Low-coherence snapshot phase-shifting diffraction phase microscope

The advantages of quantitative phase microscopy (QPM) such as label-free imaging with high spatial sensitivity, live cell compatibility and high-speed imaging makes it viable for various biological applications. The measurement accuracy of QPM strongly relies on the shape of the recorded interferograms, whether straight or curved fringes are recorded during the data acquisition. Moreover, for a single shot phase recovery high fringe density is required. The wavefront curvature for the high-density fringes over the entire field of view is difficult to be discerned with the naked eye. As a consequence, there is a quadratic phase aberration in the recovered phase images due to curvature mismatch. In the present work, we have implemented sampling moiré method for real-time sensing of the wavefront curvature mismatch between the object and the reference wavefronts and further for its correction. By zooming out the interferogram, moiré fringes are generated which helps to easily identify the curvature of the fringes. The wavefront curvature mismatch correction accuracy of the method is tested with the help of low temporal coherent light source such as a white light (temporal coherence ∼ 1.6 µm). The proposed scheme is successfully demonstrated to remove the quadratic phase aberration caused due to wavefront mismatch from an USAF resolution target and the biological tissue samples. The phase recovery accuracy of the current scheme is further compared with and found to better than the standard method called principle component analysis. The proposed method enables recording of the corrected wavefront interferogram without needing any additional optical components or modification and also does not need any post-processing correction algorithms. The proposed method of curvature compensation paves the path for a high-throughput and accurate quantitative phase imaging.

We present single-shot quantitative phase imaging with polarization differential interference contrast (PDIC) for a differential interference contrast microscope which records the unfiltered Stokes vector of the differential interference pattern with a polarization camera. PDIC enables high spatial resolution phase imaging in real-time, applicable to either absorptive or transparent samples and integrates simply with epi-fluorescence imaging. An algorithm with total variation regularization is further introduced to solve the quantitative phase map from the partial derivative along one single axis, improving the accuracy and the image quality from the Fourier transform solution. After quantifying the accuracy of PDIC phase imaging with numerical simulations and phantom measurements, we demonstrate the biomedical applications by imaging the quantitative phase of both stained and unstained histological tissue sections and visualizing the fission yeast Schizosaccharomyces pombe's cytokinesis.

We introduce an off-axis, wide-field, low-coherence and dual-channel interferometric imaging system, which is based on a simple-to-align, common-path interferometer. The system requires no optical-path-difference matching between the interferometric arms in order to obtain interference with low-coherence light source, and is capable of achieving two channels of off-axis interference with high spatial frequency. The two 180°-phase-shifted interferograms are acquired simultaneously using a single digital camera, and processed into a single, noise-reduced and DC-suppressed interferogram. We demonstrate using the proposed system for phase imaging of fingerprint templates. Due to the fact that conventional phase unwrapping algorithms cannot handle the complex and deep surface topography imposed by fingerprint templates, we experimentally implemented two-wavelength phase unwrapping using a supercontinuum laser coupled to acousto-optical tunable filter, together functioning as a low-coherence tunable light source. From the unwrapped phase map, we produced high quality depth profiles of fingerprint templates.

Noninterferometric phase determination

We have developed a modality for quantitative phase imaging within spectral domain optical coherence tomography based on using an off-axis reference beam. By tilting the propagation of the reference beam relative to that of the sample beam, a spatially varying fringe is generated. Upon detection of this fringe using a parallel spectral domain scheme, the fringe can be used to separate the interference component of the signal and obtain the complex sample field. In addition to providing quantitative phase measurements within a depth resolved measurement, this approach also allows elimination of the complex conjugate artifact, a known limitation of spectral interferometry. The principle of the approach is described here along with demonstration of its capabilities using technical samples.

Quantitative optical phase information provides an alternative method to observe biomedical properties, where conventional phase imaging fails. Phase retrieval typically requires multiple intensity measurements and iterative computations to ensure uniqueness and robustness against detection noise. To increase the measurement speed, we propose a single-shot quantitative phase imaging method with metasurface optics that can be conveniently integrated into conventional imaging systems with minimal modification. The improvement of the measurement speed is simultaneously made possible by combining deep learning with the transport-of-intensity equation. As a proof-of-concept, we demonstrate phase retrieval on both calibrated phase objects and biological specimens by using an imaging system integrated with our metasurface. When combined with the matched neural network, the system yields result with errors as low as 5% and increased space-bandwidth-product. A multitude of commercial applications can benefit from the compactness and rapid implementation of our proposed method.

