Single cell viability measurements in 3D scaffolds using in situ label free imaging by optical coherence microscopy

The focus on creating tissue engineered constructs of clinically relevant sizes requires new approaches for monitoring construct health during tissue development. A few key requirements are that the technology be in situ, non-invasive, and provide temporal and spatial information. In this work, we demonstrate that optical coherence microscopy (OCM) can be used to assess cell viability without the addition of exogenous probes in three-dimensional (3D) tissue scaffolds maintained under standard culture conditions. This is done by collecting time-lapse images of speckle generated by sub-cellular features. Image cross-correlation is used to calculate the number of features the final image has in common with the initial image. If the cells are live, the number of common features is low. The number of common features approaches 100% if the cells are dead. In control experiments, cell viability is verified by the addition of a two-photon fluorescence channel to the OCM. Green fluorescent protein transfected human bone marrow stromal cells cultured in a transparent poly(ethylene glycol) tetramethacrylate hydrogel scaffold is used as the control system. Then, the utility of this approach is demonstrated by determining L929 fibroblast cell viability in a more challenging matrix, collagen, an optical scatterer. These results demonstrate a new technique for in situ mapping of single cell viability without any exogenous probes that is capable of providing continuous monitoring of construct health.     

High-resolution optical coherence tomography imaging of the living kidney

Optical coherence tomography (OCT) is a rapidly emerging imaging modality that can provide non-invasive, cross-sectional, high-resolution images of tissue morphology in situ and in real-time. In the present series of studies, we used a high-speed OCT imaging system equipped with a frequency-swept laser light source (1.3 mum wavelength) to study living kidneys in situ. Adult, male Munich-Wistar rats were anesthetized, a laparotomy was performed and the living kidneys were exposed for in situ observation. We observed the kidneys prior to, during and following exposure to renal ischemia induced by clamping the renal artery. The effects of intravenous mannitol infusion (1.0 ml of 25%) prior to and during renal ischemia were also studied. Finally, living kidneys were flushed with a renal preservation solution, excised and observed while being stored at 0-4 degrees C. Three-dimensional OCT data sets enabled visualization of the morphology of the uriniferous tubules and the renal corpuscles. When renal ischemia was induced, OCT revealed dramatic shrinkage of tubular lumens due to swelling of the lining epithelium. Three-dimensional visualization and volumetric rendering software provided an accurate evaluation of volumetric changes in tubular lumens in response to renal ischemia. Observations of kidneys flushed with a renal preservation solution and stored at 0-4 degrees C also revealed progressive and significant loss of tubular integrity over time. Intravenous infusion of mannitol solution resulted in thinning of the tubular walls and an increase in the tubular lumen diameters. Mannitol infusion also prevented the cell swelling that otherwise resulted in shrinkage of proximal tubule lumens during ischemia. We conclude that OCT represents an exciting new approach to visualize, in real-time, pathological changes in the living kidney in a non-invasive fashion. Possible clinical applications are discussed.     

Visualization of left ventricular Purkinje fiber distribution using widefield optical coherence microscopy

Background: The distribution and connection of ventricular Purkinje fibers are known to be associated with idiopathic left ventricular arrhythmias. Unusual anatomy is one of the important factors associated with catheter ablation success rate. With the widefield high-speed, swept-source optical coherence microscopy (OCM) and light microscope, we visualized the left ventricular Purkinje fiber distribution. Methods: Left ventricular walls of five adult ovine hearts were incised from the mitral annulus to the apex. Using the widefield OCM technique and light microscopy, we observed the distribution, direction, depth, and dividing patterns of the Purkinje network with multiple tangential angles and without tissue destruction. Results: Widefield OCM was used to characterize the ovine heart Purkinje network system in a 4 × 4 mm² field. Left ventricular Purkinje fibers traveled in the sub-endocardial area near the left-sided peri-membranous septal area and ran like a wide hair bundle. The distal branching fibers penetrated to the endocardium and connected to the contractile muscle. In this distal area, Purkinje fibers were connected to each other, forming multiple layers. Some Purkinje fibers were directly connected within the false tendon between the papillary muscles or between the trabeculations. Some free-running Purkinje fibers were directly connected to the papillary muscle from the left bundle. Conclusion: Using widefield OCM, we were able to observe the left bundle and its branching patterns in ovine left ventricle without tissue destruction. This might be applied to future cardiac ablation procedures.     

