Multiscale optical phase fluctuations link disorder strength and fractal dimension of cell structure.

Optical methods for examining cellular structure based on endogenous contrast rely on analysis of refractive index changes to discriminate cell phenotype. These changes can be visualized using techniques such as phase contrast microscopy, detected by light scattering, or analyzed numerically using quantitative phase imaging. The statistical variations of refractive index at the nanoscale can be quantified using disorder strength, a metric seen to increase with neoplastic change. In contrast, the spatial organization of these variations is typically characterized using a fractal dimension, which is also seen to increase with cancer progression. Here, we seek to link these two measurements using multiscale measurements of optical phase to calculate disorder strength and in turn to determine the fractal dimension of the structures. First, quantitative phase images are analyzed to show that the disorder strength metric changes with resolution. The trend of disorder strength with length scales is analyzed to determine the fractal dimension of the cellular structures. Comparison of these metrics is presented for different cell lines with varying phenotypes including MCF10A, MCF7, BT474, HT-29, A431, and A549 cell lines, in addition to three cell populations with modified phenotypes. Our results show that disorder strength and fractal dimension can both be obtained with quantitative phase imaging and that these metrics can independently distinguish between different cell lines. Furthermore, their combined use presents a new approach for better understanding cellular restructuring during different pathways.

Localized compliance measurement of the airway wall using anatomic optical coherence elastography.

We describe an elastographic method to circumferentially-resolve airway wall compliance using endoscopic, anatomic optical coherence tomography (aOCT) combined with an intraluminal pressure catheter. The method was first demonstrated on notched silicone phantoms of known elastic modulus under respiratory ventilation, where localized compliance measurements were validated against those predicted by finite element modeling. Then, ex vivo porcine tracheas were scanned, and the pattern of compliance was found to be consistent with histological identification of the locations of (stiff) cartilage and (soft) muscle. This quantitative method may aid in diagnosis and monitoring of collapsible airway wall tissues in obstructive respiratory disorders.

Optimization of immunolabeled plasmonic nanoparticles for cell surface receptor analysis.

Noble metal nanoparticles hold great potential as optical contrast agents due to a unique feature, known as the plasmon resonance, which produces enhanced scattering and absorption at specific frequencies. The plasmon resonance also provides a spectral tunability that is not often found in organic fluorophores or other labeling methods. The ability to functionalize these nanoparticles with antibodies has led to their development as contrast agents for molecular optical imaging. In this review article, we present methods for optimizing the spectral agility of these labels. We discuss synthesis of gold nanorods, a plasmonic nanoparticle in which the plasmonic resonance can be tuned during synthesis to provide imaging within the spectral window commonly utilized in biomedical applications. We describe recent advances in our group to functionalize gold and silver nanoparticles using distinct antibodies, including EGFR, HER-2 and IGF-1, selected for their relevance to tumor imaging. Finally, we present characterization of these nanoparticle labels to verify their spectral properties and molecular specificity.

Single Cell Analysis of Stored Red Blood Cells Using Ultra-High Throughput Holographic Cytometry.

Holographic cytometry is introduced as an ultra-high throughput implementation of quantitative phase imaging of single cells flowing through parallel microfluidic channels. Here, the approach was applied for characterizing the morphology of individual red blood cells during storage under regular blood bank conditions. Samples from five blood donors were examined, over 100,000 cells examined for each, at three time points. The approach allows high-throughput phase imaging of a large number of cells, greatly extending our ability to study cellular phenotypes using individual cell images. Holographic cytology images can provide measurements of multiple physical traits of the cells, including optical volume and area, which are observed to consistently change over the storage time. In addition, the large volume of cell imaging data can serve as training data for machine-learning algorithms. For the study here, logistic regression was used to classify the cells according to the storage time points. The analysis showed that at least 5000 cells are needed to ensure accuracy of the classifiers. Overall, results showed the potential of holographic cytometry as a diagnostic tool.

Sensing Inhalation Injury-Associated Changes in Airway Wall Compliance by Anatomic Optical Coherence Elastography.

Quantitative methods for assessing the severity of inhalation (burn) injury are needed to aid in treatment decisions. We hypothesize that it is possible to assess the severity of injuries on the basis of differences in the compliance of the airway wall. Here, we demonstrate the use of a custom-built, endoscopic, anatomic optical coherence elastography (aOCE) system to measure airway wall compliance. The method was first validated using airway phantoms, then performed on ex vivo porcine tracheas under varying degrees of inhalation (steam) injury. A negative correlation between aOCE-derived compliance and severity of steam injuries is found, and spatially-resolved compliance maps reveal regional heterogeneity in airway properties.

