Dual-axis optical coherence tomography for deep tissue imaging.

We have developed dual-axis optical coherence tomography (DA-OCT) which enables deep tissue imaging by using a novel off-axis illumination/detection configuration. DA-OCT offers a 100-fold speed increase compared with its predecessor, multispectral multiple-scattering low coherence interferometry (ms2/LCI), by using a new beam scanning mechanism based on a microelectro-mechanical system (MEMS) mirror. The data acquisition scheme was altered to take advantage of this scanning speed, producing tomographic images at a rate of 4 frames (B-scans) per second. DA-OCT differs from ms2/LCI in that the dual axes intersect at a shallower depth (∼1 mm). This difference, coupled with the faster scanning speed, shifts the detection priority from multiply scattered to ballistic light. The utility of this approach was demonstrated by imaging both ex vivo porcine ear skin and in vivo rat skin from a McFarlane flap model. The enhanced penetration depth provided by the DA-OCT system will be beneficial to various clinical applications in dermatology and surgery.

In vivo analysis of burns in a mouse model using spectroscopic optical coherence tomography.

Spectroscopic analysis of biological tissues can provide insight into changes in structure and function due to disease or injury. Depth-resolved spectroscopic measurements can be implemented for tissue imaging using optical coherence tomography (OCT). Here, spectroscopic OCT is applied to in vivo measurement of burn injury in a mouse model. Data processing and analysis methods are compared for their accuracy. Overall accuracy in classifying burned tissue was found to be as high as 91%, producing an area under the curve of a receiver operating characteristic curve of 0.97. The origins of the spectral changes are identified by correlation with histopathology.

Mechanisms of bone fragility in a mouse model of glucocorticoid-treated rheumatoid arthritis: implications for insufficiency fracture risk.

OBJECTIVE: Glucocorticoid (GC) therapy is associated with increased risk of fracture in patients with rheumatoid arthritis (RA). To elucidate the cause of this increased risk, we examined the effects of chronic erosive inflammatory arthritis and GC treatment on bone quality, structure, and biomechanical properties in a murine model. METHODS: Mice with established arthritis and expressing human tumor necrosis factor α (TNFα) transgene (Tg) and their wild-type (WT) littermates were continually treated with GC (prednisolone 5 mg/kg/day via subcutaneous controlled-release pellet) or placebo for 14, 28, or 42 days. Microstructure, biomechanical properties, chemical composition, and morphology of the tibiae and lumbar vertebral bodies were assessed by micro-computed tomography, biomechanical testing, Raman spectroscopy, and histology, respectively. Serum markers of bone turnover were also determined. RESULTS: TNF-Tg and GC treatment additively decreased mechanical strength and stiffness in both the tibiae and the vertebral bodies. GC treatment in the TNF-Tg mice increased the ductility of tibiae under torsional loading. These changes were associated with significant alterations in the biochemical and structural composition of the mineral and organic components of the bone matrix, a decrease in osteoblast activity and bone formation, and an increase in osteoclast activity. CONCLUSION: Our findings indicate that the concomitant decrease in bone strength and increase in bone ductility associated with chronic inflammation and GC therapy, coupled with the significant changes in the bone quality and structure, may increase the susceptibility of the bone to failure under low-energy loading. This may explain the mechanism of symptomatic insufficiency fractures in patients with RA receiving GC therapy who do not have radiographic manifestations of fracture.

Spatially offset Raman spectroscopy for in vivo bone strength prediction.

Bone strength is a worldwide health concern. Although multiple techniques have been developed to evaluate bone quality, there are still gaps to be filled. Here we report a non-invasive approach for the prediction of bone strength in vivo using spatially offset Raman spectroscopy. Raman spectra were acquired transcutaneously from the tibiae of mice from 4 to 23 weeks old and subsequently on the exposed bones. Partial least squares regression was applied to generate predictions of the areal bone mineral density (aBMD), volumetric bone mineralization density (vBMD), and maximum torque (MT) of each tibia as quantified by dual-energy X-ray absorptiometry, microCT imaging, and biomechanical tests, respectively. Significant correlations were observed between Raman spectral predictions and the reference values in all three categories. To our knowledge, this is the first demonstration of Raman spectroscopy predicting a biomechanical bone parameter (MT) in vivo with an uncertainty much smaller than the spread in the reference values.

