Matthew T. Rinehart

Dual-interference-channel quantitative-phase microscopy of live cell dynamics

We introduce and experimentally demonstrate a fast and accurate method for quantitative imaging of the dynamics of live biological cells. Using a dual-channel interferometric setup, two phase-shifted interferograms of nearly transparent biological samples are acquired in a single digital camera exposure and digitally processed into the phase profile of the sample. Since two interferograms of the same sample are acquired simultaneously, most of the common phase noise is eliminated, enabling the visualization of millisecond-scale dynamic biological phenomena with subnanometer optical path length temporal stability.

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 Permittivity Measurements of Nanoliter Liquid Volumes in Microfluidic Channels to 40 GHz

We describe the design, fabrication, and evaluation of a new on-wafer measurement platform for the rapid and quantitative determination of the complex permittivity of nanoliter fluid volumes over the continuous frequency range from 45 MHz to 40 GHz. Our measurement platform integrates micrometer-scale poly(dimethylsiloxane) (PDMS)-based microfluidic channels with high-frequency coplanar waveguide (CPW) transmission lines to accurately place small fluid volumes at well-defined locations within planar measurement structures. We applied new on-wafer calibration techniques to accurately determine the scattering parameters of our integrated devices, and we developed a transmission-line model to extract the distributed circuit parameters of the fluid-loaded transmission line segment from the response of the overall test structure. All the necessary model parameters were experimentally determined directly from a single set of measurements without requiring a reference fluid of known permittivity. We extracted the complex permittivity of the fluid under test from the distributed capacitance and conductance per unit length of the fluid-loaded transmission line segment using finite-element analysis of the transmission line cross section. Our measurements show excellent agreement with bulk fluid permittivity determinations for methanol at room temperature and yield consistent results for the extracted fluid permittivity for the same microfluidic channel embedded in multiple CPW transmission lines of different dimensions.

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.

Simultaneous two-wavelength transmission quantitative phase microscopy with a color camera

We present a quantitative phase microscopy method that uses a Bayer mosaic color camera to simultaneously acquire off-axis interferograms in transmission mode at two distinct wavelengths. Wrapped phase information is processed using a two-wavelength algorithm to extend the range of the optical path delay measurements that can be detected using a single temporal acquisition. We experimentally demonstrate this technique by acquiring the phase profiles of optically clear microstructures without 2pi ambiguities. In addition, the phase noise contribution arising from spectral channel crosstalk on the color camera is quantified.

Broadband Permittivity of Liquids Extracted from Transmission Line Measurements of Microfluidic Channels

We present a semi-analytical procedure to extract the permittivity of fluids from measurements in our microfluidic-microelectronic platform. This platform consists of broadband coplanar waveguide transmission lines with integrated microfluidic channels for characterizing the dielectric properties of submicroliter fluid samples. On-wafer calibration techniques are used to obtain the S-parameters of the composite structure (transmission line and microfluid channel) up to 40 GHz. Using microwave network analysis theory, we isolated the response of the microfluidic channel. We modeled the microfluidic channel as a distributed transmission line segment and as a single RLCG elemental segment. Both approaches analytically yield the circuit capacitance CF and conductance GF per unit length of the segment incorporating the microfluidic channel. A finite element electromagnetic simulator is then used to obtain the permittivity of the fluid as a continuous function of frequency. We applied this technique to extract the permittivity of submicroliter liquid volumes. Here we present results on polystyrene latex beads suspended in an aqueous solution over the frequency range from 100 MHz to 40 GHz.

Influence of defocus on quantitative analysis of microscopic objects and individual cells with digital holography

Digital holography offers a unique method for studying microscopic objects using quantitative measurements of the optical phase delays of transmitted light. The optical phase may be integrated across the object to produce an optical volume measurement, a parameter related to dry mass by a simple scaling factor. While digital holography is useful for comparing the properties of microscopic objects, especially cells, we show here that quantitative comparisons of optical phase can be influenced by the focal plane of the measurement. Although holographic images can be refocused digitally using Fresnel propagation, ambiguity can result if this aspect is not carefully controlled. We demonstrate that microscopic objects can be accurately profiled by employing a digital refocusing method to analyze phase profiles of polystyrene microspheres and red blood cells.

