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.
Adequate supply of oxygen to the body is the most essential requirement. In vertebrate species this function is performed by Hemoglobin contained in red blood cells. The mass concentration of the Hb determines the oxygen carrying capacity of the blood. Thus it becomes necessary to determine its concentration in the blood, which helps in monitoring the health of a person. If the amount of Hb crosses certain range, then it is considered critical. As the Hb constitutes upto 96% of red blood cells dry content, it would be interesting to examine various physical and mechanical parameters of RBCs which depends upon its concentration. Various diseases bring about significant variation in the amount of hemoglobin which may alter certain parameters of the RBC such as surface area, volume, membrane fluctuation etc. The study of the variations of these parameters may be helpful in determining Hb content which will reflect the state of health of a human body leading to disease diagnosis. Any increase or decrease in the amount of Hb will change the density and hence the optical thickness of the RBCs, which affects the cell membrane and thereby changing its mechanical and physical properties. Here we describe the use of lateral shearing digital holographic microscope for quantifying the cell parameters for studying the change in biophysical properties of cells due to variation in hemoglobin concentration.
Measurement of rotation of plane of polarization of linearly polarized light can provide information about the concentration of the optically active system with which it interacts. For substances containing sugar, accurate measurement of rotation of linearly polarized light can provide quantitative information about concentration of sugar in the material. Measurement of sugar concentration is important in areas ranging from blood sugar level measurement in body fluids to measurement of sugar concentrations in juices and other beverages. But in many of these cases, the changes introduced to the state of polarization considering a sample of practical proportion is low and the measurement of low optical rotations becomes necessary. So methods with higher sensitivity, accuracy and resolution need to be developed for the measurement of low optical rotations. Here we describe the development of a compact, low cost, field portable, device for rotation sensing leading to sugar concentration measurements, using speckle de-correlation technique. The developed device measures rotations by determining the changes occurring to a speckle pattern generated by a laser beam passing through the medium under investigation. The device consists of a sample chamber, a diode laser module, a ground glass diffuser and a digital sensor for recording of laser speckle patterns. The device was found to have high resolution and sensitivity.
Imaging and measurement of diffusion process in liquid solutions is a challenging and interesting problem. Especially the mixing of binary liquid solutions in real-time provides an insight into the physics of diffusion as well as leads to measurement of diffusion coefficient, which is the most important parameter of a diffusing liquid solution. Accurate measurement of diffusion coefficient is important in areas ranging from oil extraction to pollution control. Interferometric methods provides very accurate measurement of diffusion coefficients albeit they impose very stringent optical conditions. Here we describe the development of a compact, easy to implement, easy to use and inexpensive device for imaging and quantification of the diffusion process. This technique does not require the stringent optical conditions of interferometric techniques. It computes the diffusivity values by measuring the amount of deflection happening to a line pattern printed on a paper and projected through the sample cell. The measured diffusivity values varied by less than 1%, with the values of diffusivities reported in literature.
Digital holographic microscope is an ideal tool for quantitative phase contrast imaging of living cells. It yields the thickness distribution of the object under investigation from a single hologram. From a series of holograms the dynamics of the cell under investigation can be obtained. But two-beam digital holographic microscopes has low temporal stability due to uncorrelated phase changes occurring in the reference and object arms. One way to overcome is to use common path techniques, in which, the reference beam is derived from the object beam itself. Both the beams travel along the same path, increasing the temporal stability of the setup. In self-referencing techniques a portion of the object beam is converted into reference beam. It could be achieved by example, using a glass plate to create two laterally sheared versions of the object beam at the sensor, which interfere to produce the holograms/interferograms. This created a common path setup, leading to high temporal stability (~0.6nm). This technique could be used to map cell membrane fluctuations with high temporal stability. Here we provide an overview of our work on the development of temporally stable quantitative phase contrast techniques for dynamic imaging of micro-objects and biological specimen including red blood cells.