Cystic fibrosis is the most common fatal genetic disorder in the US. In airways with CF, bicarbonate concentration affects airway liquid pH, which then modulates mucogenesis. Causative relationships between these chemical parameters and the functional microanatomy during pathogenesis and disease progression are unclear due to the lack of quantitative tools suitable for in situ studies. We utilized a new µOCT-FCM system to measure the airway functional microanatomy parameters and subsequent pH in the mucus in situ in a non-contact manner. We demonstrated the quantification of pH of the ASL using SNARF-1 in a spatially resolved manner. The µOCT-FCM imaging allowed us to visualize pH in the mucus compartments of the airway and achieved colocalized μOCT and fluorescence imaging. In human bronchial epithelial cells, CF cell ASL pH values were lower than those in the non-CF cells as expected. Pilot results from ex-vivo swine trachea showed that tracheal mucus is heterogeneous.
Dynamic micro-optical coherence tomography (DµOCT) is a technology that is capable of interrogating intracellular dynamics in intact, viable tissues. Towards our goal of advancing DµOCT for phenotyping cells, we imaged freshly excised human biopsies and performed correlative studies with histological results. To date, more than 30 biopsies from 17 patients with numerous types of gastrointestinal pathologies, including cancer, diverticulitis, and Crohn’s disease were imaged. In addition, using mouse models, we performed DµOCT imaging studies on tumors locally treated with chemotherapeutics delivered via custom implantable microdevices to observe the impact of those drugs on the tumor. Cyclical immunofluorescence staining was used to co-register ~20 markers on the same cross-sectional plane. We further demonstrate the utility of principal component analysis, K-means clustering, and convolutional long short-term memory (ConvLSTM) neural network for expanding the capabilities of DµOCT.
Micro-Optical Coherence Tomography (μOCT) enables non-invasive, label-free cross-sectional imaging with subcellular resolution and extended depth of focus. However, μOCT provides limited sensitivity and specificity to the molecular and genomic signatures. The combination of µOCT and Fluorescence Confocal Microscopy (FCM) would improve our capability to interrogate biological specimens. We built and characterized a µOCT-FCM that enables colocalized imaging of µOCT and fluorescence signals. Depth scanning in the fluorescence channel was enabled by using an electronically tunable lens. We report the optical design, system-level integration, and characterization, and demonstrate the quantification of pH of airway mucus in a spatially resolved manner in cell cultures and ex-vivo swine tissue.
JOM Associate Editor Dr. Hui Min Leung of Harvard Medical School and Massachusetts General Hospital interviews Dr. Guillermo (Gary) Tearney, the Remondi Family Endowed MGH Research Institute Chair, Professor of Pathology at Harvard Medical School, and an Affiliated Faculty member of the Harvard-MIT Division of Health Sciences and Technology. He maintains a lab at the Wellman Center for Photomedicine at the Massachusetts General Hospital. With the use of advanced endomicroscopy technologies, Tearney’s lab performs development and clinical validation of non-invasive, high-resolution optical imaging methods for human disease diagnosis. Through this interview, he described how he got into the field and how important and relevant optical microsystems are to those research projects. He also gave his thoughts on the future of the field and the grand challenges that remain to be tackled.
A virtual outreach program was developed specifically for the field of biophotonics. The program consisted of hands-on activities using kits sent to students in advance, real-time video lab tours and prerecorded video content.
Since the recognition that entry factors of SARS-CoV-2 are highly expressed in the nasal epithelium, a custom personal protection booth was developed to enable intranasal µOCT studies to be safely performed on Covid-positive subjects. The booth was designed to preserve familiarity of the prior established intranasal imaging techniques used by clinicians. A group of Covid-positive subjects (n=13) were imaged, shortly after onset of symptoms (7.8 ± 3.7 days). Historical data from healthy controls (n=10) were included in the study. While the blinded analysis is underway, an interim analysis of the µOCT data revealed several notable abnormalities in the nasal epithelium including delayed mucociliary transport, epithelial injury, and high inflammatory cell count in a pilot cohort. We anticipate that the pathophysiologies captured by intranasal µOCT will provide invaluable insights to the mechanisms of Covid-19 related mucociliary dysfunction.
We report the advancement of magnetomotive micro-optical coherence tomography (µOCT) technology for mapping the cystic fibrosis (CF) mucus viscoelastic properties in situ. We conducted pilot experiments in ex-vivo CF ferret trachea and wild-type swine trachea with a new magnetomotive system design. The system utilizes an electromagnet to drive magnetic nanoparticles with constant and uniform magnetomotive force in the field of view of µOCT. Preliminary results showed that the new system enhances individual magnetic micro-particle tracking and enables the measurement of the viscoelastic properties in different compartments of the mucus.
