Phase imaging microscopy under the Gabor regime in a minimally modified regular bright-field microscope

Abstract

Quantitative phase imaging (QPI) is nowadays a powerful tool for visualization and analysis of biological processes. QPI is usually attained from specifically designed optical microscopes retrieving phase information in a quantitative way. In this paper we report on an extremely simple, low cost and compact way to update a standard bright-field microscope with coherent sensing capabilities. It is based on the in-line Gabor holography concept and only needs to replace the illumination broadband source of the regular microscope with a coherent one. The proposed methodology is completed by the recording of a digital in-line Gabor hologram instead of regular imaging conditions and by the numerical processing of the recorded hologram to finally achieve QPI. The selection of the defocus distance is critical to finally achieve high quality phase imaging, so calibration considering phase and amplitude test targets is presented for the proper definition of such defocus distance. In addition, the selected configuration is experimentally validated using different samples (microbeads, cheek cells and alive spermatozoa). All the experiments are implemented in a regular Olympus BX-60 upright microscope using a 20X 0.46NA objective lens. The proposed method shows how to convert a regular microscope into a holographic one with probably the minimum needed modifications and with the main limitation coming from the Gabor imaging conditions (weak diffractive samples and twin image presence).