Snapshot Multispectral Imaging Is Not Inferior to SPY Laser Fluorescence Imaging When Predicting Murine Flap Necrosis.

BACKGROUND: Objective assessment of tissue viability is critical to improve outcomes of cosmetic and reconstructive procedures. A widely used method to predict tissue viability is indocyanine green angiography. The authors present an alternative method that determines the relative proportions of oxyhemoglobin to deoxyhemoglobin through multispectral reflectance imaging. This affordable, hand-held device is noninvasive and may be used in clinic settings. The authors hypothesize that multispectral reflectance imaging is not inferior to indocyanine green angiography in predicting flap necrosis in the murine model. METHODS: Reverse McFarlane skin flaps measuring 10 × 3 cm were raised on 300- to 400-g male Sprague-Dawley rats. Indocyanine green angiography and multispectral reflectance imaging was performed before surgery, immediately after surgery, and 30 minutes after surgery. Clinical outcome images acquired 72 hours after surgery were evaluated by three independent plastic surgeons. Objective data obtained immediately after surgery were compared to postsurgical clinical outcomes to determine which method more accurately predicted flap necrosis. RESULTS: Nine reverse McFarlane skin flaps were evaluated 72 hours after flap elevation. Data analysis demonstrated that the 95 percent confidence intervals for the sensitivity of postoperative multispectral reflectance imaging and indocyanine green angiography imaging to predict 72-hour tissue viability at a fixed specificity of 90 percent for predicting tissue necrosis were 86.3 to 91.0 and 79.1 to 86.9, respectively. CONCLUSIONS: In this experimental animal model, multispectral reflectance imaging does not appear to be inferior to indocyanine green angiography in detecting compromised tissue viability. With the advantages of noninvasiveness, portability, affordability, and lack of disposables, multispectral reflectance imaging has an exciting potential for widespread use in cosmetic and reconstructive procedures.

Fractal dimension and directional analysis of elastic and collagen fiber arrangement in unsectioned arterial tissues affected by atherosclerosis and aging.

Structural proteins like collagen and elastin are major constituents of the extracellular matrix (ECM). ECM degradation and remodeling in diseases significantly impact the microorganization of these structural proteins. Therefore, tracking the changes of collagen and elastin fiber morphological features within ECM impacted by disease progression could provide valuable insight into pathological processes such as tissue fibrosis and atherosclerosis. Benefiting from its intrinsic high-resolution imaging power and superior biochemical specificity, nonlinear optical microscopy (NLOM) is capable of providing information critical to the understanding of ECM remodeling. In this study, alterations of structural fibrillar proteins such as collagen and elastin in arteries excised from atherosclerotic rabbits were assessed by the combination of NLOM images and textural analysis methods such as fractal dimension (FD) and directional analysis (DA). FD and DA were tested for their performance in tracking the changes of extracellular elastin and fibrillar collagen remodeling resulting from atherosclerosis progression/aging. Although other methods of image analysis to study the organization of elastin and collagen structures have been reported, the simplified calculations of FD and DA presented in this work prove that they are viable strategies for extracting and analyzing fiber-related morphology from disease-impacted tissues. Furthermore, this study also demonstrates the potential utility of FD and DA in studying ECM remodeling caused by other pathological processes such as respiratory diseases, several skin conditions, or even cancer. NEW & NOTEWORTHY Textural analyses such as fractal dimension (FD) and directional analysis (DA) are straightforward and computationally viable strategies to extract fiber-related morphological data from optical images. Therefore, objective, quantitative, and automated characterization of protein fiber morphology in extracellular matrix can be realized by using these methods in combination with digital imaging techniques such as nonlinear optical microscopy (NLOM), a highly effective visualization tool for fibrillar collagen and elastic network. Combining FD and DA with NLOM is an innovative approach to track alterations of structural fibrillar proteins. The results illustrated in this study not only prove the effectiveness of FD and DA methods in extracellular protein characterization but also demonstrate their potential value in clinical and basic biomedical research where protein microstructure characterization is critical.

Near-Infrared Spectroscopy Imaging for Assessing Skin and Wound Oxygen Perfusion.

Near infrared spectroscopy (NIRS) is a technique whereby light is transmitted to the skin or the surface of a wound. Light is selectively absorbed or reflected in order to calculate the percentage of oxygenated and deoxygenated hemoglobin. By calculating this ratio, the level of tissue oxygen perfusion can be assessed. This article describes an NIRS imaging device and demonstrates its use as a clinical tool to predict future viability of skin flaps, potential for wound healing, and progress of closure following application of a biologic wound product. Several cases are examined.

Review of near-infrared methods for wound assessment.