Full depth measurement of tenofovir transport in rectal mucosa using confocal Raman spectroscopy and optical coherence tomography.

The prophylactic activity of antiretroviral drugs applied as microbicides against sexually transmitted HIV is dependent upon their concentrations in infectable host cells. Within mucosal sites of infection (e.g., vaginal and rectal mucosa), those cells exist primarily in the stromal layer of the tissue. Traditional pharmacokinetic studies of these drugs have been challenged by poor temporal and spatial specificity. Newer techniques to measure drug concentrations, involving Raman spectroscopy, have been limited by laser penetration depth into tissue. Utilizing confocal Raman spectroscopy (RS) in conjunction with optical coherence tomography (OCT), a new lateral imaging assay enabled concentration distributions to be imaged with spatial and temporal specificity throughout the full depth of a tissue specimen. The new methodology was applied in rectal tissue using a clinical rectal gel formulation of 1% tenofovir (TFV). Confocal RS revealed diffusion-like behavior of TFV through the tissue specimen, with significant partitioning of the drug at the interface between the stromal and adipose tissue layers. This has implications for drug delivery to infectable tissue sites. The new assay can be applied to rigorously analyze microbicide transport and delineate fundamental transport parameters of the drugs (released from a variety of delivery vehicles) throughout the mucosa, thus informing microbicide product design.

Label-free analysis of tenofovir delivery to vaginal tissue using co-registered confocal Raman spectroscopy and optical coherence tomography.

Vaginally applied microbicide products offer a female-controlled strategy for preventing sexual transmission of HIV. Microbicide transport processes are central to their functioning, and there is a clear need for a better understanding of them. To contribute to that end, we developed an assay to analyze mass transport rates of microbicide molecules within the epithelial and stromal layers of polarized vaginal mucosal tissue during contact with a gel vehicle. The assay utilizes a new diffusion chamber mounted in a custom instrument that combines confocal Raman spectroscopy and optical coherence tomography. This measures depth-resolved microbicide concentration distributions within epithelium and stroma. Data for a tenofovir gel were fitted with a compartmental diffusion model to obtain fundamental transport properties: the molecular diffusion and partition coefficients in different compartments. Diffusion coefficients in epithelium and stroma were computed to be 6.10 ± 2.12 x 10-8 and 4.52 ± 1.86 x 10-7 cm2/sec, respectively. The partition coefficients between epithelium and gel and between stroma and epithelium were found to be 0.53 ± 0.15 and 1.17 ± 0.16, respectively. These drug transport parameters are salient in governing the drug delivery performance of different drug and gel vehicle systems. They can be used to contrast drugs and vehicles during product design, development and screening. They are critical inputs to deterministic transport models that predict the gels' pharmacokinetic performance, which can guide improved design of products and optimization of their dosing regimens.

Sensitivity of spatially offset Raman spectroscopy (SORS) to subcortical bone tissue.

The development of spatially offset Raman spectroscopy (SORS) has enabled deep, non-invasive chemical characterization of turbid media. Here, we use SORS to measure subcortical bone tissue and depth-resolved biochemical variability in intact, exposed murine bones. We also apply the technique to study a mouse model of the genetic bone disorder osteogenesis imperfecta. The results suggest that SORS is more sensitive to disease-related biochemical differences in subcortical trabecular bone and marrow than conventional Raman measurements.

Label-Free Measurements of Tenofovir Diffusion Coefficients in a Microbicide Gel Using Raman Spectroscopy.

