Department of Biomedical Engineering Duke University Office: 2573 CIEMAS Deliveries: 136 Hudson Hall Durham, NC 27708 Phone: (919)660-5128; Fax: (919)613-9144 http://www.bme.duke.edu/faculty/izatt/index.php
Abstract: Recent advances in Doppler techniques have enabled high sensitivity imaging of biological flow to measure blood velocities and vascular perfusion. Here we compare spectrometer-based and wavelength-swept Doppler OCT implementations theoretically and experimentally, characterizing the lower and upper observable velocity limits in each configuration. We specifically characterize the washout limit for Doppler OCT, the velocity at which signal degradation results in loss of flow information, which is valid for both quantitative and qualitative flow imaging techniques. We also clearly differentiate the washout effect from the separate phenomenon of phase wrapping. We demonstrate that the maximum detectable Doppler velocity is determined by the fringe washout limit and not phase wrapping. Both theory and experimental results from phantom flow data and retinal blood flow data demonstrate the superiority of the swept-source technique for imaging vessels with high flow rates.
Abstract: Segmentation of anatomical structures in corneal images is crucial for the diagnosis and study of anterior segment diseases. However, manual segmentation is a time-consuming and subjective process. This paper presents an automatic approach for segmenting corneal layer boundaries in Spectral Domain Optical Coherence Tomography images using graph theory and dynamic programming. Our approach is robust to the low-SNR and different artifact types that can appear in clinical corneal images. We show that our method segments three corneal layer boundaries in normal adult eyes more accurately compared to an expert grader than a second grader-even in the presence of significant imaging outliers.
Abstract: PURPOSE: To determine the dynamic morphologic development of the human fovea in vivo using portable spectral domain-optical coherence tomography (SD-OCT). DESIGN: Prospective, observational case series. PATICIPANTS: Thirty-one prematurely born neonates, 9 children, and 9 adults. METHODS: Sixty-two neonates were enrolled in this study. After examination for retinopathy of prematurity (ROP), SD-OCT imaging was performed at the bedside in nonsedated infants aged 31 to 41 weeks postmenstrual age (PMA) (= gestational age in weeks + chronologic age) and at outpatient follow-up ophthalmic examinations. Thirty-one neonates met eligibility criteria. Nine children and nine adults without ocular pathology served as control groups. Semiautomatic retinal layer segmentation was performed. Central foveal thickness, foveal to parafoveal (FP) ratio (central foveal thickness divided by thickness 1000 μm from the foveal center), and 3-dimensional thickness maps were analyzed. MAIN OUTCOME MEASURES: In vivo determination of foveal morphology, layer segmentation, analysis of subcellular changes, and spatiotemporal layer shifting. RESULTS: In contrast with the adult fovea, several signs of immaturity were observed in the neonates: a shallow foveal pit, persistence of inner retinal layers (IRLs), and a thin photoreceptor layer (PRL) that was thinnest at the foveal center. Three-dimensional mapping showed displacement of retinal layers out of the foveal center as the fovea matured and the progressive formation of the inner/outer segment band in the opposite direction. The FP-IRL ratios decreased as IRL migrated before term and minimally after that, whereas FP-PRL ratios increased as PRL subcellular elements formed closer to term and into childhood. A surprising finding was the presence of cystoid macular edema in 58% of premature neonates that appeared to affect inner foveal maturation. CONCLUSIONS: This study provides the first view into the development of living cellular layers of the human retina and of subcellular specialization at the fovea in premature infant eyes using portable SD-OCT. Our work establishes a framework of the timeline of human foveal development, allowing us to identify unexpected retinal abnormalities that may provide new keys to disease activity and a method for mapping foveal structures from infancy to adulthood that may be integral in future studies of vision and visual cortex development. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.
Abstract: To demonstrate an operating microscope-mounted spectral domain optical coherence tomography (MMOCT) system for human retinal and model surgery imaging.
Abstract: Swept-source optical coherence tomography (SSOCT) provides a substantial sensitivity advantage over its time-domain counterpart, but suffers from a reduced imaging depth range due to sensitivity falloff and complex conjugate ambiguity. Heterodyne complex conjugate-resolved SSOCT (HCCR-SSOCT) has been previously demonstrated as a technique to completely resolve the complex conjugate ambiguity, effectively doubling the falloff limited imaging depth, without the reduction in imaging speed associated with other CCR techniques. However, previous implementations of this technique have employed expensive and lossy optical modulators to provide the required differential phase modulation. In this paper, we demonstrate the use of a dispersive optical delay line (D-ODL) as the reference arm of an OCT system to realize HCCR-SSOCT. This technique maintains the existing advantages of HCCR-SSOCT in that it completely resolves the complex conjugate artifact and does not reduce imaging speed, while conferring the additional advantages of being low cost, maintaining system sensitivity and resolution, not requiring any additional signal processing, and working at all wavelengths and imaging speeds. The D-ODL also allows for hardware correction of unbalanced dispersion in the reference and sample arm, adding further flexibility to system design. We demonstrate the technique using an SSOCT system operating at 100kHz with a central wavelength of 1040nm. Falloff measurements performed using a standard OCT configuration and the proposed D-ODL demonstrate a doubling of the effective imaging range with no sensitivity or resolution penalty. Feasibility of the technique for in vivo imaging was demonstrated by imaging the ocular anterior segments of healthy human volunteers.
Abstract: Spectral domain phase microscopy (SDPM) is an extension of spectral domain optical coherence tomography (SDOCT) that exploits the extraordinary phase stability of spectrometer-based systems with common-path geometry to resolve sub-wavelength displacements within a sample volume. This technique has been implemented for high resolution axial displacement and velocity measurements in biological samples, but since axial displacement information is acquired serially along the lateral dimension, it has been unable to measure fast temporal dynamics in extended samples. Depth-Encoded SDPM (DESDPM) uses multiple sample arms with unevenly spaced common path reference reflectors to multiplex independent SDPM signals from separate lateral positions on a sample simultaneously using a single interferometer, thereby reducing the time required to detect unique optical events to the integration period of the detector. Here, we introduce DESDPM and demonstrate the ability to acquire useful phase data concurrently at two laterally separated locations in a phantom sample as well as a biological preparation of spontaneously beating chick cardiomyocytes. DESDPM may be a useful tool for imaging fast cellular phenomena such as nervous conduction velocity or contractile motion.
Abstract: We present in vivo human fundus imaging using a fiber-based confocal scanning laser ophthalmoscope (SLO). Spectrally encoded confocal scanning laser ophthalmoscopy (SECSLO) utilizes a spectral encoding technique in one dimension, combined with single-axis lateral scanning, to create video-rate reflectivity maps of the fundus. This implementation of the SLO allows for high-contrast high-resolution in vivo human retinal imaging through a single-mode optical fiber. We experimentally quantify the full confocality of SECSLO in both the spectrally encoded and laterally scanned dimensions, and demonstrate 50 Hz frame rate fundus imaging.
Abstract: To describe age-related considerations and methods to improve hand-held spectral domain optical coherence tomography (HH-SD OCT) imaging of eyes of neonates, infants, and children.
