of the DNA-drug complexes increases strongly as the drug concentration increases up to some critical value. Above this critical value, the persistence length decreases abruptly and remains approximately constant for larger drug concentrations, at least in the concentration range used in our experiments. Measured intercalators critical concentrations for the persistence length transition coincide with the reported values for the helix-coil transition of DNA-drug complexes obtained from sedimentation experiments. The contour length of the molecules increases monotonically and saturates as the drug concentration increases. The neighbor exclusion model fits to our results for the total drug concentration as a function of the relative increase of the contour length. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
3. DNA-psoralen interaction: A single molecule experiment.
- Author
-
Rocha, M. S., Viana, N. B., and Mesquita, O. N.
- Subjects
PSORALENS ,DNA ,MOLECULES ,POLYSTYRENE ,ENTROPY ,COMPLEX compounds - Abstract
By attaching one end of a single λ-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 λ-DNA by measuring force versus extension as we stretch the molecule. This powerful method permits single molecule studies. We are particulary 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. © 2004 American Institute of Physics. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
4. Shape reconstruction and height fluctuations of red blood cells using defocusing microscopy
- Author
-
Siman, L., Roma, P. M. S., Amaral, F. T., Agero, U., and Mesquita, O. N.
- Subjects
Physics::Biological Physics ,Biological Physics (physics.bio-ph) ,FOS: Physical sciences ,Physics - Biological Physics ,Quantitative Biology::Cell Behavior - Abstract
In this paper the bright-field defocusing microscopy (DM) technique is presented. DM is able to obtain quantitative information of each plane/surface of pure phase objects, as live unlabeled cells, and its application to red blood cells (RBCs) is demonstrated. Based on contrast, simple methods to obtain thickness profile and three dimensional (3D) total reconstruction of RBCs are proposed and the actual height profiles of upper and lower surface-membranes (lipid bilayer$/$cytoskeleton) of discocyte and stomatocyte red cells are presented as examples. In addition, using the mean square contrast fluctuation and modeling the RBC membranes fluctuations spectra as dependent of a bending modulus $(\kappa_c)$, a surface tension $(\sigma)$ and a confining potential $(\gamma)$ term, slowly varying quantities along the cell radius, a genetic algorithm (GA) is used and the radial height fluctuations of each surface-membrane are accessed, separately. The radial behaviors of $\kappa_c$, $\sigma$ and $\gamma$ are also obtained, allowing the discussion of physical aspects of the RBC membrane.
- Published
- 2014
5. Total 3D imaging of phase objects using defocusing microscopy: application to red blood cells
- Author
-
Roma, P. M. S., Siman, L., Amaral, F. T., Agero, U., and Mesquita, O. N.
- Subjects
Biological Physics (physics.bio-ph) ,FOS: Physical sciences ,Physics - Biological Physics ,Optics (physics.optics) ,Physics - Optics - Abstract
We present Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total 3D imaging of transparent objects. By total 3D imaging we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a new methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic and hypertonic solutions. For each situation the shape of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of RBCs surfaces due to adhesion on the glass-substrate. The axial resolution of our technique allowed us to image surface-membranes separated by distances as small as 300 nm. Finally, we determine volume, superficial area, sphericity index and RBCs refractive index for each osmotic condition., 7 pages; 4 figures
- Published
- 2014
6. Towards absolute calibration of optical tweezers
- Author
-
Viana, N. B., Rocha, M. S., Mesquita, O. N., Mazolli, A., Neto, P. A. Maia, and Nussenzveig, H. M.
- Subjects
Biological Physics (physics.bio-ph) ,FOS: Physical sciences ,Physics - Biological Physics ,Optics (physics.optics) ,Physics - Optics - Abstract
Aiming at absolute force calibration of optical tweezers, following a critical review of proposed theoretical models, we present and test the results of MDSA (Mie-Debye-Spherical Aberration) theory, an extension of a previous (MD) model, taking account of spherical aberration at the glass/water interface. This first-principles theory is formulated entirely in terms of experimentally accessible parameters (none adjustable). Careful experimental tests of the MDSA theory, undertaken at two laboratories, with very different setups, are described. A detailed description is given of the procedures employed to measure laser beam waist, local beam power at the transparent microspheres trapped by the tweezers, microsphere radius and the trap transverse stiffness, as a function of radius and height in the (inverted microscope) sample chamber. We find generally very good agreement with MDSA theory predictions, for a wide size range, from the Rayleigh domain to large radii, including the values most often employed in practice, and at different chamber heights, both with objective overfilling and underfilling. The results asymptotically approach geometrical optics in the mean over size intervals, as they should, and this already happens for size parameters not much larger than unity. MDSA predictions for the trapping threshold, position of stiffness peak, stiffness variation with height, multiple equilibrium points and `hopping' effects among them are verified. Remaining discrepancies are ascribed to focus degradation, possibly arising from objective aberrations in the infrared, not yet included in MDSA theory., 15 pages, 20 figures
- Published
- 2006
7. Recoiling DNA Molecule: Simulation & Experiment
- Author
-
Neto, Jose Coelho, Dickman, Ronald, and Mesquita, O. N.
