Estudo de interação DNA-proteína

Estudo da técnica de Desfocalização

Estudo da interação proteína-membrana de canais de íons de Gramicidina A.

Estudo do transporte de potassio em macrofagos.

 
 

    Phase objects can become visible by slightly defocusing an optical microscope. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. We obtain that the image contrast is proportional to the two-dimensional 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. 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, 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 gives additional dynamical 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 the size of the inhomogeneities 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 zymozan 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 celular processes such as phagocytosis exhibited by macrophages.