Speckle reduction in optical coherence tomography imaging by affine-motion image registration

Signal-degrading speckle is one factor that can reduce the quality of optical coherence tomography images. We demonstrate the use of a hierarchical model-based motion estimation processing scheme based on an affine-motion model to reduce speckle in optical coherence tomography imaging, by image registration and the averaging of multiple B-scans. The proposed technique is evaluated against other methods available in the literature. The results from a set of retinal images show the benefit of the proposed technique, which provides an improvement in signal-to-noise ratio of the square root of the number of averaged images, leading to clearer visual information in the averaged image. The benefits of the proposed technique are also explored in the case of ocular anterior segment imaging.     

In vivo human retinal imaging by Fourier domain optical coherence tomography

We present what is to our knowledge the first in vivo tomograms of human retina obtained by Fourier domain optical coherence tomography. We would like to show that this technique might be as powerful as other optical coherence tomography techniques in the ophthalmologic imaging field. The method, experimental setup, data processing, and images are discussed.     

Dynamic focus in optical coherence tomography for retinal imaging

Imaging the human retina still represents the main field of application of optical coherence tomography (OCT). A major advantage of OCT is the decoupling of depth resolution (given by the coherence length of the light source) and transverse resolution (given by the focal spot size). This enables images of the retina with high depth resolution. On the other hand, in most OCT systems, a limited transverse resolution has been accepted to provide a sufficiently large (approximately 1 mm) depth of focus. However, to obtain images with high transverse resolution throughout the whole depth of the retina (especially in the nerve head region) a tracking of coherence gate and focus position (dynamic focus) is essential. This study realizes a dynamic focus in a time domain transversal (en face) scanning system for retinal imaging. We show that maintenance of a transverse resolution of approximately 4.4 microm can be achieved over an optical depth of 1 mm in a model eye and apply our method to imaging the human retina in vivo.     

Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy

Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy.     

Functional imaging of dye concentration in tissue phantoms by spectroscopic optical coherence tomography

We present functional imaging of the concentration of a photodynamic therapy (PDT)-related dye in scattering tissue phantoms based on spatially resolved measurements of optical properties through spectroscopic optical coherence tomography (OCT). Expressions for the OCT signal are developed, enabling estimation of depth-resolved sample optical properties. Based on these expressions, we discuss speckle statistics and speckle correlations of the OCT signal. Speckle noise reduction is performed by spatial filtering and is used to improve accuracy in the estimated optical properties at the expense of spatial resolution. An analytic expression for the precision in the estimated optical properties is derived. This expression shows that axial filtering, and thereby a reduction of axial resolution, gives a larger improvement in precision compared to the same filtering and reduction in the transversal resolution. It also shows that imaging with a shorter coherence length, or a larger numerical aperture, improves precision when the filter length determines the spatial resolution. Good agreement is obtained between experimentally determined and theoretically predicted variance in the estimated attenuation coefficients and dye concentration. Finally, we present guidelines for spectroscopic OCT systems for concentration imaging and discuss application of the method to more realistic phantoms and tissue.     

High-resolution imaging diagnosis of human fetal membrane by three-dimensional optical coherence tomography

Microscopic chorionic pseudocyst (MCP) arising in the chorion leave of the human fetal membrane (FM) is a clinical precursor for preeclampsia which may progress to fatal medical conditions (e.g., abortion) if left untreated. To examine the utility of three-dimensional (3D) optical coherence tomography (OCT) for noninvasive delineation of the morphology of human fetal membranes and early clinical detection of MCP, 60 human FM specimens were acquired from 10 different subjects undergoing term cesarean delivery for an ex vivo feasibility study. Our results showed that OCT was able to identify the four-layer architectures of human FMs consisting of high-scattering decidua vera (DV, average thickness d(DV) ≈ 92±38 μm), low-scattering chorion and trophoblast (CT, d(CT) ≈ 150±67 μm), high-scattering subepithelial amnion (A, d(A) ≈ 95±36 μm), and low-scattering epithelium (E, d(E) ≈ 29±8 μm). Importantly, 3D OCT was able to instantaneously detect MCPs (low scattering due to edema, fluid buildup, vasodilatation) and track (staging) their thicknesses d(MCP) ranging from 24 to 615 μm. It was also shown that high-frequency ultrasound was able to compliment OCT for detecting more advanced thicker MCPs (e.g., d(MCP)>615 μm) because of its increased imaging depth.     

