Application of optical coherence tomography, pulsed photoacoustic technique, and time-of-flight technique to detect changes in the scattering properties of a tissue-simulating phantom

Intralipid is a well-known emulsion used as a tissue-simulating phantom in developing optical imaging and diagnostic techniques for medical applications. The optical coherence tomography (OCT), pulsed photoacoustic (PA), and time-of-flight (TOF) techniques were used to detect glucose-induced changes in the optical properties of Intralipid. A comparison of the applicability of these techniques to register changes in the scattering properties of Intralipid samples showed that OCT is the most effective method, whereas the sensitivity of the PA technique was less pronounced. Photon migration studies with the TOF technique showed changes in pulse amplitude, pulse width, and arrival time of the pulse maximum as a function of changes in Intralipid concentration. Also the measured signal parameters showed changes when measuring high glucose concentrations.     

In vivo study of glucose-induced changes in skin properties assessed with optical coherence tomography

Recently, our in vivo studies demonstrated a strong correlation between blood glucose concentration and the slope of the optical coherence tomography (OCT) signal when the probing beam was scanned over a straight line. To improve the sensitivity of OCT for blood glucose monitoring, two-dimensional (2D) lateral scanning of the OCT probing beam was proposed. Depth-dependent changes in pig skin properties with variation of blood glucose concentration were revealed due to significant suppression of speckle noise and motion artefacts in 2D scanning mode. The correlation coefficient of the OCT signal slope with blood glucose concentration varied periodically in the range from -0.9 to +0.9 depending on depth. The period of variation of the correlation coefficient was 100-150 microm that corresponded to the distance between neighbour collagen bundles. We also observed a decrease of skin thickness by 10 +/- 7.5 microm with an increase of blood glucose concentration by 277 +/- 56 mg dl(-1). Mechanisms of glucose-induced changes in skin properties owing to tissue layer shift caused by dehydration associated with the glucose osmotic effect were considered.     

Measurement of bone mineral density via light scattering

In this study we have investigated the potential of optical techniques to monitor changes in bone mineral density (BMD) via changes in scattering coefficient. For each of five bone samples, diffuse reflection and transmission coefficients were measured over the wavelength range 520-960 nm using an integrating sphere and CCD spectrometer. These were converted into optical absorption and scattering coefficients using a Monte Carlo inversion procedure. Measurements were made on samples immersed in formic acid solution for different lengths of time in order to investigate the effect of reduction in BMD on the optical properties. After full demineralization, the optical scattering coefficient fell by a factor 4. From the observed degree of fluctuation of the measurements, we estimate that BMD could be measured with an accuracy of 7% if optical scattering can be measured with an accuracy of 10%. We also report preliminary measurements of bone scattering using optical coherence tomography (OCT). An inter-side variability of 3% is obtained on dry samples with and without overlying periosteum. These results suggest that minimally invasive techniques for measuring optical scattering, such as OCT, may have a role in monitoring regional changes in BMD. This could be an important advance in our understanding of bone remodelling and its relationship to osteoarthritis. Both the integrating sphere and OCT measurements also suggest that light transport in bone is spatially anisotropic. OCT was used to assess probability of obtaining results in vivo.     

Effect of tissue preservation on imaging using ultrahigh resolution optical coherence tomography

Ultrahigh resolution optical coherence tomography (OCT) is an emerging imaging modality that enables noninvasive imaging of tissue with 1- to 3-microm resolutions. Initial OCT studies have typically been performed using harvested tissue specimens (ex vivo). No reports have investigated postexcision tissue degradation on OCT image quality. We investigate the effects of formalin fixation and commonly used cell culture media on tissue optical scattering characteristics in OCT images at different times postexcision compared to in vivo conditions. OCT imaging at 800-nm wavelength with 1.5-mum axial resolution is used to image the hamster cheek pouch in vivo, followed by excision and imaging during preservation in phosphate-buffered saline (PBS), Dulbecco's Modified Eagle's Media (DMEM), and 10% neutral-buffered formalin. Imaging is performed in vivo and at sequential time points postexcision from 15 min to 10 to 18 h. Formalin fixation results in increases in scattering intensity from the muscle layers, as well as shrinkage of the epithelium, muscle, and connective tissue of approximately 50%. PBS preservation shows loss of optical contrast within two hours, occurring predominantly in deep muscle and connective tissue. DMEM maintains tissue structure and optical scattering characteristics close to in vivo conditions up to 4 to 6 h after excision and best preserved tissue optical properties when compared to in vivo imaging.     

