A lateral shearing interferometer is used to take holographic images and videos of red blood cells (RBCs). The extracted physical and mechanical parameters can be used to define the state-of-health of the RBCs.
Digital holography interferometry (DHI) and digital holographic interference microscopy (DHIM) are tools that provide whole field information of the wavefront interacting with the object. This imaging modality can be an ideal tool for quantitative phase imaging of technical objects such as semiconductor samples. The phase information numerically reconstructed from the holograms can lead to extraction of the thickness/height of the sample. Usually DHI and DHIM are used in transmission mode for determination of optical thickness of the sample under investigation. In reflection mode this imaging techniques provide height of the sample.
For the semiconductor industry determination of height/thickness of object structures as well as the quantification of defects in the object structures is an important issue. The thickness and defect determination can be that of semiconductor thin films, micro/nano-pillars, LED displays, liquid crystal panels, the cover glasses used for protection of these structures etc. Digital holographic interferometric method (both reflection and transmission) can be used to design devices that can act as a fast, single shot technique for quantitative phase imaging of such samples. Such devices can provide more information about the sample compared to intensity based measurement systems. Also compact the digital holographic interferometric systems can be deployed in the manufacturing line of such devices to provide real time information.
We are involved in the design and development of digital holographic devices for inspection of semi-conductor wafers, thin films, displays and glass plates covering such samples. We have implemented digital holographic devices in the lens-less mode (in the case of DHI) and also with the use of an imaging lens (in the case of DHIM) both in reflection and transmission mode. DHI provides field of view equivalent to the sensor size, while DHIM technique was implemented with different magnifications, thereby providing varying field of views of the sample. Also in the case of DHI a propagation from the hologram plane (the plane at which the digital array for recording the hologram was situated) to the best focus plane (object plane) was realized by numerical implementation of diffraction integral. In DHIM, the digital array used for recording the hologram was at the image plane of the magnifying/de-magnifying lens. So the whole numerical reconstruction process reduced to Fourier fringe analysis, making the technique less computationally exhaustive, fast and quasi real-time. The developed devices were calibrated using known objects and then tested on different samples. The obtained results are found to be encouraging. In this paper, we describe our efforts in design, development and fabrication of digital holographic devices for the inspection of semiconductor samples.
One of the drawbacks of using Lloyd’s mirror interferometer for quantitative phase contrast microscopy is that it leads to overlapping of the object information which restricts the use of field of view. One way to overcome this limitation is to prepare the sample slide in such a way that the object information is contained in only one portion of the illuminating beam, while the other acts as a separate reference beam. This geometry has excellent temporal stability and is used to examine human erythrocytes.
Nonuniform refractive index distributions in transparent mediums are of interest as it gives rise to a modification of the probe light beam passing through such mediums. Various properties of the probe beam can be used to quantify the modification happening to the probe beam. One of these properties is the deflection of the beam. This could be used to map and quantify the spatiotemporal evolution of refractive index distribution in such mediums. The deflections could be measured by imaging the deflection of structured line pattern projected through such a system. We describe the development of a compact, portable device for mapping of refractive index distributions as well measurement of the diffusion coefficient of liquid solutions. The method and device are demonstrated by the real-time display of the refractive changes as well as measurement of diffusion coefficients in diffusing binary liquid solutions.
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.
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.
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.
A noninterferometric technique used to measure the diffusion coefficients of transparent liquid solutions is reported. This technique uses a white light source and a diffusion cell, with an artificially developed fringe pattern of dark and white stripes at its entrance. As the diffusion process takes place in the cell, the light passing through this nonuniform refractive index medium will bend toward the higher refractive index region, which results in a fringe shift. This shift in the fringe pattern at different times is recorded in a personal computer (PC) using a CCD camera for the calculation of diffusion coefficients. The fringe shift is calculated after skeletonization and linear fit of the captured fringe system. The diffusion coefficient of different concentrations of ammonium dihydrogen phosphate was determined using the proposed technique and the measured values lay within 1% of the reported values. Detailed theoretical and experimental analyses with a comparison of other existing results are discussed.
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.