Wound management is a challenging and costly problem that is growing in importance as people are living longer. Instrumental methods are increasingly being relied upon to provide objective measures of wound assessment to help guide management. Technologies that employ near-infrared (NIR) light form a prominent contingent among the existing and emerging technologies. We review some of these technologies. Some are already established, such as indocyanine green fluorescence angiography, while we also speculate on others that have the potential to be clinically relevant to wound monitoring and assessment. These various NIR-based technologies address clinical wound management needs along the entire healing trajectory of a wound.

Quantitative nonlinear optical assessment of atherosclerosis progression in rabbits.

Quantification of atherosclerosis has been a challenging task owing to its complex pathology. In this study, we validated a quantitative approach for assessing atherosclerosis progression in a rabbit model using a numerical matrix, optical index for plaque burden, derived directly from the nonlinear optical microscopic images captured on the atherosclerosis-affected blood vessel. A positive correlation between this optical index and the severity of atherosclerotic lesions, represented by the age of the rabbits, was established based on data collected from 21 myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits with age ranging between new-born and 27 months old. The same optical index also accurately identified high-risk locations for atherosclerotic plaque formation along the entire aorta, which was validated by immunohistochemical fluorescence imaging.

Assessment of tissue oxygenation of periodontal inflammation in patients with coronary artery diseases using optical spectroscopy.

BACKGROUND: We have recently developed a non-invasive periodontal diagnostic tool that was validated in periodontitis patients without systemic disorders like coronary artery disease (CAD). The purpose of present study is to verify whether this optical instrument can also be used in periodontitis patients with CAD. METHODS: A total of 62 periodontitis patients with CAD were recruited along with a control group consisting of 59 age and gender matched periodontitis volunteers without systemic disorders. Using a portable optical near-infrared spectrometer, optical spectra were obtained, processed and evaluated from the two groups. A modified Beer-Lambert unmixing model that incorporates a nonparametric scattering loss function was used to determine the relative contribution of deoxygenated hemoglobin (Hb) and oxygenated hemoglobin (HbO2) to the overall spectrum. The balance between tissue oxygen delivery and utilization in periodontal tissues was then assessed. RESULTS: Tissue oxygen saturation was significantly decreased in the periodontitis sites (p < 0.01), compared to the healthy sites in those individuals with CAD. There was a trend towards increased concentration of Hb and decreased concentration of HbO2 from healthy to diseased sites, without statistical significance (p > 0.05). No statistical differences were found in tissue oxygen saturation between the CAD and control groups either in periodontal healthy or inflammatory sites. CONCLUSION: This study supports the hypothesis that optical spectroscopy can determine the periodontal inflammation in patients with certain systemic disorders like CAD. And the overall periodontal oxygenation profiles in CAD patients resemble those in non-CAD individuals either in healthy or inflammatory sites.

Caspase 3 activity in isolated fetal rat lung fibroblasts and rat periodontal ligament fibroblasts: cigarette smoke induced alterations.

BACKGROUND: Cigarette smoking is the leading cause of preventable death and has been implicated in pathogenesis of pulmonary, oral and systemic diseases. Smoking during pregnancy is a risk factor for the developing fetus and may be a major cause of infant mortality. Moreover, the oral cavity, and all cells within are the first to be exposed to cigarette smoke and may be a possible source for the spread of toxins to other organs of the body. Fibroblasts in general are morphologically heterogeneous connective tissue cells with diverse functions. Apoptosis or programmed cell death is a crucial process during embryogenesis and for the maintenance of homeostasis throughout life. Deregulation of apoptosis has been implicated in abnormal lung development in the fetus and disease progression in adults. Caspases are proteases which belong to the family of cysteine aspartic acid proteases and are key components for downstream amplification of intracellular apoptotic signals. Of 14 known caspases, caspase-3 is the key executioner of apoptosis. In the present study we explored the hypothesis that cigarette smoke (CS) extract activates caspase-3 in two types of fibroblasts, both of which would be exposed directly to cigarette smoke, isolated fetal rat lung fibroblasts and adult rat periodontal ligament (PDL) fibroblasts. METHODS: Isolated fetal rat lung fibroblasts and adult PDLs were used. Cells were exposed to different concentrations of CS for 60 min. Caspase-3 activity and its inhibition by Z-VAD-fmk were measured by caspase-3 fluorometric assay. The effect of CSE on cellular viability was measured using the MTT formazan assay. Caspase-3 expression was detected by western blot analysis and cellular localization of caspase-3 was determined by immunofluorescence using fluorescence microscopy. RESULTS: It was observed in fetal rat lung fibroblast cells that CSE extract significantly (p<0.05) increased caspase-3 activity and decrease cell proliferation. However, no significant changes in activity or viability were observed in PDLs. CONCLUSIONS: This indicates CS activates caspase-3 the key regulatory point in apoptosis in fetal rat lung fibroblast cells suggesting that smoking during pregnancy may alter the developmental program of fetal lung, jeopardizing the establishment of critical cellular mechanisms necessary to expedite pulmonary maturation at birth.of critical cellular mechanisms necessary to expedite pulmonary maturation at birth.

