Dense optical flow software to quantify cellular contractility.

Cell membrane deformation is an important feature that occurs during many physiological processes, and its study has been put to good use to investigate cardiomyocyte function. Several methods have been developed to extract information on cardiomyocyte contractility. However, no existing computational framework has provided, in a single platform, a straightforward approach to acquire, process, and quantify this type of cellular dynamics.

Model for DNA Interactions with Proteins and Other Large Ligands: Extracting Physical Chemistry from Pure Mechanical Measurements.

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.

Fluorescence of a Natural Fluorophore as a Key to Improve Fingerprint Contrast Image.

The optical and morphological properties of resveratrol were investigated. This nontoxic fluorescent natural material, emitting in the visible blue light, was used as an optical marker, enabling the enhancement of the image contrast coming from relief pictures marked on challenging surfaces. By applying appropriated imaging softwares, this marker was verified to be very useful in the latent fingerprint recognition deposited on different wood surface types, mainly those with high level of roughness, where conventional forensic materials do not allow effective fingerprint image visualization.

Measurement of the refractive index profile of waveguides using defocusing microscopy.

Defocusing microscopy (DM) is a bright-field optical microscopy technique often used to obtain structural parameters of objects with low difference in refractive index in relation to the surrounding medium (phase objects). We show a use of this technique to measure the refractive index (n) profile of waveguides produced by femtosecond laser micromachining inside the bulk of a sodalime glass. The results are used to analyze the influence of production parameters on n.

Graph analysis of cell clusters forming vascular networks.

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 of embryos from 40 to 55 h of development. In the endothelial cell clusters self-organize as a primitive and approximately regular network of capillaries.

Profiling of individual human red blood cells under osmotic stress using defocusing microscopy.

We use a quantitative phase imaging technique, defocusing microscopy (DM), to measure morphological, chemical, and mechanical parameters of individual red blood cells (RBCs) immersed in solutions with different osmolalities. We monitor the RBCs’ radius, volume, surface area, sphericity index, and hemoglobin content and concentration. The complete shape of cells is recovered and the effects of their adhesion to the glass substrate are observed.

Anti-erythrocyte antibodies may contribute to anaemia in Plasmodium vivax malaria by decreasing red blood cell deformability and increasing erythrophagocytosis.

Background: Plasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria.

Controlling Cooperativity in β-Cyclodextrin-DNA Binding Reactions.

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.

Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes.

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis.

DNA interaction with Actinomycin D: mechanical measurements reveal the details of the binding data.

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.

Quantitative assessment of the interplay between DNA elasticity and cooperative binding of ligands.

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.

Real-time study of shape and thermal fluctuations in the echinocyte transformation of human erythrocytes using defocusing microscopy.

We present a real-time method to measure the amplitude of thermal fluctuations in biological membranes by means of a new treatment of the defocusing microscopy (DM) optical technique. This approach was also applied to study the deformation of human erythrocytes to its echinocyte structure. This was carried out by making three-dimensional shape reconstructions of the cell and measuring the thermal fluctuations of its membrane, as the cell is exposed to the anti-inflammatory drug naproxen and as it recovers its original shape, when it is subsequently cleansed of the drug.

Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

Background: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization.

The long-term impaired macrophages functions are already observed early after high-dose ethanol administration.

Herein, we described an experimental model of high-dose ethanol (EtOH) administration, able to induce in vitro impairment in macrophage phagocytic capacity, already observed at 24 h after the last EtOH administration. This phenomenon was characterized by enlarged time required for adhesion and internalization events. Parallel studies documented an overall impaired production of interleukin (IL)-6 and nitric oxide (NO) production by peritoneal macrophages in EtOH-treated mice following interferon (IFN)-gamma and lipopolysaccharide (LPS) stimuli.

Transition on the entropic elasticity of DNA induced by intercalating molecules.

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.

Effect of mutalysin II on vascular recanalization after thrombosis induction in the ear of the hairless mice model.

Mutalysin II (mut-II) is an alpha-fibrinogenase isolated from Lachesis muta muta (bushmaster) snake venom. The enzyme lyses fibrin clots in vitro, and this activity does not depend on plasminogen activation. The aim of this study was to assess by intravital microscopy the effect of Mutalysin II on the recanalization of microvessels after thrombus induction in the ears of hairless mice.

Living cell motility.

The motility of living eukaryotic cells is a complex process driven mainly by polymerization and depolymerization of actin filaments underneath the plasmatic membrane (actin cytoskeleton). However, the exact mechanisms through which cells are able to control and employ 'actin-generated' mechanical forces, in order to change shape and move in a well-organized and coordinated way, are not quite established. Here, we summarize the experimental results obtained by our research group during recent years in studying the motion of living cells, such as macrophages and erythrocytes.

New tools to study biophysical properties of single molecules and single cells.

We present a review on two new tools to study biophysical properties of single molecules and single cells. A laser incident through a high numerical aperture microscope objective can trap small dielectric particles near the focus. This arrangement is named optical tweezers.

Towards absolute calibration of optical tweezers.

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.

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.

Measuring optical and mechanical properties of a living cell with defocusing microscopy.

Defocusing microscopy (DM) is a recently developed technique that allows quantitative analysis of membrane surface dynamics of living cells using a simple bright-field optical microscope. According to DM, the contrast of defocused images is proportional to cell surface curvature. Although, until now, this technique was used mainly to determine size and amount of membrane shape fluctuations, such as ruffles and small random membrane fluctuations, in macrophages, its applications on cell biology extend beyond that.

Real-time measurements of membrane surface dynamics on macrophages and the phagocytosis of Leishmania parasites.

Defocusing microscopy was used for real-time observation and quantification of membrane surface dynamics in murine bone marrow macrophages. Small random membrane fluctuations (SRMF), possibly metabolic driven, were detected uniformly over all membrane surface. Morphological and dynamical parameters of ruffles, such as shape, dimensions, and velocity of propagation, were analyzed.

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.

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.

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).