Specificity of noninvasive blood glucose sensing using optical coherence tomography technique: a pilot study

Noninvasive monitoring of blood glucose concentration in diabetic patients would significantly reduce complications and mortality associated with this disease. In this paper, we experimentally and theoretically studied specificity of noninvasive blood glucose monitoring with the optical coherence tomography (OCT) technique. OCT images and signals were obtained from skin of Yucatan micropigs and New Zealand rabbits. Obtained results demonstrate that: (1) several body osmolytes may change the refractive index mismatch between the interstitial fluid (ISF) and scattering centres in tissue, however the effect of the glucose is approximately one to two orders of magnitude higher; (2) an increase of the ISF glucose concentration in the physiological range (3-30 mM) may decrease the scattering coefficient by 0.22% mM(-1) due to cell volume change; (3) stability of the OCT signal slope is dependent on tissue heterogeneity and motion artefacts; and (4) moderate skin temperature fluctuations (+/- 1 degree C) do not decrease accuracy and specificity of the OCT-based glucose sensor, however substantial skin heating or cooling (several degrees C) significantly change the OCT signal slope. These results suggest that the OCT technique may provide blood glucose concentration monitoring with sufficient specificity under normal physiological conditions.     

Characterization of age-related effects in human skin: A comparative study that applies confocal laser scanning microscopy and optical coherence tomography

Skin structure and age-related changes in human skin were characterized in vivo by applying confocal laser scanning microscopy (CLSM) and optical coherence tomography (OCT). The overall effect of aging skin, derived from studies of volunteers belonging to two age groups, was found to be a significant decrease in the maximum thickness of the epidermis and flattening of the dermo-epidermal junction. At a certain depth in the dermis, well below the basal layer, a reflecting layer of fibrous structure is observed in CLSM images. The location of this layer strongly depends on age and is situated much deeper below the skin surface in younger than in older skin. In addition, large structural changes were observed with age. The OCT images show two bright reflecting layers. The first one is due to scattering at the skin surface. The second band appears to be caused by a layer of fibrous structure in the dermis. Direct comparison of CLSM and OCT suggests that the same fibrous layer is imaged by the two techniques. This layer might be due to the transition between the papillary and reticular dermis. A comparison of CLSM and OCT enables a better understanding of the images.     

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).     

Reduction of image artifacts in three-dimensional optical coherence tomography of skin in vivo

This paper presents results of in vivo studies on the effect of refractive index-matching media on image artifacts in optical coherence tomography (OCT) images of human skin. These artifacts present as streaks of artificially low backscatter and displacement or distortion of features. They are primarily caused by refraction and scattering of the OCT light beam at the skin surface. The impact of the application of glycerol and ultrasound gel is assessed on both novel skin-mimicking phantoms and in vivo human skin, including assessment of the epidermal thickening caused by the media. Based on our findings, recommendations are given for optimal OCT imaging of skin in vivo.     

Effect of target biological tissue and choice of light source on penetration depth and resolution in optical coherence tomography

The effectiveness of an optical coherence tomography (OCT) system depends largely on the light source chosen. Published data on the optical properties of tissues are used to quantify the exponential attenuation of broadband light on transport through tissue. The effective attenuation coefficient is taken to be the sum of the absorption and scattering coefficients. This is used to demonstrate the effect on the spectra of a wide range of published OCT sources and the change in system resolution induced, and hence to comment on the suitability of different sources for OCT. The tissues studied include skin dermis, liver, and gallbladder. Sources at higher wavelengths are shown to be capable of high-resolution OCT imaging at greater depths. Titanium:sapphire lasers would be most suited for high-resolution OCT over comparatively shallow depths into tissue. For lower-resolution applications of OCT, a semiconductor optical amplifier and ytterbium fiber sources have better powers and bandwidths than superluminescent diodes. The resolution of OCT systems is not reduced significantly with imaging depth.     