Abstract: An important feature of tumor hypoxia is its temporal instability, or "cycling hypoxia." The primary consequence of cycling hypoxia is increased tumor aggressiveness and treatment resistance beyond that of chronic hypoxia. Longitudinal imaging of tumor metabolic demand, hemoglobin oxygen saturation, and blood flow would provide valuable insight into the mechanisms and distribution of cycling hypoxia in tumors. Fluorescence imaging of metabolic demand via the optical redox ratio (fluorescence intensity of FAD/NADH), absorption microscopy of hemoglobin oxygen saturation, and Doppler optical coherence tomography of vessel morphology and blood flow are combined to noninvasively monitor changes in oxygen supply and demand in the mouse dorsal skin fold window chamber tumor model (human squamous cell carcinoma) every 6 h for 36 h. Biomarkers for metabolic demand, blood oxygenation, and blood flow are all found to significantly change with time (p<0.05). These variations in oxygen supply and demand are superimposed on a significant (p<0.05) decline in metabolic demand with distance from the nearest vessel in tumors (this gradient was not observed in normal tissues). Significant (p<0.05), but weak (r<or=0.5) correlations are found between the hemoglobin oxygen saturation, blood flow, and redox ratio. These results indicate that cycling hypoxia depends on both oxygen supply and demand, and that noninvasive optical imaging could be a valuable tool to study therapeutic strategies to mitigate cycling hypoxia, thus increasing the effectiveness of radiation and chemotherapy.
Abstract: Spectral domain-optical coherence tomography (SD-OCT) may be useful for efficient measurement of drusen in patients with age-related macular degeneration (AMD). Areas identified as drusen from semiautomated segmentation of drusen on SD-OCT were compared to those identified from review of digital color fundus photographs (CFPs).
Abstract: Scanning laser ophthalmoscopy (SLO) and spectral domain optical coherence tomography (SDOCT) have become essential clinical diagnostic tools in ophthalmology by allowing for video-rate noninvasive en face and depth-resolved visualization of retinal structure. Current generation multimodal imaging systems that combine both SLO and OCT as a means of image tracking remain complex in their hardware implementations. Here, we combine a spectrally encoded confocal scanning laser ophthalmoscope (SECSLO) with an ophthalmic SDOCT system. This novel implementation of an interlaced SECSLO-SDOCT system allows for video-rate SLO fundus images to be acquired alternately with high-resolution SDOCT B-scans as a means of image aiming, guidance, and registration as well as motion tracking. The system shares the illumination source, detection system, and scanning optics between both SLO and OCT as a method of providing a simple multimodal ophthalmic imaging system that can readily be implemented as a table-top or hand-held device.
Abstract: We demonstrate in vivo human retinal imaging using an intraoperative microscope-mounted optical coherence tomography system (MMOCT). Our optomechanical design adapts an Oculus Binocular Indirect Ophthalmo Microscope (BIOM3), suspended from a Leica ophthalmic surgical microscope, with spectral domain optical coherence tomography (SD-OCT) scanning and relay optics. The MMOCT enables wide-field noncontact real-time cross-sectional imaging of retinal structure, allowing for SD-OCT augmented intrasurgical microscopy for intraocular visualization. We experimentally quantify the axial and lateral resolution of the MMOCT and demonstrate fundus imaging at a 20Hz frame rate.
Abstract: Segmentation of anatomical and pathological structures in ophthalmic images is crucial for the diagnosis and study of ocular diseases. However, manual segmentation is often a time-consuming and subjective process. This paper presents an automatic approach for segmenting retinal layers in Spectral Domain Optical Coherence Tomography images using graph theory and dynamic programming. Results show that this method accurately segments eight retinal layer boundaries in normal adult eyes more closely to an expert grader as compared to a second expert grader.
Abstract: Capable of three-dimensional imaging of the cornea with micrometer-scale resolution, spectral domain-optical coherence tomography (SDOCT) offers potential advantages over Placido ring and Scheimpflug photography based systems for accurate extraction of quantitative keratometric parameters. In this work, an SDOCT scanning protocol and motion correction algorithm were implemented to minimize the effects of patient motion during data acquisition. Procedures are described for correction of image data artifacts resulting from 3D refraction of SDOCT light in the cornea and from non-idealities of the scanning system geometry performed as a pre-requisite for accurate parameter extraction. Zernike polynomial 3D reconstruction and a recursive half searching algorithm (RHSA) were implemented to extract clinical keratometric parameters including anterior and posterior radii of curvature, central cornea optical power, central corneal thickness, and thickness maps of the cornea. Accuracy and repeatability of the extracted parameters obtained using a commercial 859nm SDOCT retinal imaging system with a corneal adapter were assessed using a rigid gas permeable (RGP) contact lens as a phantom target. Extraction of these parameters was performed in vivo in 3 patients and compared to commercial Placido topography and Scheimpflug photography systems. The repeatability of SDOCT central corneal power measured in vivo was 0.18 Diopters, and the difference observed between the systems averaged 0.1 Diopters between SDOCT and Scheimpflug photography, and 0.6 Diopters between SDOCT and Placido topography.
Abstract: The continuing improvement of high-speed area-scan cameras has made possible the construction of parallel optical coherence tomography (OCT) systems that are competitive with the fastest demonstrated swept-source OCT systems. Unfortunately, when imaging through turbid media using a partially coherent source, parallel OCT suffers resolution loss from coherent multiple scattering, a phenomenon known as crosstalk. We demonstrate the use of a full-field OCT system employing multimode fiber in the illumination arm to reduce the spatial coherence of a partially coherent source. By reducing the spatial coherence area below the system's lateral resolution, we create a spatial coherence gate that rejects these multiply scattered photons. We quantify the image quality and resolution improvement of this method by comparing images of a USAF test chart acquired beneath turbid phantoms using both coherent and incoherent illumination and computing the resulting modulation transfer functions. We demonstrate the feasibility of this method for imaging biological specimens by imaging a Drosophila melanogaster sample.
Abstract: To evaluate the spectrum of foveal architecture in pediatric albinism and to assess the utility of spectral-domain optical coherence tomography (OCT) in ocular imaging of children with nystagmus.
Abstract: A single-camera, high-speed, polarization-sensitive, spectral-domain optical-coherence-tomography system was developed to measure the polarization properties of the in vivo human retina. A novel phase-unwrapping method in birefringent media is described to extract the total reflectivity, accumulative retardance, and fast-axis orientation from a specially designed sequence of polarization states incident on the sample. A quarter-wave plate was employed to test the performance of the system. The average error and standard deviation of retardation measurements were 3.2 degrees and 2.3 degrees , respectively, and of the fast-axis orientation 1.2 degrees and 0.7 degrees over the range of 0 degrees -180 degrees . The depolarization properties of the retinal pigment epithelium were clearly observed in both retardance and fast-axis orientation image. A normalized standard deviation of the retardance and of the fast-axis orientation is introduced to segment the polarization-scrambling layer of the retinal pigment epithelium.
Abstract: To test in vivo whether spectral domain optical coherence tomography (SD-OCT) provides adequate resolution for reproducible measurement of photoreceptor (PR) layer at the margins of geographic atrophy (GA), and if it delineates the relationship between PR layer and retinal pigment epithelium at the margins of GA.
Abstract: We demonstrate in vivo velocity-resolved, volumetric bidirectional blood flow imaging in human retina using single-pass flow imaging spectral domain optical coherence tomography (SPFI-SDOCT). This technique uses previously described methods for separating moving and non-moving scatterers within a depth by using a modified Hilbert transform. Additionally, a moving spatial frequency window is applied, creating a stack of depth-resolved images of moving scatterers, each representing a finite velocity range. The resulting velocity reconstruction is validated with and strongly correlated to velocities measured with conventional Doppler OCT in flow phantoms. In vivo velocity-resolved flow mapping is acquired in healthy human retina and demonstrate the measurement of vessel size, peak velocity, and total foveal blood flow with OCT.