Confocal Raman spectroscopy was implemented in a new label-free technique to quantify molecular diffusion coefficients within gels. A leading anti-HIV drug, tenofovir, was analyzed in a clinical microbicide gel. The gel was tested undiluted, and in 10%-50% wt/wt dilutions with vaginal fluid simulant to capture the range of conditions likely occurring in vivo. The concentration distributions of tenofovir in gel over time and space were measured and input to a mathematical diffusion model to deduce diffusion coefficients. These were 3.16 ± 0.11 × 10-6 cm2/s in undiluted gel, and increased by 11%-46% depending on the extent of dilution. Results were interpreted with respect to traditional release rate measurements in devices such as Franz cells. This comparison highlighted an advantage of our assay in that it characterizes the diffusive barrier within the gel material itself; in contrast, release rate in the traditional assay is affected by external conditions, such as drug partitioning at the gel/liquid sink interface. This new assay is relevant to diffusion in polymeric hydrogels over pharmacologically relevant length scales, for example, those characteristic of topical drug delivery. Resulting transport parameters are salient measures of drug delivery potential, and serve as inputs to computational models of drug delivery performance.

Deep imaging of absorption and scattering features by multispectral multiple scattering low coherence interferometry.

We have developed frequency domain multispectral multiple scattering low coherence interferometry (ms2/LCI) for deep imaging of absorption and scattering contrast. Using tissue-mimicking phantoms that match the full scattering phase function of human dermal tissue, we demonstrate that ms2/LCI can provide a signal/noise ratio (SNR) improvement of 15.4 dB over conventional OCT at an imaging depth of 1 mm. The enhanced SNR and penetration depth provided by ms2/LCI could be leveraged for a variety of clinical applications including the assessment of burn injuries where current clinical classification of severity only provides limited accuracy. The utility of the approach was demonstrated by imaging a tissue phantom simulating a partial-thickness burn revealing good spectroscopic contrast between healthy and injured tissue regions deep below the sample surface. Finally, healthy rat skin was imaged in vivo with both a commercial OCT instrument and our custom ms2/LCI system. The results demonstrate that ms2/LCI is capable of obtaining spectroscopic information far beyond the penetration depth provided by conventional OCT.

Functional optical coherence tomography: principles and progress.

In the past decade, several functional extensions of optical coherence tomography (OCT) have emerged, and this review highlights key advances in instrumentation, theoretical analysis, signal processing and clinical application of these extensions. We review five principal extensions: Doppler OCT (DOCT), polarization-sensitive OCT (PS-OCT), optical coherence elastography (OCE), spectroscopic OCT (SOCT), and molecular imaging OCT. The former three have been further developed with studies in both ex vivo and in vivo human tissues. This review emphasizes the newer techniques of SOCT and molecular imaging OCT, which show excellent potential for clinical application but have yet to be well reviewed in the literature. SOCT elucidates tissue characteristics, such as oxygenation and carcinogenesis, by detecting wavelength-dependent absorption and scattering of light in tissues. While SOCT measures endogenous biochemical distributions, molecular imaging OCT detects exogenous molecular contrast agents. These newer advances in functional OCT broaden the potential clinical application of OCT by providing novel ways to understand tissue activity that cannot be accomplished by other current imaging methodologies.

Evaluation of burn severity in vivo in a mouse model using spectroscopic optical coherence tomography.

Clinical management of burn injuries depends upon an accurate assessment of the depth of the wound. Current diagnostic methods rely primarily on subjective visual inspection, which can produce variable results. In this study, spectroscopic optical coherence tomography was used to objectively evaluate burn injuries in vivo in a mouse model. Significant spectral differences were observed and correlated with the depth of the injury as determined by histopathology. The relevance of these results to clinical burn management in human tissues is discussed.

Co-localized confocal Raman spectroscopy and optical coherence tomography (CRS-OCT) for depth-resolved analyte detection in tissue.

