Dual-beam manually actuated distortion-corrected imaging (DMDI): two dimensional scanning with a single-axis galvanometer.

We recently demonstrated a new two-dimensional imaging paradigm called dual-beam manually actuated distortion-corrected imaging (DMDI). This technique uses a single mechanical scanner and two spatially separated beams to determine relative sample velocity and simultaneously corrects image distortions due to manual actuation. DMDI was first demonstrated using a rotating dual-beam micromotor catheter. Here, we present a new implementation of DMDI using a single axis galvanometer to scan a pair of beams in approximately parallel lines onto a sample. Furthermore, we present a method for automated distortion correction based on frame co-registration between images acquired by the two beams. Distortion correction is possible for manually actuated motion both perpendicular and parallel to the galvanometer-scanned lines. Using en face OCT as the imaging modality, we demonstrate DMDI and the automated distortion correction algorithm for imaging a printed paper phantom, a dragon fruit, and a fingerprint.

Dual-beam manually-actuated distortion-corrected imaging (DMDI) with micromotor catheters.

We present a new paradigm for performing two-dimensional scanning called dual-beam manually-actuated distortion-corrected imaging (DMDI). DMDI operates by imaging the same object with two spatially-separated beams that are being mechanically scanned rapidly in one dimension with slower manual actuation along a second dimension. Registration of common features between the two imaging channels allows remapping of the images to correct for distortions due to manual actuation. We demonstrate DMDI using a 4.7 mm OD rotationally scanning dual-beam micromotor catheter (DBMC). The DBMC requires a simple, one-time calibration of the beam paths by imaging a patterned phantom. DMDI allows for distortion correction of non-uniform axial speed and rotational motion of the DBMC. We show the utility of this technique by demonstrating en face OCT image distortion correction of a manually-scanned checkerboard phantom and fingerprint scan.

Endoscopic high-resolution autofluorescence imaging and OCT of pulmonary vascular networks.

High-resolution imaging from within airways may allow new methods for studying lung disease. In this work, we report an endoscopic imaging system capable of high-resolution autofluorescence imaging (AFI) and optical coherence tomography (OCT) in peripheral airways using a 0.9 mm diameter double-clad fiber (DCF) catheter. In this system, AFI excitation light is coupled into the core of the DCF, enabling tightly focused excitation light while maintaining efficient collection of autofluorescence emission through the large diameter inner cladding of the DCF. We demonstrate the ability of this imaging system to visualize pulmonary vasculature as small as 12 µm in vivo.

Real-time visualization of melanin granules in normal human skin using combined multiphoton and reflectance confocal microscopy.

BACKGROUND: Recent advances in biomedical optics have enabled dermal and epidermal components to be visualized at subcellular resolution and assessed noninvasively. Multiphoton microscopy (MPM) and reflectance confocal microscopy (RCM) are noninvasive imaging modalities that have demonstrated promising results in imaging skin micromorphology, and which provide complementary information regarding skin components. This study assesses whether combined MPM/RCM can visualize intracellular and extracellular melanin granules in the epidermis and dermis of normal human skin. METHODS: We perform MPM and RCM imaging of in vivo and ex vivo skin in the infrared domain. The inherent three-dimensional optical sectioning capability of MPM/RCM is used to image high-contrast granular features across skin depths ranging from 50 to 90 µm. The optical images thus obtained were correlated with conventional histologic examination including melanin-specific staining of ex vivo specimens. RESULTS: MPM revealed highly fluorescent granular structures below the dermal-epidermal junction (DEJ) region. Histochemical staining also demonstrated melanin-containing granules that correlate well in size and location with the granular fluorescent structures observed in MPM. Furthermore, the MPM fluorescence excitation wavelength and RCM reflectance of cell culture-derived melanin were equivalent to those of the granules. CONCLUSION: This study suggests that MPM can noninvasively visualize and quantify subepidermal melanin in situ.