Abstract: Phase sensing implementations of spectral domain optical coherence tomography (SDOCT) have demonstrated the ability to measure nanometer-scale temporal and spatial profiles of samples. However, the phase information suffers from a 2pi ambiguity that limits observations of larger sample displacements to lengths less than half the source center wavelength. We introduce a synthetic wavelength phase unwrapping technique in SDOCT that uses spectral windowing and corrects the 2pi ambiguity, providing accurate measurements of sample motion with information gained from standard SDOCT processing. We demonstrate this technique by using a common path implementation of SDOCT and correctly measure phase profiles from a phantom phase object and human epithelial cheek cells which produce multiple wrapping artifacts. Using a synthetic wavelength for phase unwrapping could prove useful in Doppler or other phase based implementations of OCT.
Abstract: Detect changes in the neurosensory retina using spectral-domain optical coherence tomography (SD OCT) imaging over drusen in age-related macular degeneration (AMD). Quantitative imaging biomarkers may aid in defining risk of disease progression.
Abstract: To compare spectral domain optical coherence tomography (SDOCT) cross-sectional images of human central retina obtained from donor eyes with and without age-related macular degeneration (AMD) to corresponding histopathology from light micrographs. To establish the utility of SDOCT for localizing pathology in the posterior eyecup, for identifying ocular disease in donor eyes, or for directing subsequent sectioning of retinal lesions for research.
Abstract: We have combined hyperspectral imaging with spectral domain optical coherence tomography (SDOCT) to noninvasively image changes in hemoglobin saturation, blood flow, microvessel morphology, and sheer rate on the vessel wall with tumor growth. Changes in these hemodynamic variables were measured over 24 h in dorsal skin fold window chamber tumors. There was a strong correlation between volumetric flow and hemoglobin saturation (rho=0.89, p=9x10(-6), N=15) and a moderate correlation between shear rate on the vessel wall and hemoglobin saturation (rho=0.56, p=0.03, N=15).
Abstract: To delineate pathologic changes in retinal cross sections obtained with spectral (Fourier) domain optical coherence tomography (SDOCT), so that the findings are maintained when collapsed into a two-dimensional fundus image for comparison with conventional retinal studies.
Abstract: Progress toward understanding embryonic heart development has been hampered by the inability to image embryonic heart structure and simultaneously measure blood flow dynamics in vivo. We have developed a spectral domain optical coherence tomography system for in vivo volumetric imaging of the chicken embryo heart. We have also developed a technique called spectral Doppler velocimetry (SDV) for quantitative measurement of blood flow dynamics. We present in vivo volume images of the embryonic heart from initial tube formation to development of endocardial cushions of the same embryo over several stages of development. SDV measurements reveal the influence of heart tube structure on blood flow dynamics.
Abstract: We have demonstrated a novel Fourier-domain optical coherence tomography system and signal-processing algorithm for full-range, real-time, artifact-free quantitative imaging of the anterior chamber. Cross-sectional full-range images comprising 1024 x 800 pixels (axial x lateral) were acquired and displayed at 6.7 images/s. Volumetric data comprising 1024 x 400 x 60 pixels (axial x lateral x elevation) were acquired in 4.5 seconds with real-time visualization of individual slices and 3-dimensional reconstruction performed in postprocessing. Details of the cornea, limbus, iris, anterior lens capsule, trabecular meshwork, and Schlemm's canal were visualized. Quantitative surface height maps of the corneal epithelium and endothelium were obtained from the volumetric data and used to generate corneal thickness maps.
Abstract: Molecular imaging is a powerful tool for investigating disease processes and potential therapies in both in vivo and in vitro systems. However, high resolution molecular imaging has been limited to relatively shallow penetration depths that can be accessed with microscopy. Optical coherence tomography (OCT) is an optical analogue to ultrasound with relatively good penetration depth (1-2 mm) and resolution (approximately 1-10 microm). We have developed and characterized photothermal OCT as a molecular contrast mechanism that allows for high resolution molecular imaging at deeper penetration depths than microscopy. Our photothermal system consists of an amplitude-modulated heating beam that spatially overlaps with the focused spot of the sample arm of a spectral-domain OCT microscope. Validation experiments in tissuelike phantoms containing gold nanospheres that absorb at 532 nm revealed a sensitivity of 14 ppm nanospheres (weight/weight) in a tissuelike environment. The nanospheres were then conjugated to anti-EGFR, and molecular targeting was confirmed in cells that overexpress EGFR (MDA-MB-468) and cells that express low levels of EGFR (MDA-MB-435). Molecular imaging in three-dimensional tissue constructs was confirmed with a significantly lower photothermal signal (p<0.0001) from the constructs composed of cells that express low levels of EGFR compared to the overexpressing cell constructs (300% signal increase). This technique could potentially augment confocal and multiphoton microscopy as a method for deep-tissue, depth-resolved molecular imaging with relatively high resolution and target sensitivity, without photobleaching or cytotoxicity.
Abstract: We present in vivo human total retinal blood flow measurements using Doppler Fourier domain optical coherence tomography (OCT). The scan pattern consisted of two concentric circles around the optic nerve head, transecting all retinal branch arteries and veins. The relative positions of each blood vessel in the two OCT conic cross sections were measured and used to determine the angle between the OCT beam and the vessel. The measured angle and the Doppler shift profile were used to compute blood flow in the blood vessel. The flows in the branch veins was summed to give the total retinal blood flow at one time point. Each measurement of total retinal blood flow was completed within 2 s and averaged. The total retinal venous flow was measured in one eye each of two volunteers. The results were 52.90+/-2.75 and 45.23+/-3.18 microlmin, respectively. Volumetric flow rate positively correlated with vessel diameter. This new technique may be useful in the diagnosis and treatment of optic nerve and retinal diseases that are associated with poor blood flow, such as glaucoma and diabetic retinopathy.
Abstract: To categorize drusen ultrastructure in age-related macular degeneration (AMD) using spectral domain optical coherence tomography (SDOCT) and correlate the tomographic and photographic drusen appearances.
Abstract: We demonstrate in vivo volumetric bidirectional blood flow imaging in animal models using single-pass flow imaging spectral domain optical coherence tomography. This technique uses a modified Hilbert transform algorithm to separate moving and non-moving scatterers within a depth. The resulting reconstructed image maps the components of moving scatterers flowing into and out of the imaging axis onto opposite image half-planes, enabling volumetric bidirectional flow mapping without manual segmentation.
Abstract: Investigation of the autoregulatory mechanism of human retinal perfusion is conducted with a real-time spectral domain Doppler optical coherence tomography (SDOCT) system. Volumetric, time-sequential, and Doppler flow imaging are performed in the inferior arcade region on normal healthy subjects breathing normal room air and 100% oxygen. The real-time Doppler SDOCT system displays fully processed, high-resolution [512 (axial) x 1000 (lateral) pixels] B scans at 17 frames/sec in volumetric and time-sequential imaging modes, and also displays fully processed overlaid color Doppler flow images comprising 512 (axial) x 500 (lateral) pixels at 6 frames/sec. Data acquired following 5 min of 100% oxygen inhalation is compared with that acquired 5 min postinhalation for four healthy subjects. The average vessel constriction across the population is -16+/-26% after oxygen inhalation with a dilation of 36+/-54% after a return to room air. The flow decreases by -6+/-20% in response to oxygen and in turn increases by 21+/-28% as flow returns to normal in response to room air. These trends are in agreement with those previously reported using laser Doppler velocimetry to study retinal vessel autoregulation. Doppler flow repeatability data are presented to address the high standard deviations in the measurements.
