Full-field Hilbert phase microscopy using nearly common-path low coherence off-axis interferometry for quantitative imaging of biological cells

Abstract

We demonstrate single shot low coherence quantitative Hilbert phase microscopy (HPM) for the reconstruction of the two dimensional (2D) phase map of biological cells. The system is based on a compact and nearly common-path high magnification Mirau-interferometric objective lens. The spatial carrier frequency of the interference fringes was increased by means of introducing tilt in one of the arms of the interferometer, thus making the system off-axis. The system is user friendly as the interference fringes and imaging of objects with high lateral and axial resolution can be obtained quickly using a low cost commercially available microscope. Experimental results for the 2D phase map of polystyrene spheres and human red blood cells (RBCs) are presented. Hilbert transform fringe analysis was used for reconstructing the phase map and refractive index (RI) of the objects. For dynamic substances which change rapidly, single shot low coherence interferometric microscopy is an important method for obtaining the phase. Experimental results with increased field-of-view and large tilt angle are also presented. It is well known that on increasing the tilt angle for improved spatial phase sampling the object remains focused in only a small area even though the field-of-view is large. This limitation was overcome by means of vertical scanning low coherence interferometry. Due to the low coherence properties of the light source the interference occurs only at the desired location of the object, i.e., where the object is sharply focused. The object was vertically scanned and the single shot interferograms were recorded for every scan and analyzed by Hilbert transform. In this way a large area of the sample can be imaged quantitatively.