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We present Defocusing Microscopy (DM), a bright-field optical microscopytechnique able to perform total 3D imaging of transparent objects. By total 3Dimaging we mean the determination of the actual shapes of the upper and lowersurfaces of a phase object. We propose a new methodology using DM and apply itto red blood cells subject to different osmolality conditions: hypotonic,isotonic and hypertonic solutions. For each situation the shape of the upperand lower cell surface-membranes (lipid bilayer/cytoskeleton) are completelyrecovered, displaying the deformation of RBCs surfaces due to adhesion on theglass-substrate. The axial resolution of our technique allowed us to imagesurface-membranes separated by distances as small as 300 nm. Finally, wedetermine volume, superficial area, sphericity index and RBCs refractive indexfor each osmotic condition.

Total 3D imaging of phase objects using defocusing microscopy: application to red blood cells