Quantitative Analysis of Biological Cells Using Digital Holographic Microscopy

Biological cells are microscopic dynamic objects, continuously adjusting their threedimensional sizes, shapes and other biophysical features. Wide-field microscopy of cell dynamics can provide a powerful research tool for cell biology studies, as well as a potential means for medical diagnosis and monitoring of diseases. Biological cells, however, are mostly-transparent objects, and thus imaging them with conventional intensity-based light microscopy fails to provide adequate optical contrast between the cell and its environment. Although exogenous contrast agents such as fluorescent dyes can be used to solve this problem, they might be cytotoxic in the long run and there is a possibility they will influence the cellular behavior. Additionally, fluorescent dyes tend to photobleach, potentially limiting the imaging time. The contrast problem when imaging biological cells can also be solved by using phase microscopy, which records the optical path delays of light passing through the cells and subsequently obtains information on the cellular structure and dynamics without using any exogenous labelling. Since detectors are sensitive to intensity only, the phase of the light that has interacted with the cells must first be converted to intensity variations for detection. Widely used methods to achieve this include phase contrast microscopy and differential interference contrast (DIC) microscopy. However, these techniques are not inherently quantitative and present distinct imaging artifacts that typically prevent straightforward extraction of the entire optical path delay profile of the cell. Wide-field digital interferometry (WFDI) is a label-free holographic technique that is able to record the entire complex wavefront (amplitude and phase) of the light which has interacted with the sample (Cuche et al., 1999). From the recorded complex field, one can obtain full quantitative phase profiles of cells as well as correct for out-of-focus image features by postprocessing. WFDI microscopy (also called digital holographic microscopy) has been applied to various types of biological cell systems and has recorded a diverse range of cellular phenomena (Marquet et al., 2005; Ikeda et al., 2005; Shaked et al., 2009 b; Shaked et al., 2010 b-d). Section 2 reviews the principle of WFDI for obtaining phase profiles of cells, starting from the experimental setup, and ending in digital processing for obtaining the final quantitative phase profile of the cell. Although WFDI is a quantitative recording technique, simple quasi-three-dimensional holographic visualization of the cell phase profile need not be the end of the process.

Automated Detection of P. falciparum Using Machine Learning Algorithms with Quantitative Phase Images of Unstained Cells.

Malaria detection through microscopic examination of stained blood smears is a diagnostic challenge that heavily relies on the expertise of trained microscopists. This paper presents an automated analysis method for detection and staging of red blood cells infected by the malaria parasite Plasmodium falciparum at trophozoite or schizont stage. Unlike previous efforts in this area, this study uses quantitative phase images of unstained cells. Erythrocytes are automatically segmented using thresholds of optical phase and refocused to enable quantitative comparison of phase images. Refocused images are analyzed to extract 23 morphological descriptors based on the phase information. While all individual descriptors are highly statistically different between infected and uninfected cells, each descriptor does not enable separation of populations at a level satisfactory for clinical utility. To improve the diagnostic capacity, we applied various machine learning techniques, including linear discriminant classification (LDC), logistic regression (LR), and k-nearest neighbor classification (NNC), to formulate algorithms that combine all of the calculated physical parameters to distinguish cells more effectively. Results show that LDC provides the highest accuracy of up to 99.7% in detecting schizont stage infected cells compared to uninfected RBCs. NNC showed slightly better accuracy (99.5%) than either LDC (99.0%) or LR (99.1%) for discriminating late trophozoites from uninfected RBCs. However, for early trophozoites, LDC produced the best accuracy of 98%. Discrimination of infection stage was less accurate, producing high specificity (99.8%) but only 45.0%-66.8% sensitivity with early trophozoites most often mistaken for late trophozoite or schizont stage and late trophozoite and schizont stage most often confused for each other. Overall, this methodology points to a significant clinical potential of using quantitative phase imaging to detect and stage malaria infection without staining or expert analysis.