VALIDATION OF THE RECONSTRUCTION OF SIMULATED RED BLOOD CELLS USING DEFOCUSING MICROSCOPY

Defocusing Microscopy (DM) is an optical microscopy technique used to obtain thickness profiles and curvature of phase objects, as well as cellular and membrane dynamic properties. The main objective of this study is to demonstrate the effectiveness of the technique to reconstruct the thickness profile of red blood cells from the defocused images contrast. We started by analyzing the known geometrical models of the red blood cell and opted to use the one described by Kuchel [1999]. The defocused contrast has been constructed from the simulated surface and is given by:
C(ρ ⃗ )= Δn/n_0 [(z_f-h_1 (ρ ⃗ )) ∇^2 h_1 (ρ ⃗ )-(z_f-h_2 (ρ ⃗ )) ∇^2 h_2 (ρ ⃗ )]
In which n_0 is the refractive index of the medium, ∆n is the refraction index difference between the cell and the medium, z_f is the focal plane where the contrast is being generated from and h_1 and h_2 are the upper and lower cell membrane, respectively. From the contrast of two defocused images, we have utilized the defocusing microscopy technique to obtain the cell thickness profile and asymmetry numerically, employing a finite element method. From the results we have reconstructed the simulated red blood cell surface and have compared with the ones originally used to generate the simulated defocused contrast. The geometrical reconstruction yielded an error of 0.035 µm, showing the reliability of the technique to obtain the geometrical profile of simulated blood cells. Assuming the similarity of the generated red blood cells with the real ones, the reconstruction of real blood cells should yield equal precision, with a much simpler experimental process than those currently used, such as atomic force microscopy (AFM) or electron microscopy (EM). The next steps of the research will be the use of DM to analyze the thickness profile in both static and dynamic conditions of other kinds of cells, most notably cardiomyocytes.