Dermal reflectivity determined by optical coherence tomography is an indicator of epidermal hyperplasia and dermal edema within inflamed skin

Psoriasis is a common inflammatory skin disease resulting from genetic and environmental alterations of cutaneous immune responses. While numerous therapeutic targets involved in the immunopathogenesis of psoriasis have been identified, the in vivo dynamics of inflammation in psoriasis remain unclear. We undertook in vivo time course focus-tracked optical coherence tomography (OCT) imaging to noninvasively document cutaneous alterations in mouse skin treated topically with Imiquimod (IMQ), an established model of a psoriasis-like disease. Quantitative appraisal of dermal architectural changes was achieved through a two parameter fit of OCT axial scans in the dermis of the form A(x, y, z) = ρ(x, y)exp?[-μ(x, y)z]. Ensemble averaging over 2000 axial scans per mouse in each treatment arm revealed no significant changes in the average dermal attenuation rate, <μ>, however the average local dermal reflectivity <ρ>, decreased significantly following 1, 3, and 6 days of IMQ treatment (p < 0.001) in comparison to vehicle-treated control mice. In contrast, epidermal and dermal thickness changes were only significant when comparing controls and 6-day IMQ treated mice. This suggests that dermal alterations, attributed to collagen fiber bundle enlargement, occur prior to epidermal thickness changes due to hyperplasia and dermal thickness changes due to edema. Dermal reflectivity positively correlated with epidermal hyperplasia (r(epi)(2) = 0.78) and dermal edema (r(derm)(2) = 0.86). Our results suggest that dermal reflectivity as measured by OCT can be utilized to quantify a psoriasis-like disease in mice, and thus has the potential to aid in the quantitative assessment of psoriasis in humans.     

Use of optical coherence tomography to monitor biological tissue freezing during cryosurgery

The use of optical coherence tomography (OCT) for imaging skin during cryosurgery is evaluated. OCT provides high spatial resolution (5-10 microm) images of optical backscattering due to local variations in refractive index, such as the boundary between liquid and frozen water in tissue. Time resolved OCT images were acquired during freezing of water, Intralipid trade mark, and in vivo hamster skin. Subsurface morphological changes were evident only during freezing of Intralipid and skin. A simple thermal model was applied which predicted freezing times on the same order of magnitude as those observed in OCT images. OCT can be used as a feedback tool during cryosurgical procedures to monitor progression of the freezing front.     

Spatially confined and temporally resolved refractive index and scattering evaluation in human skin performed with optical coherence tomography

In the present applications of optical coherence tomography (OCT), parameters besides pure morphology are evaluated in skin tissue under in vivo conditions. Spatially mapped refractive indices and scattering coefficients may support tissue characterization for research and diagnostic purposes in cosmetics/pharmacy and medicine, respectively. The sample arm of our OCT setup has been arranged to permit refractive index evaluation with little mechanical adjustment of a lens within the objective. A simple algorithm has been derived. Known from atmospheric work, the Klett algorithm [J. D. Klett, "Stable analytical inversion solution for processing LIDAR returns," Appl. Opt. 20(2), 211-220 (1981)] has been applied to the same data set for retrieval of scattering coefficients. Both parameters have been measured in layered structures in skin like stratum corneum, epidermis and dermis. Significant water content in a localized sweat gland duct has been observed by refractive index evaluation. Time studies over 1.5 h permitted a first understanding about physiological changes in skin which are not obtainable by intrusive methods.     

Signal degradation by multiple scattering in optical coherence tomography of dense tissue: a Monte Carlo study towards optical clearing of biotissues

Multiple scattering is a major source that limits light penetration into biotissues, thereby preventing visualization of the deep microstructures for high-resolution optical imaging techniques. The optical clearing approach is a new adventure in biomedical optics for manipulating the optical properties of tissue; for example, the scattering coefficient and the degree of forward scattering of photons, by the use of the chemical administration method in order to improve the optical imaging depth, particularly for the recently developed optical coherence tomography (OCT). This paper investigates systematically how the multiple scattering affects signal attenuation and localization in general, and how the alterations of optical properties of tissue enhance the optical imaging depth and signal localization in particular, by the use of Monte Carlo simulations through the separate considerations of the least scattered photons (LSP) and multiple scattered photons (MSP). The LSP are those photons that contribute to the precise OCT signal, i.e. localization, and the MSP are those that degrade the OCT signal. It is shown that with either the reduction of the scattering coefficient or the increase of the degree of forward scattering, signal localization and imaging depth for OCT is enhanced. Whilst the increase of the anisotropic factor of the medium is more efficient in improving signal localization, it introduces more scattering events for the photons travelling within the tissue for both the LSP and MSP. It is also found that the OCT imaging resolution is almost reduced exponentially with the increase of the probing depth as opposed to the claimed system resolution. We demonstrate that optical clearing could be a useful tool to improve the imaging resolution when the light progressively penetrates the high scattering medium. Experimental results are also presented to show intuitively how multiple scattering affects OCT signal profiles by the use of intralipid solution and healthy human whole blood, representing moderately and highly scattering media respectively.     

