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.Standard optical microscopy imaging of phase objects is generally obtained with microscopes operating in Phase Contrast or Nomarsky configurations 1,2 . However, even for objects with uniform refractive index these techniques present difficulties for obtaining accurate full-field thickness profiles. Recently, new approaches known as quantitative phase microscopy techniques 3-7 , have adequately obtained full-field height profile of phase objects with successful application in living cells. Despite that, in the case of red blood cells (RBCs) these techniques are only able to provide the thickness profile and thickness amplitude fluctuations, such that height profile information of each particular cell surface-membrane (lipid bilayer/cytoskeleton) is not accessed.Using Defocusing Microscopy (DM), we present a new method able to perform total 3D imaging of RBC, and thus, capable of determining the actual shape of the cell's upper and lower surface-membranes, separately. New procedures to retrieve RBC volume, superficial area, sphericity index and refractive index using DM are also exposed. All developed methods are applied to data of 25 RBCs immersed in three different solutions: hypotonic (200 mOsm/kg), isotonic (300 mOsm/kg) and hypertonic (400 mOsm/kg), showing the differences in the lower membrane adhesion to the glass substrate. For assessment of bio-mechanical properties along the RBCs surfaces, nanometer height fluctuations for each interface can be obtained separately, such that the effect of adhesion to the substrate can also be evaluated 8,9 . A defocusing technique has been recently applied for 3D imaging of cells using a phase contrast microscope under white-light illumination, with transverse resolution of 350 nm and axial resolution of 900 nm 10 . This technique cannot resolve surface-membranes separated by an axial distance smaller than 900 nm, which is the case of most RBCs. Strikingly, our Defocusing Microscopy technique, using a bright-field defocused microscope and our a) E-mail me at: mesquita@fisica.ufmg.br theoretical framework, can resolve surface-membranes of RBCs separated by axial distances down to 300 nm.Transparent objects that would be...

The alternative methodology allows complete IOL characterisation with a reduced number of steps compared to those required by the Standard ANSI Z80.30, achieving satisfactory results in measurements of IOL dioptric power, spherical aberration, cylindrical power and MTF. Although MTF measurements were not performed with a model eye, they may contain additional relevant information regarding the design of ophthalmic lenses.

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Single aspheric IOLs had better optical performance than piggybacking lower-power aspheric IOLs. In the spherical lenses group, the results were the opposite, with the piggyback options having higher optical quality than the single IOL. MTF shows that single aspheric lenses provide the highest contrast sensitivity among all of the analyzed settings.

In this paper the bright-field defocusing microscopy (DM) technique is presented. DM is able to obtain quantitative information of each plane/surface of pure phase objects, as live unlabeled cells, and its application to red blood cells (RBCs) is demonstrated. Based on contrast, simple methods to obtain thickness profile and three dimensional (3D) total reconstruction of RBCs are proposed and the actual height profiles of upper and lower surface-membranes (lipid bilayer/cytoskeleton) of discocyte and stomatocyte red cells are presented as examples. In addition, using the mean square contrast fluctuation and modeling the RBC membranes fluctuations spectra as dependent of a bending modulus (κ c ), a surface tension (σ) and a confining potential (γ) term, slowly varying quantities along the cell radius, a genetic algorithm (GA) is used and the radial height fluctuations of each surface-membrane are accessed, separately. The radial behaviors of κ c , σ and γ are also obtained, allowing the discussion of physical aspects of the RBC membrane.