We report the development of a combined confocal Raman spectroscopy (CRS) and optical coherence tomography (OCT) instrument (CRS-OCT) capable of measuring analytes in targeted biological tissues with sub-100-micron spatial resolution. The OCT subsystem was used to measure depth-resolved tissue morphology and guide the acquisition of chemically-specific Raman spectra. To demonstrate its utility, the instrument was used to accurately measure depth-resolved, physiologically-relevant concentrations of Tenofovir, a microbicide drug used to prevent the sexual transmission of HIV, in ex vivo tissue samples.

Sensitivity of coded aperture Raman spectroscopy to analytes beneath turbid biological tissue and tissue-simulating phantoms.

Traditional slit-based spectrometers have an inherent trade-off between spectral resolution and throughput that can limit their performance when measuring diffuse sources such as light returned from highly scattering biological tissue. Recently, multielement fiber bundles have been used to effectively measure diffuse sources, e.g., in the field of spatially offset Raman spectroscopy, by remapping the source (or some region of the source) into a slit shape for delivery to the spectrometer. Another approach is to change the nature of the instrument by using a coded entrance aperture, which can increase throughput without sacrificing spectral resolution.In this study, two spectrometers, one with a slit-based entrance aperture and the other with a coded aperture, were used to measure Raman spectra of an analyte as a function of the optical properties of an overlying scattering medium. Power-law fits reveal that the analyte signal is approximately proportional to the number of transport mean free paths of the scattering medium raised to a power of -0.47 (coded aperture instrument) or -1.09 (slit-based instrument). These results demonstrate that the attenuation in signal intensity is more pronounced for the slit-based instrument and highlight the scattering regimes where coded aperture instruments can provide an advantage over traditional slit-based spectrometers.

Overconstrained library-based fitting method reveals age- and disease-related differences in transcutaneous Raman spectra of murine bones.

Clinical diagnoses of bone health and fracture risk typically rely on measurements of bone density or structure, but the strength of a bone is also dependent on its chemical composition. Raman spectroscopy has been used extensively in ex vivo studies to measure the chemical composition of bone. Recently, spatially offset Raman spectroscopy (SORS) has been utilized to measure bone transcutaneously. Although the results are promising, further advancements are necessary to make noninvasive, in vivo measurements of bone with SORS that are of sufficient quality to generate accurate predictions of fracture risk. In order to separate the signals from bone and soft tissue that contribute to a transcutaneous measurement, we developed an overconstrained extraction algorithm that is based on fitting with spectral libraries. This approach allows for accurate spectral unmixing despite the fact that similar chemical components (e.g., type I collagen) are present in both bone and soft tissue. The algorithm was utilized to transcutaneously detect biochemical differences in the tibiae of wild-type mice between 1 and 7 months of age and between the tibiae of wild-type mice and a mouse model of osteogenesis imperfecta. These results represent the first diagnostically sensitive, transcutaneous measurements of bone using SORS.

Bone fragility beyond strength and mineral density: Raman spectroscopy predicts femoral fracture toughness in a murine model of rheumatoid arthritis.

Clinical prediction of bone fracture risk primarily relies on measures of bone mineral density (BMD). BMD is strongly correlated with bone strength, but strength is independent of fracture toughness, which refers to the bone's resistance to crack initiation and propagation. In that sense, fracture toughness is more relevant to assessing fragility-related fracture risk, independent of trauma. We hypothesized that bone biochemistry, determined by Raman spectroscopy, predicts bone fracture toughness better than BMD. This hypothesis was tested in tumor necrosis factor-transgenic mice (TNF-tg), which develop inflammatory-erosive arthritis and osteoporosis. The left femurs of TNF-tg and wild type (WT) littermates were measured with Raman spectroscopy and micro-computed tomography. Fracture toughness was assessed by cutting a sharp notch into the anterior surface of the femoral mid-diaphysis and propagating the crack under 3 point bending. Femoral fracture toughness of TNF-tg mice was significantly reduced compared to WT controls (p=0.04). A Raman spectrum-based prediction model of fracture toughness was generated by partial least squares regression (PLSR). Raman spectrum PLSR analysis produced strong predictions of fracture toughness, while BMD was not significantly correlated and produced very weak predictions. Raman spectral components associated with mineralization quality and bone collagen were strongly leveraged in predicting fracture toughness, reiterating the limitations of mineralization density alone.

