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