Desenvolvimento de métodos em microscopia óptica para obtenção de parâmetros biomecânicos de células e moléculas únicas

Abstract

The aim of this thesis is the development of new experimental tools to improve the understanding of single cells and molecules basic processes. Two dierent methods in Optical Microscopy have been developed: the rst (Method 1) regarding the interaction between DNA molecules and drugs, and the second (Method 2) regarding mechanical properties of red blood cells. Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. A methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation is proposed. An general expression for the eective persistence length as a function of the total ligand concentration is obtained and applied to the experimental data of the DNA-cationic -cyclodextrin and to the DNA-HU protein data, the rst one obtained by the Ph. student during her master degree and the last one gotten from 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 non-monotonically as a function of ligand concentration and based on the results obtained some physical aspects of the interplay between DNA-elasticity and cooperative binding of ligands are discussed. The development of Method 1 generated the paper Quantitative assessment of the interplay between DNA elasticity and cooperative binding of ligands . The second method presented is based on contrast measurements of red blood cells (RBC) using Defocusing Microscopy (DM) technique. DM is a bright-eld microscopy technique developed in the Laboratory of Biological Systems of UFMG, used to obtain quantitative information of phase objects. In this thesis the contrast equation for a defocused microscope is deduced, and based on it very simple methodologies to obtain refractive index, thickness prole, volume and three dimensional (3D) reconstruction of red blood cells (RBC) are presented. In addition, as a new feature of the technique, the shape prole of each of the red cell surfaces (upper and lower membranes, the last one adhered to the substrate) are obtained, separately. The DM mean square contrast uctuation equation is also presented, and its relation to red cell surface membrane uctuations (RBC ickering) is shown. Modeling themembrane height uctuation spectrum as dependent of a bending modulus (c), a surface tension () and a conning potential (), all of them slowly varying along the cell radius, the surface uctuations of the cell free interface and of the one in contact with the substrate is obtained, separately. Moreover, the behaviour of the elastic parameters c, and for each interface along the cell radius is monitored and some physical aspects of the red cell membranes are discussed. Finally, all the developed methods are applied to the experimental data of 42 RBC and the average biomechanical properties are shown. The development of Method 2 generated the paper Shape reconstruction and height uctuation of phase objects using DM: application to red blood cells, which is in submission process.