High speed, wide velocity dynamic range Doppler optical coherence tomography (Part IV): split spectrum processing in rotary catheter probes.

We report a technique for blood flow detection using split spectrum Doppler optical coherence tomography (ssDOCT) that shows improved sensitivity over existing Doppler OCT methods. In ssDOCT, the Doppler signal is averaged over multiple sub-bands of the interferogram, increasing the SNR of the Doppler signal. We explore the parameterization of this technique in terms of number of sub-band windows, width and overlap of the windows, and their effect on the Doppler signal to noise in a flow phantom. Compared to conventional DOCT, ssDOCT processing has increased flow sensitivity. We demonstrate the effectiveness of ssDOCT in-vivo for intravascular flow detection within a porcine carotid artery and for microvascular vessel detection in human pulmonary imaging, using rotary catheter probes. To our knowledge, this is the first report of visualizing in-vivo Doppler flow patterns adjacent to stent struts in the carotid artery.

Fiber-optic polarization diversity detection for rotary probe optical coherence tomography.

We report a polarization diversity detection scheme for optical coherence tomography with a new, custom, miniaturized fiber coupler with single mode (SM) fiber inputs and polarization maintaining (PM) fiber outputs. The SM fiber inputs obviate matching the optical lengths of the X and Y OCT polarization channels prior to interference and the PM fiber outputs ensure defined X and Y axes after interference. Advantages for this scheme include easier alignment, lower cost, and easier miniaturization compared to designs with free-space bulk optical components. We demonstrate the utility of the detection system to mitigate the effects of rapidly changing polarization states when imaging with rotating fiber optic probes in Intralipid suspension and during in vivo imaging of human airways.

Frontiers in bronchoscopic imaging.

Bronchoscopy is a minimally invasive method for diagnosis of diseases of the airways and the lung parenchyma. Standard bronchoscopy uses the reflectance/scattering properties of white light from tissue to examine the macroscopic appearance of airways. It does not exploit the full spectrum of the optical properties of bronchial tissues. Advances in optical imaging such as optical coherence tomography (OCT), confocal endomicroscopy, autofluorescence imaging and laser Raman spectroscopy are at the forefront to allow in vivo high-resolution probing of the microscopic structure, biochemical compositions and even molecular alterations in disease states. OCT can visualize cellular and extracellular structures at and below the tissue surface with near histological resolution, as well as to provide three-dimensional imaging of the airways. Cellular and subcellular imaging can be achieved using confocal endomicroscopy or endocytoscopy. Contrast associated with light absorption by haemoglobin can be used to highlight changes in microvascular structures in the subepithelium using narrow-band imaging. Blood vessels in the peribronchial space can be displayed using Doppler OCT. Biochemical compositions can be analysed with laser Raman spectroscopy, autofluorescence or multispectral imaging. Clinically, autofluorescence and narrow-band imaging have been found to be useful for localization of preneoplastic and neoplastic bronchial lesions. OCT can differentiate carcinoma in situ versus microinvasive cancer. Endoscopic optical imaging is a promising technology that can expand the horizon for studying the pathogenesis and progression of airway diseases such as COPD and asthma, as well as to evaluate the effect of novel therapy.

Differentiation of HaCaT cell and melanocyte from their malignant counterparts using micro-Raman spectroscopy guided by confocal imaging.

BACKGROUND: Skin cancer is the most common type of cancer in humans. Current techniques for identifying normal and neoplastic tissues are either destructive or not sensitive and specific enough. Raman spectroscopy and confocal imaging may obviate many limitations of existing methods by providing noninvasive, high-resolution, and real-time morphological and biochemical analysis of living tissues and cells. METHODS: We conducted micro-Raman spectroscopy studies on HaCaT cells, melanocytes (MC) and their malignant counterparts squamous cell carcinoma (SCC) and melanoma (MM) cells, respectively. Reflectance confocal imaging is used as guidance for the spectral measurements. RESULTS: Significant differences were found between the spectra of HaCaT cells and SCC cells, MC cells and MM cells, as well as all normal cells (HaCaT and MC) and all tumor cells (SCC and MM). Approximately 90% sensitivity and specificity was achieved for all the separations that we performed. CONCLUSION: Our results demonstrated the robust capability of confocal Raman spectroscopy in separating different cell lines. The acquired Raman spectra of major types of skin cells and their malignant counterparts will be useful for the interpretation of Raman spectra from in vivo skin. We believe it will eventually help diagnosis of skin cancer and other skin disease in clinical dermatology.