Assessing tissues' inhomogeneous optical properties is helpful for diagnosis, but high-cost measurement and experimental setups limit its development, data collecting and applications. In this paper, a portable microscope is proposed to assess the inhomogeneous optical properties of the sample. With a LED illumination, accurate quantitative phase (QP) map can be recovered from 5 intensity images captured at different axial positions. Then based on the scattering-phase theorem and statistical dispersion relation (SDR), the inhomogeneous optical properties of the sample can be quantitatively assessed from these QP maps. In contrast to DHM and SLIM, our setup is cost-effective, use-flexible, and with a small amount of data acquisition, thus having the potential to promote the development of assessing tissues' inhomogeneous optical properties, especially in resource-limited areas.

Quantitative phase measurements at multiple wavelengths has created an opportunity for exploring new avenues in phase microscopy such as enhancing imaging-depth (1), measuring hemoglobin concentrations in erythrocytes (2), and more recently in tomographic mapping of the refractive index of live cells (3). To this end, quantitative phase imaging has been demonstrated both at few selected spectral points as well as with high spectral resolution (4,5). However, most of these developed techniques compromise imaging speed, field of view, or the spectral resolution to perform interferometric measurements at multiple colors. In the specific application of quantitative phase in studying blood diseases and red blood cells, current techniques lack the required sensitivity to quantify biological properties of interest at individual cell level. Recently, we have set out to develop a stable quantitative interferometric microscope allowing for measurements of such properties for red cells without compromising field of view or speed of the measurements. The feasibility of the approach will be initially demonstrated in measuring dispersion curves of known solutions, followed by measuring biological properties of red cells in sickle cell anemia.

Quantitative differential phase-contrast (DPC) imaging is one of the commonly used methods for phase retrieval. However, quantitative DPC imaging requires several pairwise intensity measurements, which makes it difficult to monitor living cells in real-time. In this study, we present a single-shot quantitative DPC imaging method based on the combination of deep learning (DL) and color-encoded illumination. Our goal is to train a model that can generate an isotropic quantitative phase image (i.e., target) directly from a single-shot intensity measurement (i.e., input). The target phase image was reconstructed using a linear-gradient pupil with two-axis measurements, and the model input was the measured color intensities obtained from a radially asymmetric color-encoded illumination pattern. The DL-based model was trained, validated, and tested using thirteen different cell lines. The total number of training, validation, and testing images was 264 (10 cells), 10 (1 cell), and 40 (2 cells), respectively. Our results show that the DL-based phase images are visually similar to the ground-truth phase images and have a high structural similarity index (>0.98). Moreover, the phase difference between the ground-truth and DL-based phase images was smaller than 13%. Our study shows the feasibility of using DL to generate quantitative phase imaging from a single-shot intensity measurement.