New method for evaluation of in vivo scattering and refractive index properties obtained with optical coherence tomography

Optical coherence tomography (OCT) provides more parameters than pure morphology does. In a recent paper [A. Knuettel and M. Boehlau-Godau, J. Biomed. Opt. 5(1) 83-92 (2000)] we have shown that the refractive index (RI) can be evaluated in a localized manner in skin tissue under in vivo conditions. Based on a theory, originally developed for light detecting and ranging applications [L. Thrane et al., J. Opt. Soc. Am. A 17(3) 484-490 (2000)], the parameter mean scattering angle (MSA) could be derived in addition to RI. The effects of hydration on MSA and RI have been evaluated in vitro in pigskin and in vivo in human skin with our OCT scanner SkinDex 300. These parameters may have a viable impact in (cosmetic) skin research and clinical diagnoses. To the best of our knowledge, this is the first time that (multiple) scattering of light has been quantified through the observation of a new scattering parameter under in vivo conditions.     

Shear flow-induced optical inhomogeneity of blood assessed in vivo and in vitro by spectral domain optical coherence tomography in the 1.3 μm wavelength range

The optical inhomogeneity of flowing blood, which appears as a waisted double fan-shaped intensity pattern inside vessels in cross-sectional optical coherence tomography (OCT) images, was investigated for the first time. High resolution spectral domain OCT in the 1.3 μm wavelength region is used to assess this inhomogeneous intravascular backscattering of light in an in vivo mouse model and flow phantom measurements. Based on a predicted alignment of the red blood cells toward laminar shear flow, an angular modulation of the corresponding backscattering cross-section inside the vessels is assumed. In combination with the signal attenuation in depth by absorption and scattering, a simple model of the intravascular intensity modulation is derived. The suitability of the model is successfully demonstrated in the in vivo experiments and confirmed by the in vitro measurements. The observed effect appears in flowing blood only and shows a strong dependency on the shear rate. In conclusion, the shear-induced red blood cell alignment in conjunction with the vessel geometry is responsible for the observed intensity distribution. This inherent effect of blood imaging has to be considered in attenuation measurements performed with OCT. Furthermore, the analysis of the intravascular intensity pattern might be useful to evaluate flow characteristics.     

Optical coherence tomography reveals in vivo cortical plasticity of adult mice in response to peripheral neuropathic pain

We examined neural plasticity in mice in vivo using optical coherence tomography (OCT) of primary somatosensory (S1) and motor (M1) cortices of mice under the influence of sciatic nerve chronic constriction injury (CCI), a model of neuropathic pain widely utilized in rats. The OCT system used in this study provided cross-sectional images of the cortical tissue of mice up to a depth of about 1mm with longitudinal resolution up to 11 microm. This is the first study to evaluate neural plasticity in vivo using OCT. CCI mice exhibited cold allodynia and spontaneous pain behaviors, which are signs of neuropathic pain, 30 days after sciatic nerve ligation, when OCT observation of S1 and M1 cortices was carried out. The scattering intensity of near-infrared light within the hind paw area of S1 and M1 regions in the contralateral hemisphere was significantly higher than in the ipsilateral hemisphere. These CCI-induced increases in scattering intensity within cortical regions associated with the hind paw probably reflect elevated neural activity associated with neuropathic pain. Synapses and mitochondria are believed to have high light scattering coefficients, since they contain remarkably high concentrations of proteins and complicated membrane structure. Number densities of mitochondria and synapses are known to increase in parallel with increases in neural activity. Our findings thus suggest that neuropathic pain gives rise to neural plasticity within the hind paw area of S1 and M1 contralateral to the ligated sciatic nerve.     