- Subjects
Quantitative Biology::Biomolecules ,Statistical Mechanics (cond-mat.stat-mech) ,Biological Physics (physics.bio-ph) ,FOS: Biological sciences ,FOS: Physical sciences ,Physics - Biological Physics ,Quantitative Biology (q-bio) ,Condensed Matter - Statistical Mechanics ,Quantitative Biology - Abstract
Single molecule DNA experiments often generate data from force versus extension measurements involving the tethering of a microsphere to one end of a single DNA molecule while the other is attached to a substrate. We show that the persistence length of single DNA molecules can also be measured based on the recoil dynamics of these DNA-microsphere complexes if appropriate corrections are made to the friction coefficient of the microsphere in the vicinity of the substrate. Comparison between computer simulated recoil curves, generated from the corresponding Langevin equation, and experimental recoils is used to assure the validity of data analysis., 14 pages (single column preprint), 7 figures. Major changes: data analysis method improved; dna-ethidium bromide results removed (dna-ethidium bromide protocol affected microspheres and coverglass behavior)
- Published
- 2002
8. Molecular dynamic simulation of directional crystal growth
- Author
-
Costa, B. V., Coura, P. Z., and Mesquita, O. N.
- Subjects
Condensed Matter - Materials Science ,Statistical Mechanics (cond-mat.stat-mech) ,Materials Science (cond-mat.mtrl-sci) ,FOS: Physical sciences ,Condensed Matter - Statistical Mechanics - Abstract
We use molecular dynamic to simulate the directional growth of binary mixtures. our results compare very well with analitical and experimental results. This opens up the possibility to probe growth situations which are difficult to reach experimentally, being an important tool for further experimental and theoretical developments in the area of crystal growth., 6 pages, 5 figures. Computer Simulation Studies in Condensed Matter Physics XII, edited by D.P. Landau, et al. (Springer, Heidelberg, in press)
- Published
- 1999
9. Variation of entropic elasticity of DNA-Psoralen complex under UV light.
- Author
-
Rocha, M. S. and Mesquita, O. N.
- Published
- 2005
- Full Text
- View/download PDF
10. Total three-dimensional imaging of phase objects using defocusing microscopy: Application to red blood cells.
- Author
-
Roma, P. M. S., Siman, L., Amara, F. T., Agero, U., and Mesquita, O. N.
- Subjects
ERYTHROCYTES ,THREE-dimensional imaging in biology ,PHASE-contrast microscopy ,HYPERTONIC solutions ,REFRACTIVE index - Abstract
We introduce Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total three-dimensional (3D) imaging of transparent objects. By total 3D imaging, we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic, and hypertonic solutions. For each situation, the shapes of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of red blood cell (RBC) surfaces due to adhesion on the glass-substrate. The axial resolution of our technique allowed us to image surface-membranes separated by distances as small as 300 nm. Finally, we determine the volume, surface area, sphericity index, and RBC refractive index for each osmotic condition. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
11. DNA interaction with Actinomycin D: mechanical measurements reveal the details of the binding data.
- Author
-
Cesconetto, E. C., Junior, F. S. A., Crisafuli, F. A. P., Mesquita, O. N., Ramos, E. B., and Rocha, M. S.
