Caracterização de parâmetros biomecânicos de hemácias via microscopia de desfocalização: imagem tridimensional total e elasticidade da membrana

Abstract

The general aim of this thesis was the development of new experimental and computational methods in light microscopy, in particular, using Defocusing Microscopy (DM) technique in the study of contrast of phase objects (transparent objects) when observed by an optical defocused microscope operating in bright _eld, which we call contrast MD. The MD was developed at the Physics Biological Systems Laboratory of UFMG and allows the direct measurement of local curvature of phase objects and the discrimination of interfaces that compose these objects. As secondary objectives, the thesis aimed to employ the developed methods in the characterization of morphological and mechanical changes of red blood cells (RBCs), which are in_uenced directly by the action of chemical and physical agents and various hematologic diseases. Therefore, the methodology was applied in 25 red blood cells subjected to di_erent osmolality solutions: isotonic - 300 mOsm/kg, hypotonic - 200 mOsm/kg and hypertonic - 400 mOsm/kg. Additionally, other forms of red blood cells (stomatocytes and echinocyte) were evaluated. The methods were based on two groups of experimental measurements: DM contrast and contrast _uctuation of red blood cells. From the DM contrast it was possible to determine parameters related to the red blood cell's morphology, such as radius, surface area, volume, sphericity index, refractive index, thickness, shape pro_le of each surface-membranes (lipid bilayer/cytoskeleton), besides the image 3D total of the cell. Through these, the changes in the upper (free) and lower(in contact with the substrate) surface-membranes of red blood cells, either due to di_erent solutions in which they were immersed or due the cell's adhesion to the substrate, were measured. The DM axial resolution allowed us to obtain images of the red blood cells' surfaces separated by a distance of down to 300 nm. From DM contrast _uctuations of red blood cells it was possible to determine the radial pro_les of _uctuations in height and the radial behaviors of the elastic parameters: bending modulus (kc), surface tension and con_ning potencial () for each surface along the cell extension. These results allowed a discussion of physical aspects of red blood cells membranes, including new interpretations for these parameters.