Intravascular atherosclerotic imaging with combined fluorescence and optical coherence tomography probe based on a double-clad fiber combiner

We developed a multimodality fluorescence and optical coherence tomography probe based on a double-clad fiber (DCF) combiner. The probe is composed of a DCF combiner, grin lens, and micromotor in the distal end. An integrated swept-source optical coherence tomography and fluorescence intensity imaging system was developed based on the combined probe for the early diagnoses of atherosclerosis. This system is capable of real-time data acquisition and processing as well as image display. For fluorescence imaging, the inflammation of atherosclerosis and necrotic core formed with the annexin V-conjugated Cy5.5 were imaged. Ex vivo imaging of New Zealand white rabbit arteries demonstrated the capability of the combined system.     

Enhanced optical clearing of skin in vivo and optical coherence tomography in-depth imaging

The strong optical scattering of skin tissue makes it very difficult for optical coherence tomography (OCT) to achieve deep imaging in skin. Significant optical clearing of in vivo rat skin sites was achieved within 15 min by topical application of an optical clearing agent PEG-400, a chemical enhancer (thiazone or propanediol), and physical massage. Only when all three components were applied together could a 15 min treatment achieve a three fold increase in the OCT reflectance from a 300 μm depth and 31% enhancement in image depth Z(threshold).     

The potential of optical coherence tomography in the engineering of living tissue

The better repair of human tissue is an urgent medical goal and in order to achieve a safe outcome there is a parallel need for sensitive, non-invasive methods of assessing the quality of the engineered tissues and organs prior to surgical implantation. Optical coherence tomography (OCT) can potentially fulfil this role. The current status of OCT as an advanced imaging tool in clinical medicine, developmental biology and material science is reviewed and the parallels to the engineering of living tissue and organs are discussed. Preliminary data are also presented for a tissue engineering bioreactor with in situ OCT imaging. The data suggest that OCT can be utilized as a real time, non-destructive, non-invasive tool to critically monitor the morphology of tissue-engineered constructs during their fabrication and growth.     

Polarization memory effect in optical coherence tomography and dental imaging application

We report the existence of polarization memory effect (PME) in optical coherence tomography and investigate its potential applications in dental imaging. We performed the study in three steps. First, microsphere scattering phantoms of different sizes were imaged in order to validate experimental results with PME theory. Both linearly and circularly polarized light were used to probe the samples. Second, healthy tooth samples were scanned and polarization memory effect was identified in dentin. In this step, specific verification and signal processing were performed to rule out possible image interpretation by birefringence effect. Third, we evaluated dentin demineralization with PME. Results show polarization memory can be useful to characterize this dynamic mineralization process for early caries detection and rehabilitation.     

Correction of distortions in optical coherence tomography imaging of the eye

Optical coherence tomography (OCT) images are affected by artefacts. These artefacts are the result of different factors such as refraction, curvature of the intermediate layers up to the depth of interest and the scanning procedure. The effect of such errors is different, depending on the way the image is acquired, either en-face or longitudinal OCT. We quantify the distortions by evaluating a lateral and an axial error. These measure the lateral and axial deviations of each image point from the object point inside the tissue. We show that the axial distortion can be larger than the achievable depth resolution in modern OCT systems. We have investigated these errors in imaging different tissue: cornea and retina in vivo and an intraocular lens in vitro.     

Investigation of optical coherence tomography as an imaging modality in tissue engineering

Monitoring cell profiles in 3D porous scaffolds presents a major challenge in tissue engineering. In this study, we investigate optical coherence tomography (OCT) as an imaging modality to monitor non-invasively both structures and cells in engineered tissue constructs. We employ time-domain OCT to visualize macro-structural morphology, and whole-field optical coherence microscopy to delineate the morphology of cells and constructs in a developing in vitro engineered bone tissue. The results show great potential for the use of OCT in non-invasive monitoring of cellular activities in 3D developing engineered tissues.     

