Yizheng Zhu

Two-step-only phase-shifting interferometry with optimized detector bandwidth for microscopy of live cells

We present a phase-shifting interferometric technique for imaging live biological cells in growth media, while optimizing spatial resolution and enabling potential real-time measurement capabilities. The technique uses slightly-off-axis interferometry which requires less detector bandwidth than traditional off-axis interferometry and fewer measurements than traditional on-axis interferometry. Experimental and theoretical comparisons between the proposed method and these traditional interferometric approaches are given. The method is experimentally demonstrated via phase microscopy of live human skin cancer cells.

Quantitative phase spectroscopy

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.

Detection of Dysplasia in Barrett's Esophagus With In Vivo Depth-Resolved Nuclear Morphology Measurements

BACKGROUND & AIMS Patients with Barretts esophagus (BE) show increased risk of developing esophageal adenocarcinoma and are routinely examined using upper endoscopy with biopsy to detect neoplastic changes. Angle-resolved low coherence interferometry (a/LCI) uses in vivo depth-resolved nuclear morphology measurements to detect dysplasia. We assessed the clinical utility of a/LCI in the endoscopic surveillance of patients with BE. METHODS Consecutive patients undergoing routine surveillance upper endoscopy for BE were recruited at 2 endoscopy centers. A novel, endoscope-compatible a/LCI system measured the mean diameter and refractive index of cell nuclei in esophageal epithelium at 172 biopsy sites in 46 patients. At each site, an a/LCI measurement was correlated with a concurrent endoscopic biopsy specimen. Each biopsy specimen was assessed histologically and classified as normal, nondysplastic BE, indeterminate for dysplasia, low-grade dysplasia (LGD), or high-grade dysplasia (HGD). The a/LCI data from multiple depths were analyzed to evaluate its ability to differentiate dysplastic from nondysplastic tissue. RESULTS Pathology characterized 5 of the scanned sites as HGD, 8 as LGD, 75 as nondysplastic BE, 70 as normal tissue types, and 14 as indeterminate for dysplasia. The a/LCI nuclear size measurements separated dysplastic from nondysplastic tissue at a statistically significant (P < .001) level for the tissue segment 200 to 300 μm beneath the surface with an accuracy of 86% (147/172). A receiver operator characteristic analysis indicated an area under the curve of 0.91, and an optimized decision point gave 100% (13/13) sensitivity and 84% (134/159) specificity. CONCLUSIONS These preliminary data suggest a/LCI is accurate in detecting dysplasia in vivo in patients with BE.

Reflective interferometric chamber for quantitative phase imaging of biological sample dynamics

We introduce a new interferometric setup for single-exposure wide-field holographic phase imaging of highly dynamic biological samples. In this setup, the interferometric signal originates from a specially designed reflective interferometric chamber (InCh), creating an off-axis interferogram on the output plane of the system. The setup only requires the InCh and a simple reflection-mode two lens imaging system, without the need for additional optical elements such as gratings in the beam path. In addition, due to the close-to-common-path geometry of the setup, phase noise is greatly reduced. We experimentally compare the inherent phase stability of the system in ambient conditions to that of a conventional interferometer. We also demonstrate use of this system for wide-field quantitative phase imaging of two different highly dynamic, optically transparent biological samples: beating myocardial cells and moving unicellular microorganisms.

Design and validation of an angle-resolved low-coherence interferometry fiber probe for in vivo clinical measurements of depth-resolved nuclear morphology

We present a novel Fourier-domain angle-resolved low-coherence interferometry (a /LCI) fiber probe designed for in vivo clinical application in gastrointestinal endoscopy. The a/LCI technique measures the depth-resolved angular scattering distribution to determine the size distribution and optical density of cell nuclei for assessing the health of epithelial tissues. Clinical application is enabled by an endoscopic fiber-optic probe that employs a 2.3-m-long coherent fiber bundle and is compatible with the standard 2.8-mm-diam biopsy channel of a gastroscope. The probe allows for real-time data acquisition by collecting the scattering from multiple angles in parallel, enabled by the Fourier domain approach. The performance of the probe is characterized through measurement of critical parameters. The depth-resolved sizing capability of the system is demonstrated using single- and double-layer microsphere phantoms with subwavelength sizing precision and accuracy achieved. Initial results from a clinical feasibility test are also presented to show in vivo application in the human esophagus.

