Microscopia de desfocalização e seus limites de validade: um estudo experimental

Abstract

Phase objects can be made visible by slight defocus in a bright-field light microscope. This property is very useful to quantify cells and other biological samples that behave as phase objects in a microscope. The Defocusing Microscopy is useful to study this systems and was developed in the Physics of Biological Systems Laboratory at UFMG. The defocus creates a phase difference between the diffracted and non-diffracted light that interfere generating a contrast in the image plane. In this work we show the validity of Defocusing Microscopy for larger defocus distances, and verify the limits defined by the paraxial approximation used in the theoretical model. We use Bessel functions to write the contrast of a defocus phase objects. Diffraction gratings with sinusoid profile and well defined, period and height, was used as phase objects. We show, as predicted by the model, for a grating with well defined wavenumber, its exact image reappears periodically, whem we change the focus position. This effect was noted for defocus distances in order of 300_m. Experimentally we find diffraction grantings amplitude and these results are very similar with results of Atomic Force Microscopy.