Quantification of fiber orientation in the canine atrial pacemaker complex using optical coherence tomography

The atrial pacemaker complex is responsible for the initiation and early propagation of cardiac impulses. Optical coherence tomography (OCT), a nondestructive imaging modality with spatial resolutions of ～1 to 15 μm, can be used to identify unique fiber orientation patterns in this region of the heart. Functionally characterized canine sinoatrial nodes (SAN) (n=7) were imaged using OCT up to ～1 mm below the endocardial tissue surface. OCT images were directly compared to their corresponding histological sections. Fiber orientation patterns unique to the crista terminalis (CT), SAN, and surrounding atrial myocardium were identified with dominant average fiber angles of 89±12 deg, 110±16 deg, and 95±35 deg, respectively. Both the CT and surrounding atrial myocardium displayed predominantly unidirectionally based fiber orientation patterns within each specimen, whereas the SAN displayed an increased amount of fiber disarray manifested quantitatively as a significantly greater standard deviation in fiber angle distribution within specimens [33±7 deg versus 23±5 deg, atrium (p=0.02); 18±3 deg, CT (p=0.0003)]. We also identified unique, local patterns of fiber orientation specific to the functionally characterized block zone. We demonstrate the ability of OCT in detecting components of the atrial pacemaker complex which are intimately involved in both normal and abnormal cardiac conduction.     

Hand-held arthroscopic optical coherence tomography for in vivo high-resolution imaging of articular cartilage

We describe a novel hand-held polarization optical coherence tomographic (OCT) probe that can be inserted into mammalian joints to permit real-time cross-sectional imaging of articular cartilage. The transverse and axial resolutions of the arthroscopic OCT device are roughly 17 and 10 microm, respectively. Two-dimensional cross-sectional images of cartilage tissue with 500 x 1000 pixels covering an area 6 mm in length and 2.8 mm in depth can be acquired at nearly five frames/s and with over 100 dB of dynamic range. Design of an OCT as a hand-held device capable of providing such an optical biopsy of articular cartilage allows eventual in vivo detection of microstructural changes in articular cartilage that are not apparent using conventional arthroscopic cameras. The OCT probe can be easily incorporated in a conventional arthroscope for cartilage site guidance. The optical arrangement in the OCT scope minimizes specular back-reflection of the probe end face and absorption of body fluid in the path and ensures in-focus OCT imaging when it is in contact with the cartilage specimen to be examined. Successful application of in vivo arthroscopy to porcine articular cartilage demonstrates sufficient resolution and practicality for use in human joints.     

Novel combined miniature optical coherence tomography ultrasound probe for in vivo intravascular imaging

We have developed a miniature integrated optical coherence tomography (OCT) ultrasound (US) probing system for intravascular imaging applications. In the OCT probe, the light coming out of a single mode fiber is focused by a gradient-index lens and then reflected by a right-angle prism from the side of the probe into the sample. It was combined with a 35 MHz PMN-PT side-viewing ultrasound transducer to obtain the ultrasound image as well. The OCT and ultrasound probes were integrated as a single probe to obtain OCT and ultrasound images simultaneously. The integrated probe has an outer diameter of 0.69 mm which, to our knowledge, is the smallest integrated OCT-US probe reported. Fast data acquisition and processing was implemented for real-time imaging. In vitro OCT and US images of human coronary artery with pathology, as well as in vivo images of normal rabbit abdominal aorta, were obtained using the integrated OCT-US probe to demonstrate its capability.     

Speckle reduction in optical coherence tomography by frequency compounding

We are investigating the possibility of a frequency compounding method for speckle reduction in optical coherence tomography. The method is based on incoherent summation of the magnitudes of two independent interferometric signals, which were recorded at two different center wavelengths simultaneously. We derive the corresponding statistics and compare the theoretical results with measurements obtained in a uniformly scattering sample. Finally we demonstrate our method by comparing images of human skin recorded in vivo with and without frequency compounding. The compounding method results in an increased contrast and improved image quality without loss of resolution.     

Retinal nerve fiber layer structure abnormalities in early Alzheimer's disease: Evidence in optical coherence tomography

Alzheimer's disease (AD) patients often have visual disorders which may be due to retinal nerve degenerative changes. The aim of the current study was to determine the thickness changes of retina nerve fibers with optical coherence tomography (OCT) in AD patients. The OCT was used to assess the thickness of retinal nerve fiber layer (RNFL) from 22 AD patients and 22 healthy age-matched controls. The corrected visual acuity and intraocular pressure were measured and the dilated fundus examination and fundus image acquisition were also performed in those subjects. Compared with healthy age-matched controls, the RNFL thickness of AD patients were much thinner (p < 0.05), especially in supra-retina and infra-retina, while no difference was found in the other retinal area. These changes were also confirmed by the fundus images. In conclusion, retinal nerve degeneration is present in the retina of AD patients and this degeneration is likely localized preferentially to the superior and inferior quadrant.