3D imaging requires the retrieval of both amplitude and phase of the wavefront interacting with the object. Quantitative
phase contrast imaging technique like digital holography uses the interference of object and a known reference wavefront
for whole field reconstructions. And for higher lateral resolution, uses of shorter wavelengths become necessary. For
short wavelength sources, due to short coherence lengths, it becomes very difficult to implement a two-beam
interferometric setup. We have developed a technique for reconstructing the amplitude and phase of object wavefront
from the volume diffraction field by sampling it at several axial positions and implementing the scalar diffraction
integral iteratively. This technique is extended to 3D microscopic imaging at 193 nanometers.
Imaging of cells is an interesting and challenging problem as they do not appreciably change the amplitude of the
electromagnetic radiation interacting with them. Phase contrast techniques can be used to overcome this hurdle.
Interferometric phase contrast techniques like digital holography can be used for quantitative phase contrast microscopic
imaging of transparent objects and it yields the three dimensional profile of the object under investigation. These
methods also have advantage of numerical focusing, allowing one to focus on to any desired object plane. But most of
the interferometric quantitative phase contrast techniques require two beams as well as the adjustment of the beams for
high fringe contrast, requiring stringent optical conditions. Here we present a single beam phase retrieval technique for
quantitative phase contrast microscopy of cells. The phase information of the object is obtained by sampling the volume
speckle field generated by the object at several axial planes. These intensity patterns are used iteratively in the diffraction
integral to retrieve the phase information about the object. The advantages of this technique include compactness,
immunity to external vibrations as well as the prospect of usage of low coherent sources.
Quantitative three-dimensional imaging of cells can provide important information about their morphology as well as their dynamics, which will be useful in studying their behavior under various conditions. There are several microscopic techniques to image unstained, semi-transparent specimens, by converting the phase information into intensity information. But most of the quantitative phase contrast imaging techniques is realized either by using interference of the object wavefront with a known reference beam or using phase shifting interferometry. A two-beam interferometric method is challenging to implement especially with low coherent sources and it also requires a fine adjustment of beams to achieve high contrast fringes. In this letter, the development of a single beam phase retrieval microscopy technique for quantitative phase contrast imaging of cells using multiple intensity samplings of a volume speckle field in the axial direction is described. Single beam illumination with multiple intensity samplings provides fast convergence and a unique solution of the object wavefront. Three-dimensional thickness profiles of different cells such as red blood cells and onion skin cells were reconstructed using this technique with an axial resolution of the order of several nanometers.
Imaging of transparent micro-objects objects, especially living cells, becomes a challenging problem since such objects
do not appreciably change the amplitude of the electromagnetic radiation interacting with it. Phase contrast techniques
can be used to overcome this hurdle. Digital holography is one of the most effective tools for phase contrast microscopic
imaging of transparent objects and it yields the three dimensional profile of the object under investigation. Another
advantage of digital holography is that of numerical focusing, allowing one to focus on to any desired object plane. This
aspect of digital holography along with interferometry is utilized to study dynamics of phase micro-objects. The changes
occurring to the object under study is determined from their three dimensional profile which is obtained from the phase
information.
Two wavelength contouring is used especially for the shape measurement and testing of steep objects. Conventional and
digital holographic techniques can be utilized for this. But both these methods require the interference of the test
wavefront with a known background or reference wavefront. This makes the adjustment of the two beams intensities to
get high contrast fringes necessary. These methods are also prone to external noise like vibrations. Phase retrieval from
intensity sampling at multiple axial planes offer an attractive alternative for whole field imaging. Diffusively reflecting
objects produce volume speckle field when exposed to highly coherent radiation like laser. This volume speckle field has
appreciable intensity variation both in the transverse as well as axial direction, which is the necessary condition to
reconstruct the complex amplitude of the object wavefront from the intensity values. The reconstruction process uses the
angular spectrum propagation approach to the scalar diffraction theory. This paper explores the use of phase retrieval in
two wavelength contouring. The method can be used for the shape measurement of steep diffuse objects and the optical
path length measurement of transparent objects. The method is explained using simulations. Some experimental
observations are also provided to validate the simulation results.