In vivo video rate multiphoton microscopy imaging of human skin.

We present a multiphoton microscopy instrument specially designed for in vivo dermatological use that is capable of imaging human skin at 27 frames per second with 256 pixels × 256 pixels resolution without the use of exogenous contrast agents. Imaging at fast frame rates is critical to reducing image blurring due to patient motion and to providing practically short clinical measurement times. Second harmonic generation and two-photon fluorescence images and videos acquired at optimized wavelengths are presented showing cellular and tissue structures from the skin surface down to the reticular dermis.

Small airway dilation measured by endoscopic optical coherence tomography correlates with chronic lung allograft dysfunction.

SIGNIFICANCE: Chronic lung allograft dysfunction (CLAD) is the leading cause of death in transplant patients who survive past the first year post-transplant. Current diagnosis is based on sustained decline in lung function; there is a need for tools that can identify CLAD onset. AIM: Endoscopic optical coherence tomography (OCT) can visualize structural changes in the small airways, which are of interest in CLAD progression. We aim to identify OCT features in the small airways of lung allografts that correlate with CLAD status. APPROACH: Imaging was conducted with an endoscopic rotary pullback OCT catheter during routine bronchoscopy procedures (n = 54), collecting volumetric scans of three segmental airways per patient. Six features of interest were identified, and four blinded raters scored the dataset on the presence and intensity of each feature. RESULTS: Airway dilation (AD) was the only feature found to significantly (p < 0.003) correlate with CLAD diagnosis (R = 0.40 to 0.61). AD could also be fairly consistently scored between raters (κinter-rater = 0.48, κintra-rater = 0.64). There is a stronger relationship between AD and the combined obstructive and restrictive (BOS + RAS) phenotypes than the obstructive-only (BOS) phenotype for two raters (R = 0.92 , 0.94). CONCLUSIONS: OCT examination of small AD shows potential as a diagnostic indicator for CLAD and CLAD phenotype and merits further exploration.

Fiber optic endoscopic optical coherence tomography (OCT) to assess human airways: The relationship between anatomy and physiological function during dynamic exercise.

Airway luminal area (Ai ) influences respiratory mechanics during dynamic exercise; however, previous studies have investigated the relationship between airway anatomy and physiological function in different groups of individuals. The purpose of this study was to determine the effect of Ai on respiratory mechanics by making in vivo measures of airway dimensions and work of breathing (Wb) in the same individuals. Healthy participants (3F/2M; 23-45 years) completed a cycle exercise test to exhaustion. During exercise, Wb was assessed using an esophageal balloon catheter, while simultaneously assessing minute ventilation ( V˙E ). On a separate day, subjects underwent a bronchoscopy procedure to capture optical coherence tomography (OCT) measures of three airways in the right lung. Each participant's Wb- V˙E data were fit to a non-linear regression equation (Wb = a V˙E3  + b V˙E2 ) that partitions Wb into its turbulent resistive (a) and viscoelastic (b) components. Measures of Ai and luminal diameter were made for the 4th-6th airway generations. A composite index of airway size was calculated as the sum of the Ai for each generation and the total area of the 4th-6th generation was calculated based on Weibel's model. Constant a was significantly correlated to the Weibel model total airway area (r = -0.94, p = 0.017) and index of airway size (r = -0.929, p = 0.023), whereas constant b was not associated with either measure (both p > 0.05). We found that individuals who had the smallest Ai had the highest resistive Wb and our findings provide the basis for further study of the relationship between airway size and respiratory mechanics during exercise.

