Imaging deformation of adherent cells due to shear stress using quantitative phase imaging.

We present a platform for detecting cellular deformations from mechanical stimuli, such as fluid shear stress, using rapid quantitative phase imaging. Rapid quantitative phase imaging was used to analyze changes in the optical path length of adherent skin cancer cells during mechanical displacement. Both the whole-cell phase displacement and the resultant shift of the cellular center of mass were calculated over the duration of the stimulus. Whole-cell phase displacement images were found to match expectation. Furthermore, center-of-mass shifts of adherent cells were found to resemble that of a one-dimensional Kelvin-Voigt (KV) viscoelastic solid. Cellular steady-state displacements from step fluid shear stimuli were found to be linearly related to the shear stress. Shear stiffness constants for cells exposed to a cytoskeletal disrupting toxin were found to be significantly lower than unexposed cells. This novel technique allows for elastographic analysis of whole-cell effective shear stiffness without the use of an exogenous force applicator, a specialized culture substrate, or tracking net perimeter movement of the cell.

Quantitative phase microscopy with off-axis optical coherence tomography.

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.

Detection of dysplasia in Barrett's esophagus with in vivo depth-resolved nuclear morphology measurements.

BACKGROUND & AIMS: Patients with Barrett's 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.

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.

Hemoglobin consumption by P. falciparum in individual erythrocytes imaged via quantitative phase spectroscopy.

Plasmodium falciparum infection causes structural and biochemical changes in red blood cells (RBCs). To quantify these changes, we apply a novel optical technique, quantitative phase spectroscopy (QPS) to characterize individual red blood cells (RBCs) during the intraerythrocytic life cycle of P. falciparum. QPS captures hyperspectral holograms of individual RBCs to measure spectroscopic changes across the visible wavelength range (475-700 nm), providing complex information, i.e. amplitude and phase, about the light field which has interacted with the cell. The complex field provides complimentary information on hemoglobin content and cell mass, which are both found to dramatically change upon infection by P. falciparum. Hb content progressively decreases with parasite life cycle, with an average 72.2% reduction observed for RBCs infected by schizont-stage P. falciparum compared to uninfected cells. Infection also resulted in a 33.1% reduction in RBC's optical volume, a measure of the cells' non-aqueous components. Notably, optical volume is only partially correlated with hemoglobin content, suggesting that changes in other dry mass components such as parasite mass may also be assessed using this technique. The unique ability of QPS to discriminate individual healthy and infected cells using spectroscopic changes indicates that the approach can be used to detect disease.

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.

Evaluation of cytomegalovirus reactivation and tolerability in seropositive umbilical cord transplant patients after implementation of an intensive prevention strategy.

OBJECTIVE/BACKGROUND: Cytomegalovirus (CMV) causes significant morbidity and mortality in CMV seropositive patients undergoing umbilical cord blood transplants (UCBT). Our study aimed to describe the incidence of CMV reactivation and burden of disease, as well as the tolerability of an intensive prevention strategy as compared to historical prevention. METHODS: This was a retrospective chart review of 33 CMV seropositive patients that underwent UCBT. The intensive prevention strategy in UCBT consisted of ganciclovir 5mg/kg/d intravenously or valganciclovir 900mg by mouth daily initiated at the beginning of the conditioning regimen until Day -2. Then from Day -1 to Day +100, patients received valacyclovir 2g by mouth three times daily, and from Day +101 to Day +365, acyclovir 800mg by mouth twice daily. Historical standard prevention was acyclovir 800mg by mouth twice daily initiated at the beginning of the conditioning regimen until Day +365. RESULTS: Thirty-three patients were included from 2008 to 2014. There were no differences in the adverse effects experienced between the two regimens (p=.4). CMV reactivation occurred significantly later with intensive prevention (p=.003). The median CMV viral titer at reactivation was lower in the intensive versus the historic prevention (1,800copies/mL and 2,700copies/mL, respectively), but was not significantly different. CMV disease occurred significantly less often in the intensive group (p=.039). CONCLUSION: The results from this study indicate that the intensive prevention strategy was well tolerated, significantly delayed CMV reactivation, and patients had less CMV disease.