Determination of parameters of the lens like the focal length, radius of curvature and refractive index are important from
its application point of view. Many interferometric as well as non interferometric methods exist for this. But most of
these methods require a visual inspection of the resulting interference pattern. Confocal imaging has very high axial
resolution and it permits three-dimensional observation of object. This same property also enables one to determine the
axial position of a specularly reflecting object. Here this capability has been applied, in reflection case, to the
determination of lens parameters. This method is explained with simulation using diffraction theory and experimental
results.
Digital holography is one of the most versatile tools for the whole field imaging of wavefronts. Since numerical
reconstruction provides both the amplitude and phase of the wavefronts it finds immense potential applications ranging
from shape measurement to microscopy. The method is useful especially for phase objects, which otherwise does not
produce any change in the amplitude of the interacting wavefront and hence is difficult to image. The wavefronts passing
through a region having non-uniform refractive index distribution will carry the information about this distribution as a
spatially varying phase. Digital holography can be used for measurement of this phase. Here the use of digital
holographic interferometry coupled with tomography is investigated for mapping of spatially as well as temporally
varying refractive index distributions.
Measurement and monitoring of diffusion process is important in many areas of physical, chemical and biological
sciences. Usually interferometric methods are used for this. Even though very accurate, they require controlled
environments (especially to be isolated from external noise) and should adhere to stringent optical considerations.
Single beam optical techniques are more suitable in noisy environments. Since a diffusing medium has a non-uniform
refractive index distribution, a ray passing through such a medium will deflect towards regions of higher refractive
index. If this deflection can be measured somehow, it can be used for finding the refractive index gradient and hence the
refractive index distribution inside the medium. Here a method is proposed to measure these deflections and hence the
diffusion coefficient using active optical elements, by converting the incident light into a spatially varying polarization
pattern.
Determination of lens parameters is important from their application point of view. These parameters include focal
length, radius of curvature and refractive index of the lens material. Of these the refractive index data will shed light on
the reflectance and transmittance of the material with which the lens is made. Digital holography can be used for the
parameter determination as well as for the test of collimation of waves emanating from lenses. Digital holograms are
recorded for various positions of the test lens from a point source and the focal length is the position of the test lens
where a single fringe results. Radius of curvature is determined by focusing a collimated beam on to the surface of the
test lens. Using the lens equation the refractive index of the material of the lens can be determined. Simulations for the
method in the case of a thin bi-convex lens are presented in the paper.
A method to test beamsplitters using Michelson interferometer is proposed. The visibility of the interference fringes is used for the beam ratio calculations. The beam ratios of all the interfering beams at the detector plane can be determined using this method.
Inteferograms are made by interfering wave fronts and hence contain important information about them. Analysis of interferogram requires the identification of all the fringes and their exterma. Here an algorithm for computer tracing of interference fringes is described. The method uses a Fourier filter for removing high frequency noise, a local averaging for binarization of images not having uniform intensity distribution, scanning the interferogram locally both horizontally and vertically to determine the type of the scan, local application of simultaneous horizontal and vertical scan for tracing of complicated fringe patterns and removal of the noise from the traces by determining the number of connected pixels. The poroposed algorithm was found to yield good result even for high noise images.
A method to measure diffusion coefficient of transparent liquid solutions using digital holographic inteferometry is described. Holograms of a diffusively reflecting object through the experimental cell containing the diffusing solutions are recorded at different time instances. The recording medium is a CCD chip. The holographic interference of the object at two instances of time is numerically carried out in a PC and is used to determine the diffusion coefficient. Holographic interference fringes can be displayed on a PC monitor. The diffusion coefficients calculated using this method matched very well with literature values.
Three simple optical methods to determine diffusion coefficients in transparent liquids are discussed. The methods use multiple beam interferometer, Michelson's interferometer and fringe projection respectively for determining the diffusion coefficients. All these methods were found to yield accurate results.
A simple technique to determine the diffusion coefficient in transparent liquids is developed. The method uses fringe projection with white light source. The diffusion coefficients obtained were compared with those obtained with other experimental methods.
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