Abstract: There is considerable interest in new methods for the assessment of retinal blood flow for the diagnosis of eye diseases. We present in vivo normal human volumetric retinal flow measurement using Fourier domain Doppler optical coherence tomography. We used a dual-plane scanning pattern to determine the angle between the blood flow and the scanning beam in order to measure total flow velocity. Volumetric flow in each blood vessel around the optic nerve head was integrated in one cardiac cycle in each measurement. Measurements were performed in the right eye of one human subject. The measured venous flow velocity ranged from 16.26 mm/s to 29.7 mm/s. The arterial flow velocity ranged from 38.35 mm/s to 51.13 mm/s. The total retinal venous and arterial flow both added up to approximately 54 microl/min. We believe this is the first demonstration of total retinal blood flow measurement using the OCT technique.
Abstract: We report on cross-sectional imaging of dynamic biological specimens using a spectral domain phase microscopy (SDPM) system capable of operating at a line rate of 19 kHz. This system combines the time-sensitive capabilities of SDPM with the multi-point acquisition features of related phase-sensitive techniques. The presented phase portraits and B-scan phase images of spontaneously beating embryonic cardiomyocytes and cytoplasmic flow in A. proteus offer insight into the nature and timing of the observed cellular phenomena, demonstrating the utility of this technique for dynamic cell studies.
Abstract: We present spectral domain phase microscopy (SDPM) as a new tool for measurements at the cellular scale. SDPM is a functional extension of spectral domain optical coherence tomography that allows for the detection of cellular motions and dynamics with nanometer-scale sensitivity in real time. Our goal was to use SDPM to investigate the mechanical properties of the cytoskeleton of MCF-7 cells. Magnetic tweezers were designed to apply a vertical force to ligand-coated magnetic beads attached to integrin receptors on the cell surfaces. SDPM was used to resolve cell surface motions induced by the applied stresses. The cytoskeletal response to an applied force is shown for both normal cells and those with compromised actin networks due to treatment with Cytochalasin D. The cell response data were fit to several models for cytoskeletal rheology, including one- and two-exponential mechanical models, as well as a power law. Finally, we correlated displacement measurements to physical characteristics of individual cells to better compare properties across many cells, reducing the coefficient of variation of extracted model parameters by up to 50%.
Abstract: We present compensating methods that address inherent errors in quantitative phase reporting for low-coherence interferometric techniques. A brief theoretical treatment of the problem and experimental validation using spectral domain phase microscopy demonstrate mitigation of the degrading effects of phase leakage on accurate measurement of optical path length in the vicinity of closely spaced reflectors. This result has direct implications for phase-sensitive interferometry techniques, such as Doppler imaging, as well as amplitude-based quantitative reporting. Corrected phase retrieval is demonstrated for conversion of interferometric phase to optical path length in cell surface deflections of beating cardiomyocytes.
Abstract: We demonstrate high-speed complex conjugate artifact (CCA) resolved imaging of human retina in vivo using spectral domain optical coherence tomography. This technique utilizes sinusoidal reference mirror modulation to implement high-speed integrating buckets acquisition and a quadrature projection reconstruction algorithm in postprocessing. This method is illustrated experimentally using sets of four integrating bucket phase scans, acquired at 52 kHz, for DC suppression of 73 dB and complex conjugate suppression of 35 dB. Densely sampled (3000 A-scans/image, acquired at 4.3 images/s) full-depth in vivo images of optic nerve head show CCA suppression for most image reflections to the noise floor.
Abstract: We present a novel molecular imaging technique which combines the 3-D tomographic imaging capability of optical coherence tomography with the molecular sensitivity of pump-probe spectroscopy. This technique, based on transient absorption, is sensitive to any molecular chromophore. It is particularly promising for the many important biomarkers, such as hemoglobin, which are poor fluorophores and therefore difficult to image with current optical techniques without chemical labeling. Previous implementations of pump-probe optical coherence tomography have suffered from inefficient pump-probe schemes which hurt the sensitivity and applicability of the technique. Here we optimize the efficiency of the pump-probe approach by avoiding the steady-state kinetics and spontaneous processes exploited in the past in favor of measuring the transient absorption of fully allowed electronic transitions on very short time scales before a steady-state is achieved. In this article, we detail the optimization and characterization of the prototype system, comparing experimental results for the system sensitivity to theoretical predictions. We demonstrate in situ imaging of tissue samples with two different chromophores; the transfectable protein dsRed and the protein hemoglobin. We also demonstrate, with a simple sample vessel and a mixture of human whole blood and rhodamine 6G, the potential to use ground state recovery time to separate the contributions of multiple chromophores to the ground state recovery signal.
Abstract: We present a full-field phase microscopy technique for quantitative nanoscale surface profiling of samples in reflection. This technique utilizes swept-source optical coherence tomography in a full-field common path interferometer for phase-stable cross-sectional acquisition without scanning. Subwavelength variations in surface sample features are measured without interference from spurious reflections by processing the interferometric phase at a selected depth plane, providing a 1.3 nm stability for high signal-to-noise ratio surface features. Nanoscale imaging was demonstrated by measuring the location of receptor sites on a DNA assay biochip and the surface topography of erythrocytes in a blood smear.
Abstract: We present a novel algorithm for full-range imaging by suppression of the complex conjugate artifact in phase-shifting Fourier domain optical coherence tomography. This technique utilizes the projection of multiple phase-shifted interferograms onto an orthogonal basis set to reconstruct the complex interferogram. Full-range imaging with >30 dB suppression of the symmetric artifact is demonstrated using a 3 x 3 fiber coupler swept source OCT system, providing a depth range of 6.6mm with -8 dB roll-off in sensitivity at the depth boundaries relative to DC. Real-time display of full-range images of the anterior segment of the human eye acquired in vivo at a line rate of 6.67 kHz are presented.
Abstract: Spectral domain phase microscopy (SDPM) is a function extension of spectral domain optical coherence tomography. SDPM achieves exquisite levels of phase stability by employing common-path interferometry. We discuss the theory and limitations of Doppler flow imaging using SDPM, demonstrate monitoring the thermal contraction of a glass sample with nanometer per second velocity sensitivity, and apply this technique to measurement of cytoplasmic streaming in an Amoeba proteus pseudopod. We observe reversal of cytoplasmic flow induced by extracellular CaCl2, and report results that suggest parabolic flow of cytoplasm in the A. proteus pseudopod.
Abstract: Drosophila melanogaster genetics provides the advantage of molecularly defined P-element insertions and deletions that span the entire genome. Although Drosophila has been extensively used as a model system to study heart development, it has not been used to dissect the genetics of adult human heart disease because of an inability to phenotype the adult fly heart in vivo. Here we report the development of a strategy to measure cardiac function in awake adult Drosophila that opens the field of Drosophila genetics to the study of human dilated cardiomyopathies. Through the application of optical coherence tomography, we accurately distinguish between normal and abnormal cardiac function based on measurements of internal cardiac chamber dimensions in vivo. Normal Drosophila have a fractional shortening of 87 +/- 4%, whereas cardiomyopathic flies that contain a mutation in troponin I or tropomyosin show severe impairment of systolic function. To determine whether the fly can be used as a model system to recapitulate human dilated cardiomyopathy, we generated transgenic Drosophila with inducible cardiac expression of a mutant of human delta-sarcoglycan (deltasg(S151A)), which has previously been associated with familial dilated cardiomyopathy. Compared to transgenic flies overexpressing wild-type deltasg, or the standard laboratory strain w(1118), Drosophila expressing deltasg(S151A) developed marked impairment of systolic function and significantly enlarged cardiac chambers. These data illustrate the utility of Drosophila as a model system to study dilated cardiomyopathy and the applicability of the vast genetic resources available in Drosophila to systematically study the genetic mechanisms responsible for human cardiac disease.