- Abstract
We have studied the interaction between the anticancer drug Actinomycin D (ActD) and the DNA molecule by performing single molecule stretching experiments and atomic force microscopy (AFM) imaging. From the stretching experiments, we determine how the mechanical properties of the DNA–ActD complexes vary as a function of drug concentration, for a fixed DNA concentration. We have found that the persistence lengths of the complexes formed behave non-monotonically: at low concentrations of ActD they are more flexible than the bare DNA molecule and become stiffer at higher concentrations. On the other hand, the contour length increases monotonically as a function of ActD concentration. Using a two-sites quenched disorder statistical model recently developed by us, we were able to extract chemical parameters such as the intrinsic binding constant and the degree of cooperativity from these pure mechanical measurements, thus performing a robust characterization of the interaction. The AFM images, otherwise, were used to measure the bending angle size distribution that ActD introduces on the double-helix structure and the average number of bendings per DNA molecule as a function of drug concentration, two quantities that cannot be determined from the stretching experiments. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
12. In situ laser power measurement at the focus of microscope objectives used in optical tweezers.
- Author
-
Viana, N. B., Rocha, M. S., and Mesquita, O. N.
- Subjects
LASERS ,OPTOELECTRONIC devices ,MICROSCOPES ,OPTICAL instruments ,MERCURY ,ELECTROSTATICS ,PHYSICS - Abstract
We discuss measurements of the laser power at the focus of high numerical aperture objectives used in optical microscopy and optical tweezers. For a given power, the focused incident laser beam heats a small mercury bead that jumps when it reaches the boiling temperature of water, the medium used in the experiments. From the size of the mercury beads, the heat conductivities of water and glass-slide, and the absorption coefficient of mercury for the laser wavelength used, the incident power can be obtained using the solution of the associated heat problem. This problem is mapped onto the problem of a charged conducting sphere embedded in a semi-infinite medium of dielectric constant ϵ
1 , separated from another semi-infinite medium of dielectric constant ϵ2 . [ABSTRACT FROM AUTHOR]- Published
- 2005
- Full Text
- View/download PDF
13. Probability distributions and thermal transport in a turbulent grid flow.
- Author
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Lane, B. R., Mesquita, O. N., Meyers, S. R., and Gollub, J. P.
- Subjects
- *
HEAT transfer , *TRANSPORT theory , *PROBABILITY theory - Abstract
Recent theoretical proposals concerning non-Gaussian statistics of passive scalars in random velocity fields are tested experimentally, by measuring the probability distributions of fluctuating temperature in an oscillating grid flow across which a steady temperature gradient is maintained. Pronounced exponential tails occur at sufficiently high Reynolds number R, and predominantly Gaussian statistics at low R. When the extended tails are present for the passive scalar, the corresponding velocity power spectrum shows reasonable scaling, and the velocity distribution is not far from Gaussian. The present paper provides a more complete characterization of the flow field than an earlier brief report [Phys. Rev. Lett. 67, 3507 (1991)], and also contains a description of additional features, such as the skewness of the distributions. Finally, the effective or eddy diffusivity of both heat and a molecular impurity are measured and compared. [ABSTRACT FROM AUTHOR]
- Published
- 1993
- Full Text
- View/download PDF
14. Molecular Dynamics Simulation of Zone Melting.
- Author
-
Coura, P. Z., Mesquita, O. N., and Costa, B. V.
- Published
- 1998
- Full Text
- View/download PDF
15. DNA-psoralen: Single-molecule experiments and first principles calculations.
- Author
-
Rocha, M. S., Lúcio, A. D., Alexandre, S. S., Nunes, R. W., and Mesquita, O. N.
- Subjects
PSORALENS ,DNA ,NUCLEIC acids ,COUMARINS ,DNA probes - Abstract
The authors measure the persistence and contour lengths of DNA-psoralen complexes, as a function of psoralen concentration, for intercalated and crosslinked complexes. In both cases, the persistence length monotonically increases until a certain critical concentration is reached, above which it abruptly decreases and remains approximately constant. The contour length of the complexes exhibits no such discontinuous behavior. By fitting the relative increase of the contour length to the neighbor exclusion model, we obtain the exclusion number and the intrinsic intercalating constant of the interaction. Ab initio calculations are employed in order to provide an atomistic picture of these experimental findings. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
16. Tomography of fluctuating biological interfaces using defocusing microscopy.
- Author
-
Glionna, G., Oliveira, C. K., Siman, L. G., Moyses, H. W., Prado, D. M. U., Monken, C. H., and Mesquita, O. N.
