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Digital holographic microscopy is the state of the art quantitative phase imaging of micro-objects including living cells. It is an ideal tool to image and quantify cell thickness profiles with nanometer thickness resolution. Digital holographic techniques usually are implemented using a two-beam setup that may be bulky and may not be field portable. Self-referencing techniques provide compact geometry but suffer from a reduction of the field of view. Here, we discuss the development of a wavefront division digital holographic microscope providing the full field of view with a compact system. The proposed approach uses a wavefront division module consisting of two lenses. The developed microscope is tested experimentally by measuring the physical and mechanical properties of red blood cells.

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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.

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Digital holographic microscopy is a single shot technique for quantitative phase imaging of objects, yielding thickness profiling of phase objects. It provides sample features based on their morphology, leading to their classification and identification. However, observing samples, especially cells, in fluids using holographic microscopes is difficult without immobilizing the object. Optical tweezers can be used for sample immobilization in fluids. The present manuscript provides an overview of our ongoing work on the development of a compact, low-cost microscopy system for digital holographic imaging of optically trapped samples. Integration of digital holographic microscopy system with tweezers is realized by using the optical pickup unit extracted from DVD burners to trap microsamples, which are then holographically imaged using a highly compact self-referencing interferometer along with a low-cost, in-house developed quadrant photodiode, providing morphological and spectral information of trapped particles. The developed integrated module was tested using polystyrene microspheres as well as human erythrocytes. The investigated system offers a multitude of sample features, including physical and mechanical parameters and corner frequency information of the sample. These features were used for sample classification. The proposed technique has vast potential in opening up new avenues for low-cost, digital holographic imaging and analysis of immobilized samples in fluids and their classification.