Abstract: To assess the accuracy of classification of narrow anterior chamber (AC) angles using quantitative imaging by optical coherence tomography (OCT) and ultrasound biomicroscopy (UBM).
Abstract: The increased sensitivity of spectral domain optical coherence tomography (OCT) has driven the development of a new generation of technologies in OCT, including rapidly tunable, broad bandwidth swept laser sources and spectral domain OCT interferometer topologies. In this work, the operation of a turnkey 1300-nm swept laser source is demonstrated. This source has a fiber ring cavity with a semiconductor optical amplifier gain medium. Intracavity mode selection is achieved with an in-fiber tunable fiber Fabry-Perot filter. A novel optoelectronic technique that allows for even sampling of the swept source OCT signal in k space also is described. A differential swept source OCT system is presented, and images of in vivo human cornea and skin are presented. Lastly, the effects of analog-to-digital converter aliasing on image quality in swept source OCT are discussed.
Abstract: Optical coherence tomography (OCT), a noninvasive optical imaging technique, provides high-resolution cross-sectional images of tissue microstructure. We developed a system for real-time endoscopic OCT (EOCT) of the human GI tract. During clinical trials, the structure of mucosa and submucosa, glands, blood vessels, pits, villi, and crypts was observed in a range of GI organs. Although EOCT images are thought to accurately depict actual histologic features, there are few data to support this assumption. Therefore, the present study correlated images acquired with an EOCT imaging system in vitro to corresponding histologic sections.
Abstract: To measure anterior chamber (AC) width and other dimensions relevant to the sizing of phakic intraocular lenses (IOLs) with a high-speed optical coherence tomography (OCT) system.
Abstract: We report that the complex conjugate artifact in Fourier domain optical coherence tomography approaches (including spectral domain and swept source OCT) may be resolved by the use of novel interferometer designs based on 3x3 and higher order fiber couplers. Interferometers built from NxN (N>2) truly fused fiber couplers provide simultaneous access to non-complementary phase components of the complex interferometric signal. These phase components may be converted to quadrature components by trigonometric manipulation, then inverse Fourier transformed to obtain A-scans and images with resolved complex conjugate artifact. We demonstrate instantaneous complex conjugate resolved Fourier domain OCT using 3x3 couplers in both spectral domain and swept source implementations. Complex conjugate artifact suppression by factors of ~20dB and ~25dB are demonstrated for spectral domain and swept source implementations, respectively.
Abstract: Optical coherence tomography (OCT) provides micrometer-scale structural imaging by coherent detection of backscattered light. Molecular contrast in OCT has been demonstrated using transient absorption, coherent anti-Stokes Raman scattering, and second-harmonic (SH) generation. The sensitivity of molecular contrast signals can be enhanced by use of Fourier domain techniques. We have constructed a spectrometer-based Fourier domain SH-OCT system for simultaneous acquisition of the fundamental and SH signals. We report a >30 dB increase in SH sensitivity over a similar time domain SH-OCT system and demonstrate contrast between cartilage and bone using collagen as the contrast agent.
Abstract: Broadband interferometry is an attractive technique for the detection of cellular motions because it provides depth-resolved phase information via coherence gating. We present a phase-sensitive technique called spectral-domain phase microscopy (SDPM). SDPM is a functional extension of spectral-domain optical coherence tomography that allows for the detection of nanometer-scale motions in living cells. The sensitivity of the technique is demonstrated, and its calibration is verified. A shot-noise limit to the displacement sensitivity of this technique is derived. Measurement of cellular dynamics was performed on spontaneously beating cardiomyocytes isolated from chick embryos.
Abstract: We have combined Fourier-domain optical coherence tomography (FD-OCT) with a closed-loop adaptive optics (AO) system using a Hartmann-Shack wavefront sensor and a bimorph deformable mirror. The adaptive optics system measures and corrects the wavefront aberration of the human eye for improved lateral resolution (~4 mum) of retinal images, while maintaining the high axial resolution (~6 mum) of stand alone OCT. The AO-OCT instrument enables the three-dimensional (3D) visualization of different retinal structures in vivo with high 3D resolution (4x4x6 mum). Using this system, we have demonstrated the ability to image microscopic blood vessels and the cone photoreceptor mosaic.
Abstract: Fourier domain (FD) techniques have increasingly gained attention in optical coherence tomography (OCT). This is primarily due to their demonstrated sensitivity of two to three orders of magnitude over conventional time-domain techniques. FDOCT images are subject to two primary sources of artifacts. First, a complex conjugate ambiguity arises because the Fourier transform of the real-valued spectral interferometric signal is Hermitian symmetric. This ambiguity leads to artifactual superposition of reflectors at positive and negative pathlength differences between the sample and reference reflectors. Second, noninterferometric and sample autocorrelation terms appear at dc, obscuring reflectors at zero pathlength difference. We show that heterodyne detection in swept-source OCT (SSOCT) enables the resolution of complex conjugate ambiguity and the removal of noninterferometric and autocorrelation artifacts. We also show that complex conjugate ambiguity resolution via frequency shifting circumvents fall-off induced by finite source linewidth in SSOCT when samples are shifted to large pathlength differences. We describe an efficient heterodyne SSOCT design that enables compensation of power losses from frequency-shifting elements. Last, we demonstrate this technique, coupled with wavenumber triggering and electronic demodulation, for in vivo imaging of the human anterior eye segment.
Abstract: We report a new molecular contrast optical coherence tomography (MCOCT) implementation that profiles the contrast agent distribution in a sample by measuring the agent's spectral differential absorption. The method, spectra triangulation MCOCT, can effectively suppress contributions from spectrally dependent scatterings from the sample without a priori knowledge of the scattering properties. We demonstrate molecular imaging with this new MCOCT modality by mapping the distribution of indocyanine green, a FDA-approved infrared red dye, within a stage 54 Xenopus laevis.
Abstract: The group index of the cornea, rather than the phase refractive index, is required for thickness calculations with optical coherence tomography. Recent advances with high-speed optical coherence tomography at 1.3 microm make index measurement at this wavelength of great interest. Group indices of three human corneas from an eye bank were measured in vitro with optical coherence domain reflectometry. Measurements were made in a calibrated cuvette filled with a preservation medium to maintain proper corneal hydration. Group indices were calculated from the optical path lengths measured. The corneal group index was 1.389 +/- 0.004 (average +/- standard deviation). The average group index of a balanced salt solution, an approximation to aqueous humor, was 1.343 +/- 0.001.
Abstract: We report the use of phytochrome A (phyA), a plant protein that can reversibly switch between two states with different absorption maxima (at 660 and 730 nm), as a contrast agent for molecular contrast optical coherence tomography (MCOCT). Our MCOCT scheme builds up a difference image revealing the distribution of phyA within a target sample from pairs of consecutive OCT A-scans acquired at a probe wavelength of 750 nm, both with and without additional illumination of the target sample with 660-nm light. We demonstrate molecular imaging with this new MCOCT modality in a target sample containing a mixture of 0.2% Intralipid and 83 microM of phyA.