Heavy metal lead exposure, osteoporotic-like phenotype in an animal model, and depression of Wnt signaling.

BACKGROUND: Exposure to lead (Pb) from environmental and industrial sources remains an overlooked serious public health risk. Elucidating the effect of Pb on bone cell function is therefore critical for understanding its risk associated with diseases of low bone mass. OBJECTIVES: We tested the hypothesis that Pb negatively affects bone mass. We also assessed the underlying mechanisms of Pb on bone signaling pathways. METHODS: We used a model of low-level Pb exposure in a rodent beginning before conception and continuing over 18 months. We characterized the effect of Pb on bone quality using dual-energy X-ray absorptiometry (DXA), micro-computed tomography, Raman spectroscopy, and histology. We assessed the effect of Pb on bone and adipocyte formation by mineral deposition, lipid droplet formation, and Western blot and RNA analysis. RESULTS: Pb-exposed animals had decreased bone mass that resulted in bones that were more susceptible to fracture. Pb decreased osteoblastic cell number leading to a depression of bone formation. Accompanying this, Pb exposure elevated sclerostin protein levels in the skeleton, and correspondingly reduced levels of ß-catenin and Runx2 in stromal precursor cells. Pb also increased skeletal expression of peroxisome proliferator-activated receptor-γ (PPAR-γ). These results indicate a shift in mesenchymal differentiation wherein Pb promoted enhanced adipogenesis and decreased osteoblastogenesis. Substantial differences in bone marrow composition were observed, highlighted by an increase in adipocytes. CONCLUSIONS: The disruption Pb has on bone mass and bone homeostasis is principally explained by inhibition of the Wnt/ß-catenin pathway, which may provide a molecular basis for novel therapeutic strategies to combat Pb-induced bone pathologies.

Raman spectroscopy detects deterioration in biomechanical properties of bone in a glucocorticoid-treated mouse model of rheumatoid arthritis.

Although glucocorticoids are frequently prescribed for the symptomatic management of inflammatory disorders such as rheumatoid arthritis, extended glucocorticoid exposure is the leading cause of physician-induced osteoporosis and leaves patients at a high risk of fracture. To study the biochemical effects of glucocorticoid exposure and how they might affect biomechanical properties of the bone, Raman spectra were acquired from ex vivo tibiae of glucocorticoid- and placebo-treated wild-type mice and a transgenic mouse model of rheumatoid arthritis. Statistically significant spectral differences were observed due to both treatment regimen and mouse genotype. These differences are attributed to changes in the overall bone mineral composition, as well as the degree of phosphate mineralization in tibial cortical bone. In addition, partial least squares regression was used to generate a Raman-based prediction of each tibia's biomechanical strength as quantified by a torsion test. The Raman-based predictions were as accurate as those produced by microcomputed tomography derived parameters, and more accurate than the clinically-used parameter of bone mineral density. These results suggest that Raman spectroscopy could be a valuable tool for monitoring bone biochemistry in studies of bone diseases such as osteoporosis, including tests of drugs being developed to combat these diseases.

Determination of ideal offset for spatially offset Raman spectroscopy.

A key design parameter in spatially offset Raman spectroscopy (SORS) is the choice of offset distance between the illumination and collection areas. To investigate this choice, we performed SORS measurements on a simple two-layer chemical phantom. We show that while the SORS ratio, or the ratio of signal from the bottom layer to the top layer, monotonically increases with spatial offset, the signal-to-noise ratio (SNR) does not. Specifically, we show that there exists a specific spatial offset that yields the best SNR for signal originating in the bottom layer of a two-layer sample. We also show that this SNR-optimal offset depends upon the strength of the particular Raman band. This work presents the considerations that should be taken into account when designing optical probes for use in SORS.