Depth-multiplexed optical coherence tomography dual-beam manually-actuated distortion-corrected imaging (DMDI) with a micromotor catheter.

We present a new micromotor catheter implementation of dual-beam manually-actuated distortion-corrected imaging (DMDI). The new catheter called a depth-multiplexed dual-beam micromotor catheter, or mDBMC, maintains the primary advantage of unlimited field-of-view distortion-corrected imaging along the catheter axis. The mDBMC uses a polarization beam splitter and cube mirror to create two beams that scan circularly with approximately constant separation at the catheter surface. This arrangement also multiplexes both imaging channels into a single optical coherence tomography channel by offsetting them in depth, requiring half the data bandwidth compared to previous DMDI demonstrations that used two parallel image acquisition systems. Furthermore, the relatively simple scanning pattern of the two beams enables a straightforward automated distortion correction algorithm. We demonstrate the imaging capabilities of this catheter with a printed paper phantom and in a section of dragon fruit.

Correction of motion artifacts in endoscopic optical coherence tomography and autofluorescence images based on azimuthal en face image registration.

We present a method for the correction of motion artifacts present in two- and three-dimensional in vivo endoscopic images produced by rotary-pullback catheters. This method can correct for cardiac/breathing-based motion artifacts and catheter-based motion artifacts such as nonuniform rotational distortion (NURD). This method assumes that en face tissue imaging contains slowly varying structures that are roughly parallel to the pullback axis. The method reduces motion artifacts using a dynamic time warping solution through a cost matrix that measures similarities between adjacent frames in en face images. We optimize and demonstrate the suitability of this method using a real and simulated NURD phantom and in vivo endoscopic pulmonary optical coherence tomography and autofluorescence images. Qualitative and quantitative evaluations of the method show an enhancement of the image quality.

Wide-field in vivo oral OCT imaging.

We have built a polarization-sensitive swept source Optical Coherence Tomography (OCT) instrument capable of wide-field in vivo imaging in the oral cavity. This instrument uses a hand-held side-looking fiber-optic rotary pullback catheter that can cover two dimensional tissue imaging fields approximately 2.5 mm wide by up to 90 mm length in a single image acquisition. The catheter spins at 100 Hz with pullback speeds up to 15 mm/s allowing imaging of areas up to 225 mm(2) field-of-view in seconds. A catheter sheath and two optional catheter sheath holders have been designed to allow imaging at all locations within the oral cavity. Image quality of 2-dimensional image slices through the data can be greatly enhanced by averaging over the orthogonal dimension to reduce speckle. Initial in vivo imaging results reveal a wide-field view of features such as epithelial thickness and continuity of the basement membrane that may be useful in clinic for chair-side management of oral lesions.

Reproducibility of optical coherence tomography airway imaging.

Optical coherence tomography (OCT) is a promising imaging technique to evaluate small airway remodeling. However, the short-term insertion-reinsertion reproducibility of OCT for evaluating the same bronchial pathway has yet to be established. We evaluated 74 OCT data sets from 38 current or former smokers twice within a single imaging session. Although the overall insertion-reinsertion airway wall thickness (WT) measurement coefficient of variation (CV) was moderate at 12%, much of the variability between repeat imaging was attributed to the observer; CV for repeated measurements of the same airway (intra-observer CV) was 9%. Therefore, reproducibility may be improved by introduction of automated analysis approaches suggesting that OCT has potential to be an in-vivo method for evaluating airway remodeling in future longitudinal and intervention studies.

Endoscopic Doppler optical coherence tomography and autofluorescence imaging of peripheral pulmonary nodules and vasculature.

