THREE DIMENSIONAL RECONSTRUCTION OF PHASE OBJECTS VIA DEFOCUSING MICROSCOPY

Defocusing Microscopy (DM) is a label-free quantitative phase contrast technique that allows the determination of viscoelastic parameters of the cell membrane using standard bright-field microscopy. DM has been used for the study of macrophages and red blood cells, and its main advantage over other techniques is that it requires only a conventional optical microscope, making it cost-effective and widely accessible. Earlier studies have shown that DM allows the calculation of the height of the upper and lower parts of a red blood cell membrane with respect to the substrate, resulting in the three dimensional reconstruction of the cell. The approach used to perform these calculations was the main limiting factor for DM application, the time needed for the reconstruction of a single cell starting from two images with 128 X 128 pixels was of the order of 5 hours. The objective of the present study was to make the reconstruction process faster, enabling reconstruction of larger cells and real-time image processing. The research led to the proposal of a new model of image formation to avoid the previous model's sensitivity to the system's boundary conditions. Using the first-order Born approximation for light scattering and propagating the light through the defocused microscope, we demonstrated that the equation that relates the image contrast to the object shape is a Poisson equation and not a Helmholtz equation as previously proposed. This development simplified the reconstruction process significantly, reducing the influence of boundary conditions on the solution as well as reducing the time required from ~5 hours to ~10 ms. The new model was used to reconstruct red blood cells in isotonic and hypotonic medium, showing that DM can reveal cell shape changes due to osmotic pressure. Future work aims at real-time cell imaging and extending the technique beyond the first-order Born approximation limit.