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.

Co-localized confocal Raman spectroscopy and optical coherence tomography (CRS-OCT) for depth-resolved analyte detection in tissue.

We report the development of a combined confocal Raman spectroscopy (CRS) and optical coherence tomography (OCT) instrument (CRS-OCT) capable of measuring analytes in targeted biological tissues with sub-100-micron spatial resolution. The OCT subsystem was used to measure depth-resolved tissue morphology and guide the acquisition of chemically-specific Raman spectra. To demonstrate its utility, the instrument was used to accurately measure depth-resolved, physiologically-relevant concentrations of Tenofovir, a microbicide drug used to prevent the sexual transmission of HIV, in ex vivo tissue samples.

Analysis of vaginal microbicide film hydration kinetics by quantitative imaging refractometry.

We have developed a quantitative imaging refractometry technique, based on holographic phase microscopy, as a tool for investigating microscopic structural changes in water-soluble polymeric materials. Here we apply the approach to analyze the structural degradation of vaginal topical microbicide films due to water uptake. We implemented transmission imaging of 1-mm diameter film samples loaded into a flow chamber with a 1.5×2 mm field of view. After water was flooded into the chamber, interference images were captured and analyzed to obtain high resolution maps of the local refractive index and subsequently the volume fraction and mass density of film material at each spatial location. Here, we compare the hydration dynamics of a panel of films with varying thicknesses and polymer compositions, demonstrating that quantitative imaging refractometry can be an effective tool for evaluating and characterizing the performance of candidate microbicide film designs for anti-HIV drug delivery.

Fast wide-field photothermal and quantitative phase cell imaging with optical lock-in detection.

We present a fast, wide-field holography system for detecting photothermally excited gold nanospheres with combined quantitative phase imaging. An interferometric photothermal optical lock-in approach (POLI) is shown to improve SNR for detecting nanoparticles (NPs) on multiple substrates, including a monolayer of NPs on a silanized coverslip, and NPs bound to live cells. Furthermore, the set up allowed for co-registered quantitative phase imaging (QPI) to be acquired in an off-axis holographic set-up. An SNR of 103 was obtained for NP-tagging of epidermal growth factor receptor (EGFR) in live cells with a 3 second acquisition, while an SNR of 47 was seen for 20 ms acquisition. An analysis of improvements in SNR due to averaging multiple frames is presented, which suggest that residual photothermal signal can be a limiting factor. The combination of techniques allows for high resolution imaging of cell structure via QPI with the ability to identify receptor expression via POLI.

Comparative review of interferometric detection of plasmonic nanoparticles.

Noble metal nanoparticles exhibit enhanced scattering and absorption at specific wavelengths due to a localized surface plamson resonance. This unique property can be exploited to enable the use of plasmonic nanoparticles as contrast agents in optical imaging. A range of optical techniques have been developed to detect nanoparticles in order to implement imaging schemes. Here we review several different approaches for using optical interferometry to detect the presence and concentration of nanoparticles. The strengths and weaknesses of the various approaches are discussed and quantitative comparisons of the achievable signal to noise ratios are presented. The benefits of each approach are outlined as they relate to specific application goals.

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.

Phase-sensitive OCT imaging of multiple nanoparticle species using spectrally multiplexed single pulse photothermal excitation.

We apply phase-sensitive optical coherence tomography to image multiple nanoparticle species with two excitation wavelengths matched to their distinct absorption peaks. Using different modulation frequencies, multiple species collocated within the sample can be distinguished. In addition, we characterize single-pulse excitation schemes as a method to minimize bulk heating of the sample. We demonstrate this new scheme with B-mode photothermal measurements of tissue phantoms.

Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy.

Quantitative phase microscopy is applied to image temporal changes in the refractive index (RI) distributions of solutions created by microbicidal films undergoing hydration. We present a novel method of using an engineered polydimethylsiloxane structure as a static phase reference to facilitate calibration of the absolute RI across the entire field. We present a study of dynamic structural changes in microbicidal films during hydration and subsequent dissolution. With assumptions about the smoothness of the phase changes induced by these films, we calculate absolute changes in the percentage of film in regions across the field of view.

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.