Quantitative three-dimensional (3-D) imaging of living cells provides important information about the cell morphology and its time variation. Off-axis, digital holographic interference microscopy is an ideal tool for 3-D imaging, parameter extraction, and classification of living cells. Two-beam digital holographic microscopes, which are usually employed, provide high-quality 3-D images of micro-objects, albeit with lower temporal stability. Common-path digital holographic geometries, in which the reference beam is derived from the object beam, provide higher temporal stability along with high-quality 3-D images. Self-referencing geometry is the simplest of the common-path techniques, in which a portion of the object beam itself acts as the reference, leading to compact setups using fewer optical elements. However, it has reduced field of view, and the reference may contain object information. Here, we describe the development of a common-path digital holographic microscope, employing a shearing plate and converting one of the beams into a separate reference by employing a pin-hole. The setup is as compact as self-referencing geometry, while providing field of view as wide as that of a two-beam microscope. The microscope is tested by imaging and quantifying the morphology and dynamics of human erythrocytes.
Development of devices for automatic identification of diseases is desired especially in developing countries. In the case of malaria, even today the gold standard is the inspection of chemically treated blood smears through a microscope. This requires a trained technician/microscopist to identify the cells in the field of view, with which the labeling chemicals gets attached. Bright field microscopes provide only low contrast 2D images of red blood cells and cell thickness distribution cannot be obtained. Quantitative phase contrast microscopes can provide both intensity and phase profiles of the cells under study. The phase information can be used to determine thickness profile of the cell. Since cell morphology is available, many parameters pertaining to the 3D shape of the cell can be computed. These parameters in turn could be used to decide about the state of health of the cell leading to disease diagnosis. Here the investigations done on digital holographic microscope, which provides quantitative phase images, for comparison of parameters obtained from the 3D shape profile of objects leading to identification of diseased samples is described.