Deep imaging with 1.3†µm dual-axis optical coherence tomography and an enhanced depth of focus.

For many clinical applications, such as dermatology, optical coherence tomography (OCT) suffers from limited penetration depth due primarily to the highly scattering nature of biological tissues. Here, we present a novel implementation of dual-axis optical coherence tomography (DA-OCT) that offers improved depth penetration in skin imaging at 1.3 µm compared to conventional OCT. Several unique aspects of DA-OCT are examined here, including the requirements for scattering properties to realize the improvement and the limited depth of focus (DOF) inherent to the technique. To overcome this limitation, our approach uses a tunable lens to coordinate focal plane selection with image acquisition to create an enhanced DOF for DA-OCT. This improvement in penetration depth is quantified experimentally against conventional on-axis OCT using tissue phantoms and mouse skin. The results presented here suggest the potential use of DA-OCT in situations where a high degree of scattering limits depth penetration in OCT imaging.

Geometric Validation of Continuous, Finely Sampled 3-D Reconstructions From aOCT and CT in Upper Airway Models.

Identification and treatment of obstructive airway disorders (OADs) are greatly aided by imaging of the geometry of the airway lumen. Anatomical optical coherence tomography (aOCT) is a promising high-speed and minimally invasive endoscopic imaging modality for providing micrometer-resolution scans of the upper airway. Resistance to airflow in OADs is directly caused by the reduction in luminal cross-sectional area (CSA). It is hypothesized that aOCT can produce airway CSA measurements as accurate as that from computed tomography (CT). Scans of machine hollowed cylindrical tubes were used to develop methods for segmentation and measurement of airway lumen in CT and aOCT. Simulated scans of virtual cones were used to validate 3-D resampling and reconstruction methods in aOCT. Then, measurements of two segments of a 3-D printed pediatric airway phantom from aOCT and CT independently were compared to ground truth CSA. In continuous unobstructed regions, the mean CSA difference for each phantom segment was 2.2 ± 3.5 and 1.5 ± 5.3 mm2 for aOCT, and -3.4 ± 4.3 and -1.9 ± 1.2 mm2 for CT. Because of the similar magnitude of these differences, these results support the hypotheses and underscore the potential for aOCT as a viable alternative to CT in airway imaging, while offering greater potential to capture respiratory dynamics.

Combined anatomical optical coherence tomography and intraluminal pressure reveal viscoelasticity of the in vivo airway.

It is hypothesized that the local, viscoelastic (time-dependent) properties of the airway are important to accurately model and ultimately predict dynamic airway collapse in airway obstruction. Toward this end, we present a portable, endoscopic, swept-source anatomical optical coherence tomography (aOCT) system combined with a pressure catheter to capture local airway dynamics in vivo during respiration. aOCT scans were performed in the airways of a mechanically ventilated pig under paralysis with dynamic and static ventilation protocols. Validation of dynamic aOCT luminal cross-sectional area (CSA) measurements against Cine CT, obtained during the same exam, showed an aggregate difference of 15 % ± 3 % . aOCT-derived CSA obtained in the in vivo trachea also exhibited hysteresis as a function of pressure, depicting the viscoelastic nature of the airway wall. The volumetric imaging capabilities were validated by comparing aOCT- and CT-derived geometries of the porcine airway spanning nine generations from the trachea to the bronchioles. The ability to delineate regional differences in airway viscoelastic properties, by measuring airway deformation using aOCT combined with intraluminal pressure, paves the way to patient-specific models of dynamic airway collapse.

Multi-modal anatomical Optical Coherence Tomography and CT for in vivo Dynamic Upper Airway Imaging.