Assessment of dermal wound repair after collagen implantation with optical coherence tomography

We present an animal study to examine the utility and potential limitations of optical coherence tomography (OCT) for noninvasive evaluation of biomaterial scaffold-assisted wound healing. The transverse and axial resolutions of the OCT system at the wavelength of 1.3 microm were 12 and 10 microm, respectively. A murine full-thickness transcutaneous wound model was employed, in which a phi 10 mm full-thickness wound was created on the back of each male Balb/cJ mouse and a porous collagen scaffold was implanted in the wound bed followed by coverage with a Tegaderm film. Sequential cross-sectional OCT scans were performed at different time points postsurgical intervention to track morphological changes during wound recovery, and the captured OCT images were validated by their corresponding histological specimens. The results indicated that with removal of the high-scattering skin, OCT was capable of imaging to a depth of over 1.5 mm into the wound bed and differentiating various features evolved during wound healing at a high resolution approaching histopathology. OCT was able to not only delineate the epidermis and dermis of normal mouse skin, but also differentiate collagen implant from the underlying subcutaneous tissue; besides, it could track the wound size changes in both lateral and vertical directions. More importantly, OCT was able to detect inflammation, early re-epithelialization, and resorption of the collagen scaffold. These findings suggested the potential of OCT for noninvasive and high-resolution monitoring of assisted wound healing in vivo, longitudinally, and instantaneously.     

Novel algorithm of processing optical coherence tomography images for differentiation of biological tissue pathologies

A numerical algorithm based on a small-angle approximation of the radiative transfer equation (RTE) is developed to reconstruct scattering characteristics of biological tissues from optical coherence tomography (OCT) images. According to the algorithm, biological tissue is considered to be a layered random medium with a set of scattering parameters in each layer: total scattering coefficient, variance of a small-angle scattering phase function, and probability of backscattering, which fully describe the OCT signal behavior versus probing depth. The reconstruction of the scattering parameters is performed by their variation to fit the experimental OCT signal by the theoretical one using a time-saving genetic algorithm. The proposed reconstruction procedure is tested on model media with known scattering parameters. The possibility to estimate scattering parameters from OCT images is studied for various regimes of OCT signal decay. The developed algorithm is applied to reconstruct optical characteristics of epithelium and stroma for normal cervical tissue and its pathologies, and the potential to distinguish between the types of pathological changes in epithelial tissue by its OCT images is demonstrated.     

Optical probes and techniques for molecular contrast enhancement in coherence imaging

Optics has played a key role in the rapidly developing field of molecular imaging. The spectroscopic nature and high-resolution imaging capabilities of light provide a means for probing biological morphology and function at the cellular and molecular levels. While the use of bioluminescent and fluorescent probes has become a mainstay in optical molecular imaging, a large number of other optical imaging modalities exist that can be included in this emerging field. In vivo imaging technologies such as optical coherence tomography and reflectance confocal microscopy have had limited use of molecular probes. In the last few years, novel nonfluorescent and nonbioluminescent molecular imaging probes have been developed that will initiate new directions in coherent optical molecular imaging. Classes of probes reviewed in this work include those that alter the local optical scattering or absorption properties of the tissue, those that modulate these local optical properties in a predictable manner, and those that are detected utilizing spectroscopic optical coherence tomography (OCT) principles. In addition to spectroscopic OCT, novel nonlinear interferometric imaging techniques have recently been developed to detect endogenous molecules. Probes and techniques designed for coherent molecular imaging are likely to improve the detection and diagnostic capabilities of OCT.     

Changes in tissue optical properties due to radio-frequency ablation of myocardium

The optical properties of pig heart tissue were measured after in vivo ablation therapy had been performed during open-heart surgery. In vitro samples of normal and ablated tissue were subjected to measurements with an optically integrating sphere set-up in the region 470–900 nm. Three independent measurements were made: total transmittance, total reflectance and collimated transmittance, which made it possible to extract the absorption and scattering coefficients and the scattering anisotropy factor g, using an inverse Monte Carlo model. Between 470 and 700 nm, only the reduced scattering coefficient and absorption could be evaluated. The absorption spectra were fitted to known tissue chromophore spectra, so that the concentrations of haemoglobin and myoglobin could be estimated. The reduced scattering coefficient was compared with Mie computations to provide Mie equivalent average radii. Most of the absorption was from myoglobin, whereas haemoglobin absorption was negligible. Metmyoglobin was formed in the ablated tissue, which could yield a spectral signature to distinguish the ablated tissue with a simple optical probe to monitor the ablation therapy. The reduced scattering coefficient increased by, on average, 50% in the ablated tissue, which corresponded to a slight decrease in the Mie equivalent radius.