We report the advancement of magnetomotive μOCT microrheology for measuring viscoelastic parameters of mucus in air-liquid interface cystic fibrosis (CF) cell cultures. We dispersed magnetic microbeads into the mucus and conducted a creep test by applying a constant magnetomotive force to the sample, while imaging the resulting mucus deformation using μOCT. The deformation of mucus compartments was then analyzed using ImageJ. The viscoelastic parameters were extracted by fitting the deformation to a power-law rheology model. Preliminary results showed that magnetomotive μOCT microrheology can probe the heterogeneous viscoelasticity of CF mucus in a spatially resolved manner.
Living cells exhibit active intracellular molecular motion that reflect their functional states. Traditional microscopy techniques that solely capture high resolution static images of cells misses out on the opportunity to tap into the wealth of information provided by complex intracellular activity. We recently developed dynamic micro-OCT (DμOCT), an extension of μOCT that achieves near-isotropic sub-cellular resolution in all three dimensions (2 µm lateral x 1 µm axial). DμOCT substantially enhanced the contrast of cells and organelles while revealing stratified, depth-dependent dynamics in the epithelial layers. In this work, we have expanded the application DμOCT to encompass imaging of human skin in vivo, and detection of pharmacologic-induced changes in human melanoma spheroids and murine tissues.
Coronary arteries are covered by a thin layer of endothelial cells (ECs). Impairment of ECs is at the origin of coronary atherosclerosis and its clinical manifestations. However, the study of ECs in humans remains elusive because of a lack of an imaging tool with sufficient resolution. We have developed a light-based 1-µm-resolution microscopic imaging technology termed micro-optical coherence tomography (µOCT) that can be implemented in a coronary catheter. In this study, we investigated the capability of µOCT to visualize EC morphology. We stripped the endothelium from 36 fresh swine coronary segments with cyanoacrylate glue. Histology showed that the stripping procedure successfully removed ECs from the swine coronary arteries. Coronary segments were then imaged in 3D with µOCT, and were processed for histology and scanning electron microscopy (SEM). µOCT images of stripped vs. intact sites were volume rendered in 3D and visually compared. 3D-µOCT allowed visualization of EC pavementing on intact artery surfaces that was strongly correlated to that seen by SEM. EC pavementing disappeared, and surface roughness calculated by computed root mean squared error diminished significantly at the sites with stripped EC compared with intact sites. µOCT was also utilized in human cadaver coronary arteries, showing its capability of identifying EC morphology of human coronary plaque harboring leukocyte adhesion, EC stent strut tissue coverage, and lack of ECs at lesions with necrotic core or superficial nodular calcifications. In conclusion, µOCT enables EC visualization in coronary arteries, suggesting that it could be useful in patients with coronary artery disease to better understand the role of ECs in the pathogenesis of coronary artery disease.
The intestinal epithelial barrier provides protection from external threats that enter the digestive system and persist beyond passage through the stomach. The effects of toxic agents on the intestinal epithelial cell monolayer have not been fully characterized at a cellular level as live imaging of this dynamic interplay at sufficient resolution to interpret cellular responses presents technological challenges. Using a high-resolution native contrast modality called Micro-Optical Coherence Tomography (μOCT), we generated real-time 3D images depicting the impact of the chemical agent EDTA on polarized intestinal epithelial monolayers. Within minutes following application of EDTA, we observed a change in the uniformity of epithelial surface thickness and loss of the edge brightness associated with the apical surface. These observations were measured by generating computer algorithms which quantify imaged-based events changing over time, thus providing parallel graphed data to pair with video. The imaging platform was designed to monitor epithelial monolayers prior to and following application of chemical agents in order to provide a comprehensive account of monolayer behavior at baseline conditions and immediately following exposure. Furthermore, the platform was designed to simultaneously measure continuous trans-epithelial electric resistance (TEER) in order to define the progressive loss of barrier integrity of the cell monolayer following exposure to toxic agents and correlate these findings to image-based metrics. This technological image-based experimental platform provides a novel means to characterize mechanisms that impact the intestinal barrier and, in future efforts, can be applied to study the impact of disease relevant agents such as enteric pathogens and enterotoxins.