We present the first endoscopic Doppler optical coherence tomography and co-registered autofluorescence imaging (DOCT-AFI) of peripheral pulmonary nodules and vascular networks in vivo using a small 0.9 mm diameter catheter. Using exemplary images from volumetric data sets collected from 31 patients during flexible bronchoscopy, we demonstrate how DOCT and AFI offer complementary information that may increase the ability to locate and characterize pulmonary nodules. AFI offers a sensitive visual presentation for the rapid identification of suspicious airway sites, while co-registered OCT provides detailed structural information to assess the airway morphology. We demonstrate the ability of AFI to visualize vascular networks in vivo and validate this finding using Doppler and structural OCT. Given the advantages of higher resolution, smaller probe size, and ability to visualize vasculature, DOCT-AFI has the potential to increase diagnostic accuracy and minimize bleeding to guide biopsy of pulmonary nodules compared to radial endobronchial ultrasound, the current standard of care.

Coregistered autofluorescence-optical coherence tomography imaging of human lung sections.

Autofluorescence (AF) imaging can provide valuable information about the structural and metabolic state of tissue that can be useful for elucidating physiological and pathological processes. Optical coherence tomography (OCT) provides high resolution detailed information about tissue morphology. We present coregistered AF-OCT imaging of human lung sections. Adjacent hematoxylin and eosin stained histological sections are used to identify tissue structures observed in the OCT images. Segmentation of these structures in the OCT images allowed determination of relative AF intensities of human lung components. Since the AF imaging was performed on tissue sections perpendicular to the airway axis, the results show the AF signal originating from the airway wall components free from the effects of scattering and absorption by overlying layers as is the case during endoscopic imaging. Cartilage and dense connective tissue (DCT) are found to be the dominant fluorescing components with the average cartilage AF intensity about four times greater than that of DCT. The epithelium, lamina propria, and loose connective tissue near basement membrane generate an order of magnitude smaller AF signal than the cartilage fluorescence.

A high-efficiency fiber-based imaging system for co-registered autofluorescence and optical coherence tomography.

We present a power-efficient fiber-based imaging system capable of co-registered autofluorescence imaging and optical coherence tomography (AF/OCT). The system employs a custom fiber optic rotary joint (FORJ) with an embedded dichroic mirror to efficiently combine the OCT and AF pathways. This three-port wavelength multiplexing FORJ setup has a throughput of more than 83% for collected AF emission, significantly more efficient compared to previously reported fiber-based methods. A custom 900 µm diameter catheter ‒ consisting of a rotating lens assembly, double-clad fiber (DCF), and torque cable in a stationary plastic tube ‒ was fabricated to allow AF/OCT imaging of small airways in vivo. We demonstrate the performance of this system ex vivo in resected porcine airway specimens and in vivo in human on fingers, in the oral cavity, and in peripheral airways.

Imaging directed photothermolysis through two-photon absorption demonstrated on mouse skin - a potential novel tool for highly targeted skin treatment.

One-photon absorption based traditional laser treatment may not necessarily be selective at the microscopic level, thus could result in un-intended tissue damage. Our objective is to test whether two-photon absorption (TPA) could provide highly targeted tissue alteration of specific region of interest without damaging surrounding tissues. TPA based laser treatments (785 nm, 140 fs pulse width, 90 MHz) were performed on ex vivo mouse skin using different average power levels and irradiation times. Reflectance confocal microscopy (RCM) and combined second-harmonic-generation (SHG) and two-photon fluorescence (TPF) imaging channels were used to image before, during, and after each laser treatment. The skin was fixed, sectioned and H & E stained after each experiment for histological assessment of tissue alterations and for comparison with the non-invasive imaging assessments. Localized destruction of dermal fibers was observed without discernible epidermal damage on both RCM and SHG + TPF images for all the experiments. RCM and SHG + TPF images correlated well with conventional histological examination. This work demonstrated that TPA-based light treatment provides highly localized intradermal tissue alteration. With further studies on optimizing laser treatment parameters, this two-photon absorption photothermolysis method could potentially be applied in clinical dermatology.