Carolin Unglert

In vivo imaging of airway cilia and mucus clearance with micro-optical coherence tomography

We have designed and fabricated a 4 mm diameter rigid endoscopic probe to obtain high resolution micro-optical coherence tomography (µOCT) images from the tracheal epithelium of living swine. Our common-path fiber-optic probe used gradient-index focusing optics, a selectively coated prism reflector to implement a circular-obscuration apodization for depth-of-focus enhancement, and a common-path reference arm and an ultra-broadbrand supercontinuum laser to achieve high axial resolution. Benchtop characterization demonstrated lateral and axial resolutions of 3.4 μm and 1.7 μm, respectively (in tissue). Mechanical standoff rails flanking the imaging window allowed the epithelial surface to be maintained in focus without disrupting mucus flow. During in vivo imaging, relative motion was mitigated by inflating an airway balloon to hold the standoff rails on the epithelium. Software implemented image stabilization was also implemented during post-processing. The resulting image sequences yielded co-registered quantitative outputs of airway surface liquid and periciliary liquid layer thicknesses, ciliary beat frequency, and mucociliary transport rate, metrics that directly indicate airway epithelial function that have dominated in vitro research in diseases such as cystic fibrosis, but have not been available in vivo.

Four-dimensional visualization of subpleural alveolar dynamics in vivo during uninterrupted mechanical ventilation of living swine.

Pulmonary alveoli have been studied for many years, yet no unifying hypothesis exists for their dynamic mechanics during respiration due to their miniature size (100-300 μm dimater in humans) and constant motion, which prevent standard imaging techniques from visualizing four-dimensional dynamics of individual alveoli in vivo. Here we report a new platform to image the first layer of air-filled subpleural alveoli through the use of a lightweight optical frequency domain imaging (OFDI) probe that can be placed upon the pleura to move with the lung over the complete range of respiratory motion. This device enables in-vivo acquisition of four-dimensional microscopic images of alveolar airspaces (alveoli and ducts), within the same field of view, during continuous ventilation without restricting the motion or modifying the structure of the alveoli. Results from an exploratory study including three live swine suggest that subpleural alveolar air spaces are best fit with a uniform expansion (r (2) = 0.98) over a recruitment model (r (2) = 0.72). Simultaneously, however, the percentage change in volume shows heterogeneous alveolar expansion within just a 1 mm x 1 mm field of view. These results signify the importance of four-dimensional imaging tools, such as the device presented here. Quantification of the dynamic response of the lung during ventilation may help create more accurate modeling techniques and move toward a more complete understanding of alveolar mechanics.

Evaluation of optical reflectance techniques for imaging of alveolar structure

Abstract. Three-dimensional (3-D) visualization of the fine structures within the lung parenchyma could advance our understanding of alveolar physiology and pathophysiology. Current knowledge has been primarily based on histology, but it is a destructive two-dimensional (2-D) technique that is limited by tissue processing artifacts. Micro-CT provides high-resolution three-dimensional (3-D) imaging within a limited sample size, but is not applicable to intact lungs from larger animals or humans. Optical reflectance techniques offer the promise to visualize alveolar regions of the large animal or human lung with sub-cellular resolution in three dimensions. Here, we present the capabilities of three optical reflectance techniques, namely optical frequency domain imaging, spectrally encoded confocal microscopy, and full field optical coherence microscopy, to visualize both gross architecture as well as cellular detail in fixed, phosphate buffered saline-immersed rat lung tissue. Images from all techniques were correlated to each other and then to corresponding histology. Spatial and temporal resolution, imaging depth, and suitability for in vivo probe development were compared to highlight the merits and limitations of each technology for studying respiratory physiology at the alveolar level.

Reflectance microscopy techniques for 3D imaging of the alveolar structure

Lung disease involving the alveoli and distal bronchioles are poorly understood and most commonly studied indirectly via lung function tests. Available imaging tools for the non-destructive assessment of the alveolar structure include X-ray computed tomography, intra-vital fluorescence microscopy and Optical Coherence Tomography, which are either limited by long acquisition time, inadequate resolution and contrast, or shallow imaging depth. In this study, we investigated the potential of two high-resolution reflectance microscopy imaging techniques, Spectrally Encoded Confocal Microscopy (SECM; 1µm (x) x 1µm (y) x 5µm (z) resolution) and Full Field Optical Coherence Microscopy (FFOCM; 1µm (x) x 1µm (y) x 1µm (z) resolution), for imaging alveolar microstructural detail. Two mouse lung samples were imaged with both SECM and FFOCM. The specimens were inflation-fixed using a modified Heitzman fixation technique at 20 cm H2O pressure. They were cut in 500mm thick slices and water immersed for imaging. Images were obtained and analyzed to determine whether or not the resolution and contrast of these techniques are sufficient to visualize the fine structures of the alveolar wall. Alveolar microstructure could be resolved in three dimensions in images obtained by both technologies. Alveolar septal walls from multiple layers could be clearly identified while sub-cellular structures such as nuclei were also visible in the SECM technique. In conclusion, we have demonstrated that two imaging technologies provide important sub-cellular detail that is required to study alveolar microstructure. Future research to develop these imaging modalities further so that they may be used in vivo is merited.