Abstract: We describe a novel imaging technique, second-harmonic-generation optical coherence tomography (SHOCT). This technique combines the spatial resolution and depth penetration of optical coherence tomography (OCT) with the molecular sensitivity of second-harmonic-generation spectroscopy. As a consequence of the coherent detection required for OCT, polarization-resolved images arise naturally. We demonstrate this new technique on a skin sample from the belly of Icelandic salmon, acquiring polarization-resolved SHOCT and OCT images simultaneously.
Abstract: Endoscopic optical coherence tomography provides images of the GI mucosa and submucosa in microscopic detail. It is unknown whether endoscopic optical coherence tomography can reliably detect dysplasia. Colon polyps were used as a model to determine whether dysplasia in GI tissue has characteristic optical coherence tomography imaging features.
Abstract: Compact electrostatic micromirror structures for use in the scanning arm of an optical coherence tomography (OCT) system are described. These devices consist of millimeter-scale mirrors resting upon micrometer-scale polyimide hinges that are tilted by a linear micromachine actuator, the integrated force array (IFA). The IFA is a network of deformable capacitor cells that electrostatically contract with an applied voltage. The support structures, hinges, and actuators are fabricated by photolithography from polyimide-upon-silicon wafers. These devices were inserted into the scanning arm of an experimental OCT imaging system to produce in vitro and in vivo images at frame rates of 4 to 8 Hz.
Abstract: We describe a novel technique for contrast enhancement in optical coherence tomography (OCT) that makes possible molecular-specific imaging for what is believed to be the first time. A pump-probe technique is employed in which a pulsed pump laser is tuned to ground-state absorption in a molecule of interest. The location of the target molecule population is derived from the resulting transient absorption of OCT sample-arm light acting as probe light. A signal processing technique for three-dimensional localization of the transient absorption signal is described, and preliminary results exhibiting OCT contrast from methylene blue dye in multilayer and scattering phantoms are presented.
Abstract: Color Doppler optical coherence tomography (CDOCT) combines laser Doppler velocimetry and optical coherence tomography for simultaneous micron-scale resolution cross-sectional imaging of tissue microstructure and blood flow. Recently, CDOCT was adapted to a slitlamp biomicroscope for imaging structure and blood flow in the human retina.
Abstract: We present a novel method for obtaining depth-resolved spectra for determining scatterer size through elastic-scattering properties. Depth resolution is achieved with a white-light source in a Michelson interferometer with the mixed signal and reference fields dispersed by a spectrograph. The spectrum is Fourier transformed to yield the axial spatial cross correlation between the signal and reference fields with near 1-microm depth resolution. Spectral information is obtained by windowing to yield the scattering amplitude as a function of wave number. The technique is demonstrated by determination of the size of polystyrene microspheres in a subsurface layer with subwavelength accuracy. Application of the technique to probing the size of cell nuclei in living epithelial tissues is discussed.
Abstract: We describe fiber-based quadrature low-coherence interferometers that exploit the inherent phase shifts of 3 x 3 and higher-order fiber-optic couplers. We present a framework based on conservation of energy to account for the interferometric shifts in 3 x 3 interferometers, and we demonstrate that the resulting interferometers provide the entire complex interferometric signal instantaneously in homodyne and heterodyne systems. In heterodyne detection we demonstrate the capability for extraction of the magnitude and sign of Doppler shifts from the complex data. In homodyne detection we show the detection of subwavelength sample motion. N x N (N > 2) low-coherence interferometer topologies will be useful in Doppler optical coherence tomography (OCT), optical coherence microscopy, Fourier-domain OCT, optical frequency domain reflectometry, and phase-referenced interferometry.
Abstract: Color Doppler optical coherence tomography (CDOCT) is a functional extension of optical coherence tomography (OCT) that can image flow in turbid media. We have developed a CDOCT system capable of imaging flow in real time. Doppler processing of the analog signal is accomplished in hardware in the time domain using a novel autocorrelation technique. This Doppler processing method is compatible with a high speed OCT system capable of imaging in real time. Using this system, we demonstrate cross-sectional imaging of bidirectional flow with CDOCT at four frames per second in a tissue-simulating phantom consisting of intralipid solution flowing in glass capillaries. As a demonstration of real-time imaging of blood flow in vivo we imaged pulsatible blood flow in a rat femoral artery at eight frames per second. Issues of velocity sensitivity, imaging speed, and range of velocity measurement are discussed, as well as potential applications of real-time CDOCT.
Abstract: Color Doppler optical coherence tomography (CDOCT) is a noninvasive optical imaging technique for micrometer-scale physiological flow mapping simultaneously with morphological optical coherence tomography imaging. We have developed a novel CDOCT signal-processing strategy capable of imaging physiological flow rates at 8 frames/s. Our new strategy features hardware-implemented digital autocorrelation across subsequent scans, permitting us to measure 300-Hz-8-kHz Doppler shifts upon signals of 0.6-MHz bandwidth. The performance of the CDOCT system was demonstrated in a flow phantom and in vivo in Xenopus laevis.
Abstract: Optical coherence tomography (OCT) is a depth-resolved, noninvasive, non-destructive imaging modality, the use of which has yet to be fully realized in developmental biology.
Abstract: By using the Born approximation deconvolved inverse scattering method instead of the traditional pulse-echo method for analyzing ultrasound pulse reflections from plastic phantoms and soft tissue specimens, improvement in image resolution is shown to be possible provided these targets are fair approximations to layered media. These images are free of speckle and are more vivid than the usual pulse-echo images.
Abstract: Doppler optical coherence tomography (DOCT) allows simultaneous micrometer-scale resolution cross-sectional imaging of tissue structure and blood flow. We demonstrate a fiber-optic polarization-diversity-based differential phase contrast DOCT system as a method to perform self-referenced velocimetry in highly scattering media. Using this strategy, we reduced common-mode interferometer noise to <1 Hz and improved Doppler estimates in a scattering flow phantom by a factor of 5.
Abstract: Recent advances in high-speed scanning technology have enabled a new generation of optical coherence tomographic (OCT) systems to perform imaging at video rate. Here, a handheld OCT probe capable of imaging the anterior segment of the eye at high frame rates is demonstrated for the first time.
Abstract: Both optical coherence tomography (OCT) and catheter probe EUS (CPEUS) are candidates for high-resolution imaging of the GI wall, but their potential roles in this clinical context have not been investigated.
Abstract: We report a method for extracting the birefringence properties of biological samples with micrometer-scale resolution in three dimensions, using a new form of polarization-sensitive optical coherence tomography. The method measures net retardance, net fast axis, and total reflectivity as a function of depth into the sample. Polarization sensing is accomplished by illumination of the sample with at least three separate polarization states during consecutive acquisitions of the same pixel, A scan, or B scan. The method can be implemented by use of non-polarization-maintaining fiber and a single detector. In a calibration test of the system, net retardance was measured with an average error of 7.5 degrees (standard deviation 2.2 degrees ) over the retardance range 0 degrees to 180 degrees , and a fast axis with average error of 4.8 degrees over the range 0 degrees to 180 degrees .