- Subjects
SURFACE chemistry ,TOMOGRAPHY ,MEDICAL radiography ,BIOCHEMISTRY ,OPTICAL resolution - Abstract
We show that a bright-field defocused microscope is effectively a phase-contrast microscope, but with advantages over the conventional one and maintaining the same optical resolution. In a multilayered transparent object, the height amplitude (static and dynamic) of each interface can be measured separately with nanometer sensitivity. By scanning the position of the objective focal plane in relation to the surfaces of a red blood cell, we obtain quantitative information on height fluctuations from each surface individually, which can be analyzed with our model of a defocused microscope and compared with theoretical models. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
17. Absolute calibration of optical tweezers.
- Author
-
Viana, N. B., Mazolli, A., Neto, P. A. Maia, Nussenzveig, H. M., Rocha, M. S., and Mesquita, O. N.
- Subjects
GEOMETRICAL optics ,MICROSPHERES ,GEOMETRICAL diffraction ,POLYSTYRENE ,REFRACTIVE index ,GAUSSIAN beams - Abstract
As a step toward absolute calibration of optical tweezers, a first-principles theory of trapping forces with no adjustable parameters, corrected for spherical aberration, is experimentally tested. Employing two very different setups, we find generally very good agreement for the transverse trap stiffness as a function of microsphere radius for a broad range of radii, including the values employed in practice, and at different sample chamber depths. The domain of validity of the WKB (“geometrical optics”) approximation to the theory is verified. Theoretical predictions for the trapping threshold, peak position, depth variation, multiple equilibria, and “jump” effects are also confirmed. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
- View/download PDF
18. In situ measurement of laser power at the focus of a high numerical aperture objective using a microbolometer.
- Author
-
Viana, N. B., Mesquita, O. N., and Mazolli, A.
- Subjects
- *
BOLOMETERS , *LASERS - Abstract
We built a simple mercury-based microbolometer to measure "in situ" laser power at the focus of high numerical aperture objectives used in optical microscopy and optical tweezers. With an optical microscope equipped with a high numerical aperture objective, we visualize a mercury droplet immersed in water that is heated by an infrared laser, whose power we want to determine. For a given laser power the mercury droplet reaches the boiling temperature of the water and then jumps, making a precise determination of this point very easy. We solve the heat equation for this system and, using known material parameters and the size of the mercury droplet, we determine the local power of the infrared laser. [ABSTRACT FROM AUTHOR]
- Published
- 2002
- Full Text
- View/download PDF
19. Photon correlation spectroscopic analysis of a natural electret material: Carnauba wax.
- Author
-
Barbosa, G. A., Russi, R., Pires, A. S. T., and Mesquita, O. N.
- Published
- 1981
- Full Text
- View/download PDF
20. Quantitative Assessment of the Interplay Between DNA Elasticity and Cooperative Binding of Ligands.
- Author
-
Siman, L., Carrasco, I. S. S., Silva, J. K. L. da, de Oliveira, M. C., Rocha, M. S., and Mesquita, O. N.
- Subjects
- *
DNA-binding proteins , *LIGANDS (Biochemistry) , *ELASTICITY , *MATHEMATICAL physics , *MATHEMATICAL statistics - Abstract
Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We propose a methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation. We obtain an expression for the effective persistence length as a function of the total ligand concentration, which we apply to our data of the DNA-cationic β-cyclodextrin and to the DNA-HU protein data available in the literature, determining the values of the local persistence lengths, the dissociation constant, and the degree of cooperativity for each set of data. In both cases the persistence length behaves nonmonotonically as a function of ligand concentration and based on the results obtained we discuss some physical aspects of the interplay between DNA elasticity and cooperative binding of ligands. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
21. Model for DNA Interactions with Proteins and Other Large Ligands: Extracting Physical Chemistry from Pure Mechanical Measurements.
- Author
-
Alves PS, Mesquita ON, and Rocha MS
- Subjects
- Chemistry, Physical, Ligands, Mechanical Phenomena, Models, Molecular, Muramidase metabolism, DNA, Viral chemistry, Muramidase chemistry
- Abstract
We present a new model to describe DNA interactions with large ligands such as proteins, based on a quenched-disorder equation for ligand binding along the double helix and on Manning's description for the local changes of the persistence length at the binding sites. Such a model allows one to extract the physical chemistry of the interactions from pure mechanical measurements, such as those typically performed with DNA-protein complexes in force spectroscopy assays. We have tested the proposed methodology here to investigate the DNA interaction with the protein lysozyme, determining binding parameters such as the equilibrium association constant, the cooperativity degree of the binding reaction, and the fraction of neutralized charges on the phosphate backbone. The model also allows one to get information on the size and positional conformation of the bound proteins.