Collagen morphology and texture analysis: from statistics to classification.

In this study we present an image analysis methodology capable of quantifying morphological changes in tissue collagen fibril organization caused by pathological conditions. Texture analysis based on first-order statistics (FOS) and second-order statistics such as gray level co-occurrence matrix (GLCM) was explored to extract second-harmonic generation (SHG) image features that are associated with the structural and biochemical changes of tissue collagen networks. Based on these extracted quantitative parameters, multi-group classification of SHG images was performed. With combined FOS and GLCM texture values, we achieved reliable classification of SHG collagen images acquired from atherosclerosis arteries with >90% accuracy, sensitivity and specificity. The proposed methodology can be applied to a wide range of conditions involving collagen re-modeling, such as in skin disorders, different types of fibrosis and muscular-skeletal diseases affecting ligaments and cartilage.

Real-time control of angioplasty balloon inflation based on feedback from intravascular optical coherence tomography: experimental validation on an excised heart and a beating heart model.

We report on real-time control of balloon inflation inside porcine arteries. In the first step, experiments were done in a coronary artery of an excised heart. In the second step, experiments were done in a beating heart setup providing conditions very close to in vivo conditions without the complications. A programmable syringe pump was used to inflate a compliant balloon in arteries, while intravascular optical coherence tomography (IVOCT) monitoring was performed. In a feedback loop, IVOCT images were processed to provide the balloon diameter values in real time to control the pump action in order to achieve a target diameter. In different experiments, various flow rates and target diameters were used. In the excised heart experiment, there was good convergence to target diameters resulting in a satisfactory balloon inflation control. In the beating heart experiment, there were oscillations in the diameter values due to cyclic arterial contractions. In these experiments, the control system maintained diameter averages satisfactorily close to predetermined target values. Real-time control of balloon inflation could not only provide a safer outcome for angioplasty procedures, but could also provide additional information for diagnostics since it implicitly provides information about the artery response to the inflation process.

Nonlinear optical microscopy in decoding arterial diseases.

Pathological understanding of arterial diseases is mainly attributable to histological observations based on conventional tissue staining protocols. The emerging development of nonlinear optical microscopy (NLOM), particularly in second-harmonic generation, two-photon excited fluorescence and coherent Raman scattering, provides a new venue to visualize pathological changes in the extracellular matrix caused by atherosclerosis progression. These techniques in general require minimal tissue preparation and offer rapid three-dimensional imaging. The capability of label-free microscopic imaging enables disease impact to be studied directly on the bulk artery tissue, thus minimally perturbing the sample. In this review, we look at recent progress in applications related to arterial disease imaging using various forms of NLOM.

A comparison of methods using optical coherence tomography to detect demineralized regions in teeth.

Optical coherence tomography (OCT) is a three- dimensional optical imaging technique that can be used to identify areas of early caries formation in dental enamel. The OCT signal at 850 nm back-reflected from sound enamel is attenuated stronger than the signal back-reflected from demineralized regions. To quantify this observation, the OCT signal as a function of depth into the enamel (also known as the A-scan intensity), the histogram of the A-scan intensities and three summary parameters derived from the A-scan are defined and their diagnostic potential compared. A total of 754 OCT A-scans were analyzed. The three summary parameters derived from the A-scans, the OCT attenuation coefficient as well as the mean and standard deviation of the lognormal fit to the histogram of the A-scan ensemble show statistically significant differences (p < 0.01) when comparing parameters from sound enamel and caries. Furthermore, these parameters only show a modest correlation. Based on the area under the curve (AUC) of the receiver operating characteristics (ROC) plot, the OCT attenuation coefficient shows higher discriminatory capacity (AUC = 0.98) compared to the parameters derived from the lognormal fit to the histogram of the A-scan. However, direct analysis of the A-scans or the histogram of A-scan intensities using linear support vector machine classification shows diagnostic discrimination (AUC = 0.96) comparable to that achieved using the attenuation coefficient. These findings suggest that either direct analysis of the A-scan, its intensity histogram or the attenuation coefficient derived from the descending slope of the OCT A-scan have high capacity to discriminate between regions of caries and sound enamel.