High resolution micro-optical coherence tomography (µOCT) technology has been demonstrated to be useful for imaging respiratory epithelial functional microanatomy relevant to the study of pulmonary diseases such as cystic fibrosis and COPD. We previously reported the use of a benchtop μOCT imaging technology to image several relevant respiratory epithelial functional microanatomy at 40 fps and at lateral and axial resolutions of 2 and 1.3μm, respectively. We now present the development of a portable μOCT imaging system with comparable optical and imaging performance, which enables the μOCT technology to be translated to the clinic for in vivo imaging of human airways.
KEYWORDS: In vivo imaging, Biology, Medical research, Biological research, Optical coherence tomography, Tomography, Image resolution, Cystic fibrosis, Coherence (optics), Real time imaging
Chronic dysregulated influx of neutrophil into the airway increases neutrophil burden and augments the inflammatory processes often observed in diseases such as cystic fibrosis. The quantification of neutrophil influx is often accomplished with the use of destructive tests such as imaging cytometry and myeloperoxidase assay. However, those methods are unable to capture information about the cascade of events that precede trans-epithelium migration. In this work, we employed a high resolution micro-optical coherence tomography (µOCT) technology to perform real time imaging of neutrophil activity across airway epithelial cells grown on the underside of Transwell permeable supports. This inverted configuration allows the creation of an air-liquid interface at the apical side of the cells. The µOCT imaging technology, based on the principles of spectral-domain OCT, has a lateral and axial resolution of 2 and 1.3µm, respectively. In addition, it has an axial range of approximately 300µm and is capable of recording cross-sectional images at 40 fps. By raster scanning the illumination beam, the behavior of the neutrophils across a 3D volume can be recorded over time. Thus, this imaging modality is capable of resolving individual neutrophils and, potentially, capturing the cascading events involving neutrophil tethering, subsequent adhesion to activated epithelial cells and the ultimate passage through the epithelial cells to the air space on the apical side. As a result, not only can the amount of neutrophil migration be quantified, how neutrophils behave, organize and interact with the epithelial cells and each other can also be more closely analyzed by µOCT imaging.
The human respiratory system is protected by a defense mechanism termed mucociliary clearance (MCC). Deficiency in MCC leads to respiratory obstruction and pulmonary infection, which often are the main causes of morbidity and mortality in diseases such as cystic fibrosis and chronic obstructive pulmonary disease (COPD). Studying key parameters that govern MCC, including ciliary beat frequency, velocity and volume of airway mucus transport, as well as periciliary liquid layer thickness are therefore of great importance in understanding human respiratory health. However, direct, in vivo visualization of ciliary function and MCC has been challenging, hindering the diagnosis of disease pathogenesis and mechanistic evaluation of novel therapeutics.
Our laboratory has previously developed a 1-µm resolution optical coherence tomography method, termed Micro-OCT, which is a unique tool for visualizing the spatiotemporal features of ciliary function and MCC. We have previously described the design of a flexible 2.5 mm Micro-OCT probe that is compatible with standard flexible bronchoscopes. This device utilizes a common-path interferometer and annular sample arm apodization to attain a sharply focused spot over an extended depth of focus.
Here, we present the most recent iteration of this probe and demonstrate its imaging performance in a mouse trachea tissue culture model. In addition, we have developed an ergonomic assembly for attaching the probe to a standard bronchoscope. The ergonomic assembly fixes the Micro-OCT probe’s within the bronchoscope and contains a means transducing linear motion through the sheath so that the Micro-OCT beam can be scanned along the trachea. We have tested the performance of these devices for Micro-OCT imaging in an anatomically correct model of the human airway. Future studies are planned to use this technology to conduct Micro-OCT in human trachea and bronchi in vivo.
An orthotopic mouse mammary window chamber (MWC) model has been developed for multimodal in-vivo functional and anatomical imaging of breast cancer xenografts. Capabilities to image numerous physiological aspects of the same tumor microenvironment over time has important applications such as in experiments studying the efficacies of therapeutic interventions, improvement of cancer detection and investigating basic cancer biology. The compatibility of this MWC model with optical, nuclear and magnetic resonance imaging (MRI) makes it possible to perform a multitude of studies ranging from cellular imaging to whole body imaging. Thus, the MWC represents a powerful tool for breast cancer research. Here, two imaging applications are highlighted, namely the nuclear imaging of glycolytic metabolism with 18FFDG and MRI of tissue perfusion. Nuclear imaging is performed with the use of a 3μm thin phosphor scintillator placed directly in contact with the tissue and visible light from the scintillation is directly detected in a low noise, light tight imaging system. Tissue perfusion is imaged either qualitatively with a dynamic contrast enhancement (DCE) MRI technique or quantitatively with an arterial spin labeling flow-sensitive alternating inversion recovery-rapid acquisition with relaxation enhancement (FAIR-RARE) technique.