- Published
- 2020
- Full Text
- View/download PDF
22. Graph analysis of cell clusters forming vascular networks.
- Author
-
Alves AP, Mesquita ON, Gómez-Gardeñes J, and Agero U
- Abstract
This manuscript describes the experimental observation of vasculogenesis in chick embryos by means of network analysis. The formation of the vascular network was observed in the area opaca of embryos from 40 to 55 h of development. In the area opaca endothelial cell clusters self-organize as a primitive and approximately regular network of capillaries. The process was observed by bright-field microscopy in control embryos and in embryos treated with Bevacizumab (Avastin
® ), an antibody that inhibits the signalling of the vascular endothelial growth factor (VEGF). The sequence of images of the vascular growth were thresholded, and used to quantify the forming network in control and Avastin-treated embryos. This characterization is made by measuring vessels density, number of cell clusters and the largest cluster density. From the original images, the topology of the vascular network was extracted and characterized by means of the usual network metrics such as: the degree distribution, average clustering coefficient, average short path length and assortativity, among others. This analysis allows to monitor how the largest connected cluster of the vascular network evolves in time and provides with quantitative evidence of the disruptive effects that Avastin has on the tree structure of vascular networks., Competing Interests: We declare we have no competing interests.- Published
- 2018
- Full Text
- View/download PDF
23. Controlling Cooperativity in β-Cyclodextrin-DNA Binding Reactions.
- Author
-
Alves PS, Mesquita ON, and Rocha MS
- Subjects
- Allosteric Site, DNA chemistry, beta-Cyclodextrins chemistry
- Abstract
We have investigated the interaction between the native neutral β-cyclodextrin (CD) and the DNA molecule by performing single-molecule stretching experiments with optical tweezers. In particular, we have monitored the changes of the mechanical properties of the CD-DNA complexes as a function of the CD concentration in the sample. By using a quenched disorder statistical model, we were also capable to extract important physicochemical information (equilibrium binding constants, cooperativity degree) of such interaction from the mechanical data. In addition, we have found that the interaction occurs by two different mechanisms, first with the formation of relatively large CD clusters along the double helix, which thereafter can locally denature the DNA molecule by forming hydrogen bonds with the base pairs that eventually flip out. A prediction of our quenched disorder model was that cooperativity could be controlled by adjusting the surface charge of β-CD molecules. This prediction is confirmed in the present work.
- Published
- 2015
- Full Text
- View/download PDF
24. Towards absolute calibration of optical tweezers.
- Author
-
Viana NB, Rocha MS, Mesquita ON, Mazolli A, Maia Neto PA, and Nussenzveig HM
- Subjects
- Brazil, Computer Simulation, Stress, Mechanical, Algorithms, Calibration standards, Equipment Failure Analysis methods, Equipment Failure Analysis standards, Models, Theoretical, Optical Tweezers standards
- Abstract
Aiming at absolute force calibration of optical tweezers, following a critical review of proposed theoretical models, we present and test the results of Mie-Debye-spherical aberration (MDSA) theory, an extension of a previous (MD) model, taking account of spherical aberration at the glass-water interface. This first-principles theory is formulated entirely in terms of experimentally accessible parameters (none adjustable). Careful experimental tests of the MDSA theory, undertaken at two laboratories, with very different setups, are described. A detailed description is given of the procedures employed to measure laser beam waist, local beam power at the transparent microspheres trapped by the tweezers, microsphere radius, and the trap transverse stiffness, as a function of radius and height in the (inverted microscope) sample chamber. We find generally very good agreement with MDSA theory predictions, for a wide size range, from the Rayleigh domain to large radii, including the values most often employed in practice, and at different chamber heights, both with objective overfilling and underfilling. The results asymptotically approach geometrical optics in the mean over size intervals, as they should, and this already happens for size parameters not much larger than unity. MDSA predictions for the trapping threshold, position of stiffness peak, stiffness variation with height, multiple equilibrium points, and "hopping" effects among them are verified. Remaining discrepancies are ascribed to focus degradation, possibly arising from objective aberrations in the infrared, not yet included in MDSA theory.