Quantitative phase microscopy (QPM) is a label-free imaging technique often employed for long-term, high-contrast imaging of live bio-samples. Yet, QPM is not specific to a certain subcellular organelle. As a remedy, fluorescence microscopy can visualize specific subcellular organelles once being labeled with fluorescent markers. In this paper, a high-resolution phase/fluorescence dual-modality microscopic imaging method based on structured light illumination is proposed. In a dual-modality microscopic system, periodic stripes are generated by a digital micromirror array (DMD), and are used as the common illumination for both modalities. For QPM imaging, the holograms of the sample under structured light illuminations from different directions and phase shifts are recorded, from which a quantitative phase image with resolution enhancement can be reconstructed via a synthetic aperture procedure. Furthermore, a numerical approach is proposed to compensate for the environmental disturbances that often challenge aperture synthesis of phase imaging. This method determines each time the phase distortions caused by environmental disturbances through using the spectrum of the 0th order of the structured light illumination, and the phase distortions are removed from the phase distributions of the waves along the 0th and the ±1st diffraction orders. Resolution enhancement of QPM imaging is realized by synthesizing the spectra of all the waves along different diffraction orders of the structured light illuminations of different orientations. With phase images, three-dimensional shapes, inner structures, or refractive index distributions of transparent and translucent samples can be obtained. For fluorescence imaging, intensity images (morie patterns) of the sample under different structured light illuminations are recorded. The spectra along different diffraction orders are separated by using a phase shifting reconstruction algorithm, and are shifted to their original positions, forming a synthesized spectrum that is much broader than the spectra of raw intensity images (NA-limited spectra). An inverse Fourier transform on the synthesized spectrum yields a super-resolution fluorescence image of the sample. With the reconstructed fluorescence images, specific subcellular organelles labeled with fluorescent markers can be visualized. The combination of quantitative phase microscopy and fluorescence microscopy can obtain multidimensional information about the sample. In this dual-mode imaging system, the spatial resolution of quantitative phase imaging and fluorescence imaging are 840 nm and 440 nm, respectively. The proposed dual-mode microscopy imaging technique has been demonstrated for imaging fluorescent beads, fly wings, spring/rice leaves, mouse tail transection, and fluorescence-stained SiHa cells. We envisage that this method can be further applied to many fields, such as biomedicine, industry, and chemistry.

Microscopy combined with the transport of intensity equation is capable of retrieving both intensity and phase distributions of samples from both in-focus and defocus intensities. However, during measurements, the focal plane is often decided artificially and the improper choice may induce errors in quantitative intensity and phase retrieval. In order to obtain accurate in-focus information, quantitative intensity and phase imaging with the numerical focusing transport of intensity equation method combined with cellular duty ratio criterion and numerical wavefront propagation is introduced in this paper. Both numerical simulations and experimental measurements are provided proving this designed method can increase both retrieved in-focus intensity and phase accuracy and reduce dependence of focal plane determination in transport of intensity equation measurements. It is believed that the proposed method can be potentially applied in various fields as in-focus compensation for quantitative phase imaging and automatic focal plane determination, etc.

Quantitative phase spectroscopy is presented as a novel method of measuring the wavelength-dependent refractive index of microscopic volumes. Light from a broadband source is filtered to an ~5 nm bandwidth and rapidly tuned across the visible spectrum in 1 nm increments by an acousto-optic tunable filter (AOTF). Quantitative phase images of semitransparent samples are recovered at each wavelength using off-axis interferometry and are processed to recover relative and absolute dispersion measurements. We demonstrate the utility of this approach by (i) spectrally averaging phase images to reduce coherent noise, (ii) measuring absorptive and dispersive features in microspheres, and (iii) quantifying bulk hemoglobin concentrations by absolute refractive index measurements. Considerations of using low coherence illumination and the extension of spectral techniques in quantitative phase measurements are discussed.

