Sai Siva Gorthi

Phase imaging flow cytometry using a focus-stack collecting microscope

This letter introduces a fluidics-based focus-stack collecting microscope. A microfluidic device transports cells through the focal plane of a microscope, resulting in an efficient method to collect focus stacks of large collections of single cells. Images from the focus stacks are used to reconstruct the quantitative phase of cells with the transport-of-intensity-equation method. Using the phase imaging flow cytometer, we measure three-dimensional shape variations of red blood and leukemia cells.

Fluorescence imaging of flowing cells using a temporally coded excitation

Imaging fluorescence in moving cells is fundamentally challenging because the exposure time is constrained by motion-blur, which limits the available signal. We report a method to image fluorescently labeled leukemia cells in fluid flow that has an effective exposure time of up to 50 times the motion-blur limit. Flowing cells are illuminated with a pseudo-random excitation pulse sequence, resulting in a motion-blur that can be computationally removed to produce near diffraction-limited images. This method enables observation of cellular organelles and their behavior in a fluid environment that resembles the vasculature.

Structured illumination microscopy

Illumination plays an important role in optical microscopy. Kohler illumination, introduced more than a century ago, has been the backbone of optical microscopes. The last few decades have seen the evolution of new illumination techniques meant to improve certain imaging capabilities of the microscope. Most of them are, however, not amenable for wide-field observation and hence have restricted use in microscopy applications such as cell biology and microscale profile measurements. The method of structured illumination microscopy has been developed as a wide-field technique for achieving higher performance. Additionally, it is also compatible with existing microscopes. This method consists of modifying the illumination by superposing a well-defined pattern on either the sample itself or its image. Computational techniques are applied on the resultant images to remove the effect of the structure and to obtain the desired performance enhancement. This method has evolved over the last two decades and has emerged as a key illumination technique for optical sectioning, super-resolution imaging, surface profiling, and quantitative phase imaging of microscale objects in cell biology and engineering. In this review, we describe various structured illumination methods in optical microscopy and explain the principles and technologies involved therein.

Piecewise polynomial phase approximation approach for the analysis of reconstructed interference fields in digital holographic interferometry

This paper proposes a new approach for the analysis of reconstructed interference fields in digital holographic interferometry. In the proposed approach the interference phase to be estimated is conceived as a piecewise polynomial signal; consequently, each segment of the reconstructed interference field is modeled as a polynomial phase signal (PPS) with constant or slowly varying amplitude. The unwrapped phase distribution is then directly computed using the maximum likelihood estimation. Salient features of the proposed approach are: it provides accurate phase estimation from a single record of the interference field; it avoids cumbersome and error-prone filtering and 2D unwrapping procedures; and it paves the way to adapt well-established PPS analysis tools available in signal processing literature for the phase estimation in holographic interferometry.

Strain estimation in digital holographic interferometry using piecewise polynomial phase approximation based method

Measurement of strain is an important application of digital holographic interferometry. As strain relates to the displacement derivative, it depends on the derivative of the interference phase corresponding to the reconstructed interference field. The paper proposes an elegant method for direct measurement of unwrapped phase derivative. The proposed method relies on approximating the interference phase as a piecewise cubic polynomial and subsequently evaluating the polynomial coefficients using cubic phase function algorithm. The phase derivative is constructed using the evaluated polynomial coefficients. The methods performance is demonstrated using simulation and experimental results.

Estimation of multiple phases from a single fringe pattern in digital holographic interferometry

Simultaneous measurement of multidimensional displacements using digital holographic interferometry involves multi-directional illumination of the deformed object and requires the reliable estimation of the resulting multiple interference phase distributions. The paper introduces an elegant method to simultaneously estimate the desired multiple phases from a single fringe pattern. The proposed method relies on modeling the reconstructed interference field as a piecewise multicomponent polynomial phase signal. Effectively, in a given region or segment, the reconstructed interference field is represented as the sum of different components i.e. complex signals with polynomial phases. The corresponding polynomial coefficients are estimated using the product high-order ambiguity function. To ensure proper matching of the estimated coefficients with the corresponding components, an amplitude based discrimination criterion is used. The main advantage of the proposed method is direct retrieval of multiple phases without the application of spatial carrier based filtering operations.

A new approach for simple and rapid shape measurement of objects with surface discontinuities

A new approach for unwrapping phase maps, obtained during the measurement of 3-D surfaces using sinusoidal structured light projection technique, is proposed. “Takedas method” is used to obtain the wrapped phase map. Proposed method of unwrapping makes use of an additional image of the object captured under the illumination of a specifically designed color-coded pattern. The new approach demonstrates, for the first time, a method of producing reliable unwrapping of objects even with surface discontinuities from a single-phase map. It is shown to be significantly faster and reliable than temporal phase unwrapping procedure that uses a complete exponential sequence. For example, if a measurement with the accuracy obtained by interrogating the object with S fringes in the projected pattern is carried out with both the methods, new method requires only 2 frames as compared to (log2S +1) frames required by the later method.

Simultaneous multidimensional deformation measurements using digital holographic moiré

This paper proposes an elegant technique for the simultaneous measurement of in-plane and out-of-plane displacements of a deformed object in digital holographic interferometry. The measurement relies on simultaneously illuminating the object from multiple directions and using a single reference beam to interfere with the scattered object beams on the CCD plane. Numerical reconstruction provides the complex object wave-fields or complex amplitudes corresponding to prior and postdeformation states of the object. These complex amplitudes are used to generate the complex reconstructed interference field whose real part constitutes a moiré interference fringe pattern. Moiré fringes encode information about multiple phases which are extracted by introducing a spatial carrier in one of the object beams and subsequently using a Fourier transform operation. The information about the in-plane and out-of-plane displacements is then ascertained from the estimated multiple phases using sensitivity vectors of the optical configuration.

Windowed high-order ambiguity function method for fringe analysis

This paper introduces a windowed high-order ambiguity function (WHAF) method for the demodulation of fringe patterns recorded in holographic interferometry. It first obtains the analytic signal of the fringe pattern and models it as a piecewise polynomial phase signal. A parametric estimation procedure based on HAF is then employed to calculate the polynomial coefficients of the phase over each window of the segmented analytic signal. A salient feature of the proposed method is that it provides an accurate and direct estimation of the unwrapped phase distribution from a single fringe pattern, even when the patterns phase is rapidly varying. WHAFs application to both digital and classical holographic interferometry is demonstrated by simulation and experimental results.

Improved high-order ambiguity-function method for the estimation of phase from interferometric fringes

Interferometers often encode the information on the measurand in the phase of a fringe pattern, which is usually recorded by an imaging device. Accuracy of measurements carried out by interferometric techniques is thus strongly dependent on the accuracy with which the underlying phase distribution of these fringe patterns is estimated. Fringe analysis methods, which have been developed to accomplish this task, are in general characterized by their performance in terms of both accuracy of phase estimation and associated computational complexity. We propose an improved high-order ambiguity-function-based fringe-analysis method that is demonstrated to provide an accurate and direct estimation of the unwrapped phase distribution in a highly computationally efficient manner. Presented simulation and experimental results in digital holographic interferometry depict the potential utility of the proposed method.