Abstract: Optical-thermal models that can accurately predict temperature rise and damage in blood vessels and surrounding tissue may be used to improve the treatment of vascular disorders. Verification of these models has been hampered by the lack of time- and depth-resolved experimental data. In this preliminary study, an optical coherence tomography system operating at 4-30 frames per second was used to visualize laser irradiation of cutaneous (hamster dorsal skin flap) blood vessels. An argon laser was utilized with the following parameters: pulse duration 0.1-2.0 s, spot size 0.1-1.0 mm, power 100-400 mW. Video microscopy images were obtained before and after irradiations, and optical-thermal modelling was performed on two irradiation cases. Time-resolved optical coherence tomography and still images were compared with predictions of temperature rise and damage using Monte Carlo and finite difference techniques. In general, predicted damage agreed with the actual blood vessel and surrounding tissue coagulation seen in images. However, limitations of current optical-thermal models were identified, such as the inability to model the dynamic changes in blood vessel diameter that were seen in the optical coherence tomography images.
Abstract: Photodynamic therapy (PDT) is a novel cancer therapy that uses light-activated drugs (photosensitizers) to destroy tumor tissue. Reactive oxygen species produced during PDT are thought to cause the destruction of tumor tissue. However, the precise mechanism of PDT is not completely understood. To provide insight into the in vitro mechanisms of PDT, we studied the subcellular localization of the photosensitizer HOSiPcOSi(CH3)2-(CH2)3N(CH3)2 (Pc 4) in mouse lymphoma (LY-R) cells using double-label confocal fluorescence microscopy. This technique allowed us to observe the relative distributions of Pc 4 and an organelle-specific dye within the same cell via two, spectrally distinct, fluorescence images. To quantify the localization of Pc 4 within different organelles, linear correlation coefficients from the fluorescence data of Pc 4 and the organelle-specific dyes were calculated. Using this measurement, the subcellular spatial distributions of Pc 4 could be successfully monitored over an 18 h period. At early times (0-1 h) after introduction of Pc 4 to LY-R cells, the dye was found in the mitochondria, lysosomes and Golgi apparatus, as well as other cytoplasmic membranes, but not in the plasma membrane or the nucleus. Over the next 2 h, there was some loss of Pc 4 from the lysosomes as shown by the correlation coefficients. After an additional incubation period of 2 h Pc 4 slowly increased its accumulation in the lysosomes. The highest correlation coefficient (0.65) was for Pc 4 and BODIPY-FL C5 ceramide, which targets the Golgi apparatus, and also binds to other cytoplasmic membranes. The correlation coefficient was also high (0.60) for Pc 4 and a mitochondria-targeting dye (Mitotracker Green FM). Both of these correlation coefficients were higher than that for Pc 4 with the lysosome-targeting dye (Lysotracker Green DND-26). The results suggest that Pc 4 binds preferentially and strongly to mitochondria and Golgi complexes.
Abstract: Optical coherence tomography (OCT) has demonstrated the microscopic structure of the gastrointestinal (GI) tract mucosa and submucosa in vitro. We evaluated a prototype OCT system and assessed the feasibility of OCT in the human GI tract.
Abstract: Noninvasive monitoring of blood flow in retinal microcirculation may elucidate the progression and treatment of ocular disorders, including diabetic retinopathy, age-related macular degeneration, and glaucoma. Color Doppler optical coherence tomography (CDOCT) is a technique that allows simultaneous micrometer-scale resolution cross-sectional imaging of tissue microstructure and blood flow in living tissues. CDOCT is demonstrated for the first time in living human subjects for bidirectional blood-flow mapping of retinal vasculature.
Abstract: Color Doppler optical coherence tomography (CDOCT) is capable of precise velocity mapping in turbid media. Previous CDOCT systems based on the short-time Fourier transform have been limited to maximum flow velocities of the order of tens of millimeters per second. We describe a technique, based on interference signal demodulation at multiple frequencies, to extend the physiological relevance of CDOCT by increasing the dynamic range of measurable velocities to hundreds of millimeters per second. The physiologically important parameter of shear rate is also derived from CDOCT measurements. The measured flow-velocity profiles and shear-rate distributions correlate very well with theoretical predictions. The multiple demodulation technique, therefore, may be useful to monitor blood flow in vivo and to identify regions with high and low shear rates.
Abstract: We report on the design and initial clinical experience with a real-time endoscopic optical coherence tomography (EOCT) imaging system. The EOCT unit includes a high-speed optical coherence tomography interferometer, endoscope-compatible catheter probes, and real-time data capture and display hardware and software. Several technological innovations are introduced that improve EOCT efficiency and performance. In initial clinical studies using the EOCT system, the esophagus, stomach, duodenum, ileum, colon, and rectum of patients with normal endoscopic findings were examined. In these initial investigations, EOCT imaging clearly delineated the substructure of the mucosa and submucosa in several gastrointestinal organs; microscopic structures such as glands, blood vessels, pits, villi, and crypts were also observed.
Abstract: Current laser treatment for vascular disorders such as port wine stains can have incomplete or unacceptable results. A customized treatment strategy based on knowledge of the patient's blood vessel structure may effect an improved clinical outcome.
Abstract: We introduce a family of power-conserving fiber-optic interferometer designs for low-coherence reflectometry that use optical circulators, unbalanced couplers, and (or) balanced heterodyne detection. Simple design equations for optimization of the signal-to-noise ratio of the interferometers are expressed in terms of relevant signal and noise sources and measurable system parameters. We use the equations to evaluate the expected performance of the new configurations compared with that of the standard Michelson interferometer that is commonly used in optical coherence tomography (OCT) systems. The analysis indicates that improved sensitivity is expected for all the new interferometer designs, compared with the sensitivity of the standard OCT interferometer, under high-speed imaging conditions.
Abstract: Laser scanning confocal autofluorescence microscopy (LSCAM) using 351- to 364-nm excitation light was used to quantitatively compare fluorescent spectral emission of unstained, frozen histological sections of normal, premalignant, and malignant colonic tissues. To identify the spatial origins of fluorescent signals accurately, the same frozen section slides used for microscopy were fixed and histochemically stained immediately following LSCAM imaging. Tissue fluorescence emission was quantified in terms of the intrinsic fluorescence coefficient beta (lambda), defined as the fluorescence power per unit tissue volume per unit wavelength (centered at lambda) divided by the incident light irradiance. Over all emission wavelengths, colonic tissues emitted autofluorescence ranging from beta (lambda) approximately 10(-1.5) to 10(-3.0) cm-1. In the 530- to 610-nm spectral region, markedly increased autofluorescence (beta up to 10(-2.5)) was observed in the dysplastic cells of adenomatous polyps, as compared to normal epithelial cells. Compared to adenomatous polyps, decreased dysplastic cell autofluorescence was observed in adenocarcinoma. The brightest fluorescence in the lamina propria, which was attributed to eosinophils (beta approximately 10(-2.5)) in previous studies, was also observed in other granular structures (beta up to 10(-1.4)). LSCAM reveals quantitative significant differences in fluorescence emission between normal and diseased colonic tissues.
Abstract: Color Doppler optical coherence tomography (CDOCT) is a recent innovation that allows spatially localized flow-velocity mapping simultaneously with microstructural imaging. We present a theoretical model for velocity-image formation in CDOCT. The proportionality between the heterodyne detector current Doppler power spectrum in CDOCT and the optical source power spectrum is established. We show that stochastic modifications of the Doppler spectrum by fluctuating scatterer distributions in the flow field give rise to unavoidable velocity-estimation inaccuracies as well as to a fundamental trade-off between image-acquisition rate and velocity precision. Novel algorithms that permit high-fidelity depth-resolved measurements of velocities in turbid media are also reported.