We describe a novel, multi-modal imaging protocol for validating quantitative dynamic airway imaging performed using anatomical Optical Coherence Tomography (aOCT). The aOCT system consists of a catheter-based aOCT probe that is deployed via a bronchoscope, while a programmable ventilator is used to control airway pressure. This setup is employed on the bed of a Siemens Biograph CT system capable of performing respiratory-gated acquisitions. In this arrangement the position of the aOCT catheter may be visualized with CT to aid in co-registration. Utilizing this setup we investigate multiple respiratory pressure parameters with aOCT, and respiratory-gated CT, on both ex vivo porcine trachea and live, anesthetized pigs. This acquisition protocol has enabled real-time measurement of airway deformation with simultaneous measurement of pressure under physiologically relevant static and dynamic conditions- inspiratory peak or peak positive airway pressures of 10-40 cm H2O, and 20-30 breaths per minute for dynamic studies. We subsequently compare the airway cross sectional areas (CSA) obtained from aOCT and CT, including the change in CSA at different stages of the breathing cycle for dynamic studies, and the CSA at different peak positive airway pressures for static studies. This approach has allowed us to improve our acquisition methodology and to validate aOCT measurements of the dynamic airway for the first time. We believe that this protocol will prove invaluable for aOCT system development and greatly facilitate translation of OCT systems for airway imaging into the clinical setting.

Airway compliance measured by anatomic optical coherence tomography.

Quantification of airway compliance can aid in the diagnosis and treatment of obstructive airway disorders by detecting regions vulnerable to collapse. Here we evaluate the ability of a swept-source anatomic optical coherence tomography (SSaOCT) system to quantify airway cross-sectional compliance (CC) by measuring changes in the luminal cross-sectional area (CSA) under physiologically relevant pressures of 10-40 cmH2O. The accuracy and precision of CC measurements are determined using simulations of non-uniform rotation distortion (NURD) endemic to endoscopic scanning, and experiments performed in a simplified tube phantom and ex vivo porcine tracheas. NURD simulations show that CC measurements are typically more accurate than that of the CSAs from which they are derived. Phantom measurements of CSA versus pressure exhibit high linearity (R2>0.99), validating the dynamic range of the SSaOCT system. Tracheas also exhibited high linearity (R2 = 0.98) suggestive of linear elasticity, while CC measurements were obtained with typically ± 12% standard error.

Swept-Source Anatomic Optical Coherence Elastography of Porcine Trachea.

Quantitative endoscopic imaging is at the vanguard of novel techniques in the assessment upper airway obstruction. Anatomic optical coherence tomography (aOCT) has the potential to provide the geometry of the airway lumen with high-resolution and in 4 dimensions. By coupling aOCT with measurements of pressure, optical coherence elastography (OCE) can be performed to characterize airway wall stiffness. This can aid in identifying regions of dynamic collapse as well as informing computational fluid dynamics modeling to aid in surgical decision-making. Toward this end, here we report on an anatomic optical coherence tomography (aOCT) system powered by a wavelength-swept laser source. The system employs a fiber-optic catheter with outer diameter of 0.82 mm deployed via the bore of a commercial, flexible bronchoscope. Helical scans are performed to measure the airway geometry and to quantify the cross-sectional-area (CSA) of the airway. We report on a preliminary validation of aOCT for elastography, in which aOCT-derived CSA was obtained as a function of pressure to estimate airway wall compliance. Experiments performed on a Latex rubber tube resulted in a compliance measurement of 0.68±0.02 mm2/cmH2O, with R2=0.98 over the pressure range from 10 to 40 cmH2O. Next, ex vivo porcine trachea was studied, resulting in a measured compliance from 1.06±0.12 to 3.34±0.44 mm2/cmH2O, (R2>0.81). The linearity of the data confirms the elastic nature of the airway. The compliance values are within the same order-of-magnitude as previous measurements of human upper airways, suggesting that this system is capable of assessing airway wall compliance in future human studies.

Quantitative upper airway endoscopy with swept-source anatomical optical coherence tomography.

Minimally invasive imaging of upper airway obstructions in children and adults is needed to improve clinical decision-making. Toward this goal, we demonstrate an anatomical optical coherence tomography (aOCT) system delivered via a small-bore, flexible endoscope to quantify the upper airway lumen geometry. Helical scans were obtained from a proximally-scanned fiber-optic catheter of 820 µm outer diameter and >2 mm focal length. Coupled with a long coherence length wavelength-swept light source, the system exhibited an SNR roll-off of < 10 dB over a 10 mm range. Operating at 10 rotations/s, the average accuracy of segmented cross-sectional areas was found to be -1.4 ± 1.0%. To demonstrate the capability of this system, aOCT was performed on a pediatric airway phantom and on ex vivo swine trachea. The ability for quantitative endoscopy afforded by this system can aid in diagnosis, medical and surgical decision making, and predictive modeling of upper airway obstructive disorders.