Digital holographic microscopy (DHM) is an important technique that may be used for quantitative phase imaging of unstained biological cell samples. Since the DHM technology is not commonly used in clinics or bioscience research labs, at present there is no well-accepted focusing criterion for unstained samples that users can follow while recording image plane digital holograms of cells. The usual sharpness metrics that are useful for auto-focusing of stained cells do not work well for unstained cells as there is no amplitude contrast. In this work, we report a practical method for estimating the best focus plane for unstained cells in the digital hologram domain. The method is based on an interesting observation that for the best focus plane the fringe pattern associated with individual unstained cells predominantly shows phase modulation effect in the form of bending of fringes and minimal amplitude modulation. This criterion when applied to unstained red blood cells shows that the central dip in the doughnut-like phase profile of cells is maximal in this plane. The proposed methodology is helpful for standardizing the usage of DHM technology across different users and application developmentefforts. LAY DESCRIPTION: Digital holographic microscopy (DHM) is slowly but steadily becoming an important microscopy modality and gaining acceptability for basic bio-science research as well as clinical usage. One of the important features of DHM is that it allows users to perform quantitative imaging of unstained transparent cells. Instead of using dyes or fluorescent labelling, DHM systems use quantitative phase as a contrast mechanism which depends on the natural refractive index variation within the cell samples. Since minimal wet lab processing is required in order to image cell samples with a DHM, cells can be imaged in their natural state. While DHM is gaining popularity among users, the imaging protocols across the labs or users need to be standardized in order to make sure that the same quantitative phase parameters are used for tasks such as quantitative phased based cellclassification. One of the important operational tasks for any microscopy work is to focus the sample under study. While focusing comes naturally to users of brightfield microscopes based on image contrast, the focusing is not straightforward when samples are unstained so that they do not offer any amplitude contrast. When performing quantitative phase imaging, defocus can actually change the phase profile of the cell due to near-zone (Fresnel) diffraction effects. So unless a standardized focusing methodology is used, it will be difficult for multiple DHM users (potentially at different sites) to agree on quantitative results out of their phase images. DHM literature has prior works which perform numerical focusing of recovered complex wave-field in the hologram plane to find the best focus plane. However such methods are not user friendly and do not allow user the same focusing experience as in a brightfield microscope. The numerical focusing is therefore a reasonably good method for an optics researcher but not necessarily so for a microscopy technician looking at cell samples with a DHM system in a clinicalsetting. The present work provides a simple focusing criterion for unstained samples that works directly in the hologram domain. The technique is based on an interesting observation that the when an unstained cell sample is in the best-focus plane, its corresponding hologram (or fringe pattern) predominantly shows phase modulation manifested by bending of fringes at the location of the cell. This criterion can be converted into a simple numerical method as we have used to find the best-focus plane using a stack of through focus holograms. We believe that the technique can be used manually by visually observing the holograms or can be converted to an auto-focus algorithm for a motorized DHMsystem.

Magnetic resonance imaging at 7 Tesla produces high-resolution gradient-echo phase images of patients with multiple sclerosis (MS) that quantify the local field shifts from iron in the basal ganglia and lesions. Phase imaging is easily integrated into clinical examinations because it is a postprocessing technique and does not require additional scanning. The purpose of this study was to quantify local field shifts in MS and to investigate their relation to disease duration and disability status. Thirty-two subjects including 19 patients with MS and 13 age- and sex-matched control subjects were scanned at a spatial resolution of up to 195 x 260 microm. Data were postprocessed to produce anatomical quantitative phase images of local field shifts, as well as conventional magnitude images. The phase images showed an increased local field in the caudate, putamen, and globus pallidus of patients relative to control subjects (p < 0.01). The local field in the caudate was strongly correlated with disease duration (r(2) = 0.77; p < 0.001). Phase images showed contrast in 74% of the 403 lesions, increasing the total lesion count by more than 30% and showing distinct peripheral rings and a close association with vasculature. The increased field in the basal ganglia and correlation with disease duration suggest pathological iron content increases in MS. The peripheral phase rings are consistent with histological data demonstrating iron-rich macrophages at the periphery of a subset of lesions. The clearly defined vessels penetrating MS lesions should increase our ability to detect focal vascular abnormalities specifically related to demyelinating processes.

A fast phase measuring profilometry (PMP) using structured illumination with a digital micro-mirror device (DMD) has been extensively studied because of its advantage of the programmable projection. In the traditionary PMP, the phase-shift device is mechanical based and is controlled by step-motor which may cause phase errors more or less and several frames of deformed fringe patterns should be capture, which may take a longer time. In the system a DMD is used for projecting only one complex color distribution on the surface of the measured object. In the distribution three interlaced RGB based tricolor sinusoidal chroma two-dimensional fringe patterns are synchronously coded by which the phase difference of the patterns are 120 degree one another. A 24-bit color CCD digital camera is used as the detector to capture only one frame of the deformed complex color distributions. By using image separating techniques the three deformed interlaced tricolor sinusoidal chroma two-dimensional fringe patterns can be decoded individually. Using the 3-step phase shift algorithm the three-dimensional topography can be reconstructed from these decoded deformed fringe patterns. To improve the phase measurement accuracy the RGB tricolor based chroma transfer function (CTF) of the measuring system is introduced. The experiments show that by a reverse correction method with the CTF the phase measurement accuracy has been improved obviously.