Abstract: Optical coherence tomography (OCT) is a novel technique for noninvasive cross-sectional imaging with high spatial resolution (10 to 20 microm). OCT is similar to B-mode ultrasound except that it uses infrared light rather than ultrasound. We studied OCT imaging of the gastrointestinal (GI) tract in vitro to analyze the potential of this technique for endoscopic applications.
Abstract: The combined excited-state phosphorescence life-times of an alexandrite crystal and platinum tetraphenylporphyrin Pt(TPP) in a single-fiber sensor are used to monitor temperature and oxygen concentration in the physiological range from 15-45 degrees C and 0-50% O2 with precision of 0.24 degree C and 0.15% O2 and accuracy of 0.28 degree C and 0.2% O2. A 500-micron cubic alexandrite crystal bound to the distal end of a 750-micron-diameter optical fiber core and the Pt(TPP) coated circumferentially with a length of 1 cm from the end of the same fiber are excited with pulsed super-bright blue LED light. This apparatus uses a 125-kHz sampler for data acquisition and frequency domain methods for signal processing. The instrument amplifies both the dc and ac components of the photomultiplier output and band limits the signal to 20 kHz. The fundamental frequency of the excitation is set to 488.3 Hz and the highest harmonic used is the 35th. This bandlimited signal is sampled and averaged over a few hundred cycles in the time domain. The frequency domain representation of the data is obtained by employing fast Fourier transform algorithms. The phase delay and the modulation ratio of each sampled harmonic are then computed. At least four log-spaced harmonic phases or modulations are averaged before decoding the two lifetimes of temperature and oxygen phosphorescent sensors. A component of zero lifetime is introduced to account for the excitation backscatter leakage through optical interference filters seen by the photodetector. Linear and second-order empirical polynomials are employed to compute the temperatures and oxygen concentrations from the inverse lifetimes. In the situation of constant oxygen concentration, the lifetime of Pt(TPP) changes with temperature but can be compensated using the measured temperature lifetime. The system drift is 0.24 degree C for the temperature measurement and 0.59% for the oxygen concentration measurement over 30 h of continuous operation. The instrumentation and methods allow for 6-s update times and 90-s full-response times.
Abstract: We describe a novel optical system for bidirectional color Doppler imaging of flow in biological tissues with micrometer-scale resolution and demonstrate its use for in vivo imaging of blood flow in an animal model. Our technique, color Doppler optical coherence tomography (CDOCT), performs spatially localized optical Doppler velocimetry by use of scanning low-coherence interferometry. CDOCT is an extension of optical coherence tomography (OCT), employing coherent signal-acquisition electronics and joint time-frequency analysis algorithms to perform flow imaging simultaneous with conventional OCT imaging. Cross-sectional maps of blood flow velocity with <50-microm spatial resolution and <0.6-mm/s velocity precision were obtained through intact skin in living hamster subdermal tissue. This technology has several potential medical applications.
Abstract: Optical coherence tomography (OCT) is an recently developed medical diagnostic technology that uses back-reflected infrared light to perform in situ micron scale tomographic imaging. In this work, we investigate the ability of OCT to perform micron scale tomographic imaging of the internal microstructure of in vitro atherosclerotic plaques.
Abstract: Quantitative assessment of nerve fiber layer (NFL) thickness in normal and glaucomatous eyes, and correlation with conventional measurements of the optic nerve structure and function.
Abstract: To assess the potential of a new diagnostic technique called optical coherence tomography for imaging macular disease. Optical coherence tomography is a novel noninvasive, noncontact imaging modality which produces high depth resolution (10 microns) cross-sectional tomographs of ocular tissue. It is analogous to ultrasound, except that optical rather than acoustic reflectivity is measured.
Abstract: To demonstrate optical coherence tomography for high-resolution, noninvasive imaging of the human retina. Optical coherence tomography is a new imaging technique analogous to ultrasound B scan that can provide cross-sectional images of the retina with micrometer-scale resolution.
Abstract: To demonstrate a new diagnostic technique, optical coherence tomography, for high-resolution cross-sectional imaging of structures in the anterior segment of the human eye in vivo. Optical coherence tomography is a new, noninvasive, noncontact optical imaging modality that has spatial resolution superior to that of conventional clinical ultrasonography (< 20 microns) and high sensitivity (dynamic range, > 90 dB).
Abstract: We describe a novel technique, based on optical coherence tomography, for enhanced optical sectioning in confocal microscopy. Confocal imaging deep into highly scattering media is demonstrated and compared with the predictions of a single-backscatter theory.
Abstract: We describe a new technique, femtosecond transillumination optical coherence tomography, for time-gated imaging of objects embedded in scattering media. Time gating is performed with a fiber-optic interferometer with femtosecond pulses and coherent heterodyne detection to achieve a 130-dB dynamic range. A confocal imaging arrangement provides additional spatial discrimination against multiply scattered light. By time gating ballistic photons, we achieve 125-microm-resolution images of absorbing objects in media 27 scattering mean free paths thick. We derive a fundamental limit on ballistic imaging thickness based on quantum noise considerations.
Abstract: We describe what are to our knowledge the first in vivo measurements of human retinal structure with optical coherence tomography. These images represent the highest depth resolution in vivo retinal images to date. The tomographic system, image-processing techniques, and examples of high-resolution tomographs and their clinical relevance are discussed.
Abstract: Pulses as short as 2.3 ps have been generated by passive mode locking of a lamp-pumped Nd:YLF laser with a microdot mirror mode locker for Kerr-lens mode-locking (KLM) saturable absorber action and a compact Gires-Tournois interferometer for dispersion compensation. KLM was initiated with an acousto-optic modulator. Average output powers of 800 mW have been achieved. This result demonstrates the potential use of KLM for generating near-bandwidth-limited pulses from high-power lamp-pumped sources.
Abstract: Pulsed laser ablation of calcified biological tissue was studied at several wavelengths in the near-ultraviolet, visible, near- and mid-infrared regions of the spectrum. The primary tissue model was bovine shank bone, while human arterial calcified plaque and normal human artery wall were also studied at selected wavelengths for comparison. Laser irradiances were on the order of MW/mm2, fluences ranged up to 1000 mJ/mm2, and repetition rates varied between 0.3-10 Hz. Spot sizes on the tissue surface ranged from 150 to 850 microns. Laser craters made with wavelengths between lambda = 295 nm and lambda = 375 nm and in the lambda = 3 microns region exhibited the highest quality ablation with clean, sharp cuts following closely the spatial contour of the incident beam. Craters drilled with visible wavelengths between lambda = 450 nm and lambda = 590 nm were generally larger than the incident laser beam spot, irregular in shape and often surrounded by large flakes of tissue debris. Ablation fluence thresholds increased with wavelength through the visible wavelengths and into the mid-infrared, but dropped to their lowest values near lambda = 3 microns. Fluence thresholds obtained with the tissue under a 1 mm depth of saline were approximately twice air thresholds. Ablation yields also varied with wavelength, probably due to increased scattering in the visible region, and were the same under saline as in air.
Abstract: We present a theory of thermal laser ablation based on the heat equation and on an energy balance equation derived from it. Ablation is assumed to be brought about by the heating and evaporation of tissue water. The model is three-dimensional, and scattering and the water-steam phase transition are explicitly taken into account. The model predicts threshold parameters and a steady-state ablation velocity in terms of the optical and thermal properties of the tissue and the laser beam intensity and spot diameter.