Detection of intestinal dysplasia using angle-resolved low coherence interferometry

Angle-resolved low coherence interferometry (a/LCI) is an optical biopsy technique that allows for depth-resolved, label-free measurement of the average size and optical density of cell nuclei in epithelial tissue to assess the tissue health. a/LCI has previously been used clinically to identify the presence of dysplasia in Barretts Esophagus patients undergoing routine surveillance. We present the results of a pilot, ex vivo study of tissues from 27 patients undergoing partial colonic resection surgery, conducted to evaluate the ability of a/LCI to identify dysplasia. Performance was determined by comparing the nuclear morphology measurements with pathological assessment of co-located physical biopsies. A statistically significant correlation between increased average nuclear size, reduced nuclear density, and the presence of dysplasia was noted at the basal layer of the epithelium, at a depth of 200 to 300 μm beneath the tissue surface. Using a decision line determined from a receiver operating characteristic, a/LCI was able to separate dysplastic from healthy tissues with a sensitivity of 92.9% (13/14), a specificity of 83.6% (56/67), and an overall accuracy of 85.2% (69/81). The study illustrates the extension of the a/LCI technique to the detection of intestinal dysplasia, and demonstrates the need for future in vivo studies.

Size and shape determination of spheroidal scatterers using two-dimensional angle resolved scattering

We demonstrate accurate determination of the size and shape of spherical and spheroidal scatterers through inverse analysis of two-dimensional solid-angle and depth resolved backscattered light intensities. Intensity of scattered light is measured over a wide range of solid angles using a novel scanning fiber optic interferometer from both individual and ensembles of scatterers. T-matrix based inverse analysis of these two-dimensional angular measurements yields completely unique size and aspect ratio determinations with subwavelength precision over a large range of possible scatterer geometries.

Detection of early colorectal cancer development in the azoxymethane rat carcinogenesis model with Fourier domain low coherence interferometry

Fourier domain low coherence interferometry (fLCI) is an emerging optical technique used to quantitatively assess cell nuclear morphology in tissue as a means of detecting early cancer development. In this work, we use the azoxymethane rat carcinogenesis model, a well characterized and established model for colon cancer research, to demonstrate the ability of fLCI to distinguish between normal and preneoplastic ex-vivo colon tissue. The results show highly statistically significant differences between the measured cell nuclear diameters of normal and azoxymethane-treated tissues, thus providing strong evidence that fLCI may be a powerful tool for non-invasive, quantitative detection of early changes associated with colorectal cancer development.

Optical Spectroscopy of Biological Cells

Optical spectroscopy has seen expanding use for the study of biological cells in recent years. An overview of relevant spectroscopic techniques is presented, and applications to biological cells are reviewed.

Scanning fiber angle-resolved low coherence interferometry

We present a fiber-optic probe for Fourier-domain angle-resolved low coherence interferometry for the determination of depth-resolved scatterer size. The probe employs a scanning single-mode fiber to collect the angular scattering distribution of the sample, which is analyzed using the Mie theory to obtain the average size of the scatterers. Depth sectioning is achieved with low coherence Mach-Zehnder interferometry. In the sample arm of the interferometer, a fixed fiber illuminates the sample through an imaging lens and a collection fiber samples the backscattered angular distribution by scanning across the Fourier plane image of the sample. We characterize the optical performance of the probe and demonstrate the ability to execute depth-resolved sizing with subwavelength accuracy by using a double-layer phantom containing two sizes of polystyrene microspheres.