Validation of two-dimensional and three-dimensional measurements of subpleural alveolar size parameters by optical coherence tomography

Abstract. Optical coherence tomography (OCT) has been increasingly used for imaging pulmonary alveoli. Only a few studies, however, have quantified individual alveolar areas, and the validity of alveolar volumes represented within OCT images has not been shown. To validate quantitative measurements of alveoli from OCT images, we compared the cross-sectional area, perimeter, volume, and surface area of matched subpleural alveoli from microcomputed tomography (micro-CT) and OCT images of fixed air-filled swine samples. The relative change in size between different alveoli was extremely well correlated (r>0.9, P<0.0001), but OCT images underestimated absolute sizes compared to micro-CT by 27% (area), 7% (perimeter), 46% (volume), and 25% (surface area) on average. We hypothesized that the differences resulted from refraction at the tissue–air interfaces and developed a ray-tracing model that approximates the reconstructed alveolar size within OCT images. Using this model and OCT measurements of the refractive index for lung tissue (1.41 for fresh, 1.53 for fixed), we derived equations to obtain absolute size measurements of superellipse and circular alveoli with the use of predictive correction factors. These methods and results should enable the quantification of alveolar sizes from OCT images in vivo.

High-speed three-dimensional imaging of the pulmonary alveoli

Investigating the structure and function of pulmonary alveoli in vivo is crucial for understanding the normal and diseased lung. In particular, understanding the three-dimensional geometry and relationship of the terminal alveoli to their neighboring alveoli, alveolar ducts and acini during respiration would be a major advance. However, the lung is an inherently difficult organ to image in vivo and the peripheral lung has many compounding challenges not limited to its highly scattering micro architecture, large motion artifacts and difficult access through the bronchial tree. In this study, we image the alveoli of fixed pig lungs using a high-speed high-resolution optical frequency domain imaging (OFDI) system that is endoscopically compatible for future in vivo imaging of human alveoli. Core imaging components include a rapidly swept wavelength source centered at 1310nm resulting in an A-line depth scan rate of 62,500Hz, a polarization diverse dual balanced receiver, and a high speed data acquisition system. Whole lungs were excised from normal piglets and inflation fixed at 15cm H2O pressure using a modified Heitzman fixation technique. Lungs were air dried in a heated oven and sectioned into 500µm slices. Three-dimensional datasets were acquired from lung slices with 512x512x1024 voxels and a voxel dimension of 5x5x8µm. Datasets were acquired at 122 frames per second and 0.23 volumes per second - indicating the potential to acquire a three-dimensional volume within a single human respiratory cycle. OFDI images reveal clear delineation of alveolar septal walls, demonstrating that high-speed three-dimensional visualization of air filled alveoli is feasible. The fixed lung data provides a strong foundation for investigating the 3D structure and function of alveoli in vivo and suggests great promise for advancing our knowledge of the functional unit of the lung.

Flexible micro-OCT endobronchial probe for imaging of mucociliary transport (Conference Presentation)

Mucociliary clearance (MCC) plays a significant role in maintaining the health of human respiratory system by eliminating foreign particles trapped within mucus. Failure of this mechanism in diseases such as cystic fibrosis and chronic obstructive pulmonary disease (COPD) leads to airway blockage and lung infection, causing morbidity and mortality. The volume of airway mucus and the periciliary liquid encapsulating the cilia, in addition to ciliary beat frequency and velocity of mucociliary transport, are vital parameters of airway health. However, the diagnosis of disease pathogenesis and advances of novel therapeutics are hindered by the lack of tools for visualization of ciliary function in vivo. Our laboratory has previously developed a 1-µm resolution optical coherence tomography method, termed Micro-OCT, which is capable of visualizing mucociliary transport and quantitatively capturing epithelial functional metrics. We have also miniaturized Micro-OCT optics in a first-generation rigid 4mm Micro-OCT endoscope utilizing a common-path design and an apodizing prism configuration to produce an annular profile sample beam, and reported the first in vivo visualization of mucociliary transport in swine. We now demonstrate a flexible 2.5 mm Micro-OCT probe that can be inserted through the instrument channel of standard flexible bronchoscopes, allowing bronchoscopic navigation to smaller airways and greatly improving clinical utility. Longitudinal scanning over a field of view of more than 400 µm at a frame rate of 40 Hz was accomplished with a driveshaft transduced by a piezo-electric stack motor. We present characterization and imaging results from the flexible micro-OCT probe and progress towards clinical translation. The ability of the bronchoscope-compatible micro-OCT probe to image mucus clearance and epithelial function will enable studies of cystic fibrosis pathogenesis in small airways, provide diagnosis of mucociliary clearance disorders, and allow individual responses to treatments to be monitored.