- Published
- 2007
- Full Text
- View/download PDF
25. Characterization of objective transmittance for optical tweezers.
- Author
-
Viana NB, Rocha MS, Mesquita ON, Mazolli A, and Maia Neto PA
- Subjects
- Computer Simulation, Equipment Design, Equipment Failure Analysis, Micromanipulation methods, Radiation Dosage, Scattering, Radiation, Stress, Mechanical, Lasers, Micromanipulation instrumentation, Models, Theoretical, Radiometry methods
- Abstract
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.
- Published
- 2006
- Full Text
- View/download PDF
26. Defocusing microscopy.
- Author
-
Agero U, Mesquita LG, Neves BR, Gazzinelli RT, and Mesquita ON
- Subjects
- Animals, Calibration, Cell Movement, Cytoskeleton physiology, Cytoskeleton ultrastructure, Macrophages physiology, Macrophages ultrastructure, Mice, Mice, Inbred C57BL, Microscopy, Atomic Force, Models, Theoretical, Optics and Photonics, Refractometry, Image Processing, Computer-Assisted, Microscopy methods
- Abstract
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., (2004 Wiley-Liss, Inc.)
- Published
- 2004
- Full Text
- View/download PDF
27. Measurements and modeling of water transport and osmoregulation in a single kidney cell using optical tweezers and videomicroscopy.
- Author
-
Lúcio AD, Santos RA, and Mesquita ON
- Subjects
- Adaptation, Physiological drug effects, Adaptation, Physiological physiology, Animals, Biological Transport, Active physiology, Cell Culture Techniques instrumentation, Cell Membrane drug effects, Cell Membrane Permeability drug effects, Cell Membrane Permeability physiology, Cells, Cultured, Computer Simulation, Dogs, Equipment Design, Kidney drug effects, Lasers, Microscopy, Video methods, Osmotic Pressure drug effects, Physical Stimulation instrumentation, Physical Stimulation methods, Vasopressins pharmacology, Water-Electrolyte Balance drug effects, Cell Culture Techniques methods, Cell Membrane physiology, Flow Cytometry methods, Kidney cytology, Kidney metabolism, Models, Biological, Water metabolism, Water-Electrolyte Balance physiology
- Abstract
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.
- Published
- 2003
- Full Text
- View/download PDF
28. Cell surface fluctuations studied with defocusing microscopy.
- Author
-
Agero U, Monken CH, Ropert C, Gazzinelli RT, and Mesquita ON
- Subjects
- Actins metabolism, Animals, Cell Membrane metabolism, Cytochalasin D pharmacology, Cytoskeleton metabolism, Glass, Image Processing, Computer-Assisted, Macrophages metabolism, Mice, Mice, Inbred C57BL, Microscopy, Phase-Contrast methods, Microscopy, Video, Models, Statistical, Normal Distribution, Phagocytosis, Time Factors, Zymosan pharmacology, Cell Membrane pathology, Microscopy methods
- Abstract
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.
- Published
- 2003
- Full Text
- View/download PDF
29. Dynamic light scattering from an optically trapped microsphere.
- Author
-
Viana NB, Freire RT, and Mesquita ON
- Subjects
- Bacteriophage lambda, DNA, Viral, Elasticity, Microscopy instrumentation, Microspheres, Oscillometry, Polystyrenes, Scattering, Radiation, Colloids, Light, Microscopy methods, Thermodynamics
- Abstract
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.
- Published
- 2002
- Full Text
- View/download PDF
30. Anomalous capillary length in cellular nematic-isotropic interfaces.
- Author
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Gomes OA, Falcão RC, and Mesquita ON
- Abstract
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.
- Published
- 2001
- Full Text
- View/download PDF
31. Experimental test of the Warren-Langer model in nematic-isotropic planar interfaces.
- Author
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Gomes OA, Viana NB, Figueiredo JM, and Mesquita ON
- Abstract
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.
- Published
- 1999
- Full Text
- View/download PDF
32. Gramicidin channel kinetics under tension.
- Author
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Goulian M, Mesquita ON, Fygenson DK, Nielsen C, Andersen OS, and Libchaber A
- Subjects
- Dimerization, Electrochemistry, Gramicidin analogs & derivatives, Ion Channels physiology, Models, Molecular, Patch-Clamp Techniques instrumentation, Phosphatidylcholines chemistry, Probability, Stress, Mechanical, Thermodynamics, Gramicidin chemistry, Ion Channels chemistry, Lipid Bilayers, Models, Biological
- Abstract
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.
- Published
- 1998
- Full Text
- View/download PDF
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