KEYWORDS: Tumors, Tissues, Breast cancer, Oxygen, Blood, In vivo imaging, Magnetic resonance imaging, Mirrors, Tumor growth modeling, Multispectral imaging
Cancer cells are characterized by adaptive features that allow them to evade apoptosis and proliferate in an unchecked
manner in the host tissue. Therapeutic strategies often involve targeting those adaptive molecular pathways leading to
downstream effects such as changes in perfusion, metabolic rate, and/or oxygen utilization in the malignant tissue. Such
surrogate biomarkers can be used to monitor therapeutic response, optimize treatment protocols, or assist in development
of new therapeutic approaches. In this study, we present an optical methodology to make in vivo measurements of
oxygen saturation as a surrogate biomarker in breast cancer xenografts within a mouse mammary window chamber
(MWC) model. By using multi-spectral measurements of the reflectance off the tissue under the coverslip of the window
chamber, we are able to obtain high resolution maps of the variation of oxygenation levels of the tissue, which allow
continuous tracking of the level of tissue oxygenation during tumor growth and following treatment. The MWC, which
was designed and fabricated in-house, is compatible with multiple imaging modalities such as MRI and high resolution
intravital microscopy, providing the capability for cross validation of oxygenation measurements on multiple imaging
platforms.
Upregulate levels of expression and activity of membrane H+ ion pumps in cancer cells drives the extracellular pH (pHe,) to values lower than normal. Furthermore, disregulated pH is indicative of the changes in glycolytic metabolism in tumor cells and has been shown to facilitate extracellular tissue remodeling during metastasis Therefore, measurement of pHe could be a useful cancer biomarker for diagnostic and therapy monitoring evaluation. Multimodality in-vivo imaging of pHe in tumorous tissue in a mouse dorsal skin fold window chamber (DSFWC) model is described. A custom-made plastic window chamber structure was developed that is compatible with both imaging optical and MR imaging modalities and provides a model system for continuous study of the same tissue microenvironment on multiple imaging platforms over a 3-week period. For optical imaging of pHe, SNARF-1 carboxylic acid is injected intravenously into a SCID mouse with an implanted tumor. A ratiometric measurement of the fluorescence signal captured on a confocal microscope reveals the pHe of the tissue visible within the window chamber. This imaging method was used in a preliminary study to evaluate sodium bicarbonate as a potential drug treatment to reverse tissue acidosis. For MR imaging of pHe the chemical exchange saturation transfer (CEST) was used as an alternative way of measuring pHe in a DSFWC model. ULTRAVIST®, a FDA approved x-ray/CT contrast agent has been shown to have a CEST effect that is pH dependent. A ratiometric analysis of water saturation at 5.6 and 4.2 ppm chemical shift provides a means to estimate the local pHe.
Window chamber models have been utilized for many years to investigate cancer development and the tumor
microenvironment. Orthotopic mammary window chamber model have been developed for detailed study of breast
cancer. Orthotopic window chamber models, due to the native environment, support more realistic growth and tumor
behavior than ectopic models. The work by other groups thus far utilizing mammary window chamber models has
focused solely on optical imaging techniques, limited to probing the first millimeter or less of tissue. These techniques
do not take full advantage of the unrestricted, three-dimensional tumor growth the model supports. We have developed a
custom plastic structure compatible with multimodality imaging. We present in this work the implementation of our
custom window chamber in a mouse model and the successful imaging of the window chamber cancer model with MRI,
nuclear imaging, and optical techniques. MRI provides a full three-dimensional view of the tumor growth and allows for
additional, potentially clinically translatable, approaches to be utilized in investigating the cancer microenvironment.
Nuclear imaging is accomplished using the Beta Imager, which is a novel approach to nuclear imaging of window
chambers. The Beta Imager detects photons after the interaction of a single positron with a scintillator, instead of the
coincidence detection of annihilation gamma ray pairs. We utilized the radioisotope glucose analog, 2-deoxy-2-
(18F)fluoro-D-glucose or FDG, with the Beta Imager to obtain information on the glycolytic metabolism of the tumor and
surrounding region.
In this paper, a novel liquid lens design is presented, in which a diffractive surface with an aspherical phase contour,
combined with the spherical-like refractive surface, is adopted to improve the inherent chromatic and spherical
aberration performance when compared to conventional pure refractive-type configurations. Single-point diamond
turning together with soft lithography is used to realize this structure. Both simulation and test measurement results agree
well with each other and demonstrate significantly improved chromatic and spherical aberration within the tunable range
of the lens.
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