Evaluation of texture parameters for the quantitative description of multimodal nonlinear optical images from atherosclerotic rabbit arteries.

The composition and structure of atherosclerotic lesions can be directly related to the risk they pose to the patient. Multimodal nonlinear optical (NLO) microscopy provides a powerful means to visualize the major extracellular components of the plaque that critically determine its structure. Textural features extracted from NLO images were investigated for their utility in providing quantitative descriptors of structural and compositional changes associated with plaque development. Ten texture parameters derived from the image histogram and gray level co-occurrence matrix were examined that highlight specific structural and compositional motifs that distinguish early and late stage plaques. Tonal-texture parameters could be linked to key histological features that characterize vulnerable plaque: the thickness and density of the fibrous cap, size of the atheroma, and the level of inflammation indicated through lipid deposition. Tonal and texture parameters from NLO images provide objective metrics that correspond to structural and biochemical changes that occur within the vessel wall in early and late stage atherosclerosis.

Assessment of local hemodynamics in periodontal inflammation using optical spectroscopy.

BACKGROUND: Among the newly emerging diagnostic approaches for periodontitis, optical spectroscopy is a promising complementary diagnostic tool. The objective of this study is to verify the reproducibility of this method at a geographically distinct location (Suzhou, China) to a broader patient population using similar instrumentation to that in a previous report. METHODS: Using a portable optical near-infrared spectrometer, optical spectra were obtained, processed, and evaluated from healthy (n = 62), gingivitis (n = 98), and periodontitis (n = 47) sites from a total of 51 patients. A modified Beer-Lambert unmixing model that incorporates a non-parametric scattering loss function was used to determine the relative contribution of oxyhemoglobin and deoxyhemoglobin to the overall spectrum. The balance between tissue oxygen delivery and oxygen use in periodontal tissues was then assessed. RESULTS: Tissue oxygenation decreased significantly from healthy sites to sites with gingivitis (P <0.01) and between gingivitis and periodontitis (P = 0.015). This is largely caused by a significant increase in deoxyhemoglobin between normal and gingivitis (P <0.01) and a concomitant decrease in oxyhemoglobin between gingivitis and periodontitis (P = 0.02). CONCLUSION: This study supports previous findings that tissue oxygenation as measured by optical spectroscopy is significantly decreased in periodontitis and that optical spectroscopy can simultaneously determine multiple inflammatory indices related to periodontal disease directly in gingival tissues in vivo.

Optical coherence tomography: fundamental principles, instrumental designs and biomedical applications.

The advances made in the last two decades in interference technologies, optical instrumentation, catheter technology, optical detectors, speed of data acquisition and processing as well as light sources have facilitated the transformation of optical coherence tomography from an optical method used mainly in research laboratories into a valuable tool applied in various areas of medicine and health sciences. This review paper highlights the place occupied by optical coherence tomography in relation to other imaging methods that are used in medical and life science areas such as ophthalmology, cardiology, dentistry and gastrointestinal endoscopy. Together with the basic principles that lay behind the imaging method itself, this review provides a summary of the functional differences between time-domain, spectral-domain and full-field optical coherence tomography, a presentation of specific methods for processing the data acquired by these systems, an introduction to the noise sources that plague the detected signal and the progress made in optical coherence tomography catheter technology over the last decade.

Intravascular optical coherence tomography on a beating heart model.

The advantages and limitations of using a beating heart model in the development of intravascular optical coherence tomography are discussed. The model fills the gap between bench experiments, performed on phantoms and excised arteries, and whole animal in-vivo preparations. The beating heart model is stable for many hours, allowing for extended measurement times and multiple imaging sessions under in-vivo conditions without the complications of maintaining whole-animal preparation. The perfusate supplying the heart with nutrients can be switched between light scattering blood to a nonscattering perfusate to allow the optical system to be optimized without the need of an efficient blood displacement strategy. Direct access to the coronary vessels means that there is no need for x-ray fluoroscopic guidance of the catheter to the heart, as is the case in whole animal preparation. The model proves to be a valuable asset in the development of our intravascular optical coherence tomography technology.

Determining the effect of calculus, hypocalcification, and stain on using optical coherence tomography and polarized Raman spectroscopy for detecting white spot lesions.

Optical coherence tomography (OCT) and polarized Raman spectroscopy (PRS) have been shown as useful methods for distinguishing sound enamel from carious lesions ex vivo. However, factors in the oral environment such as calculus, hypocalcification, and stain could lead to false-positive results. OCT and PRS were used to investigate extracted human teeth clinically examined for sound enamel, white spot lesion (WSL), calculus, hypocalcification, and stain to determine whether these factors would confound WSL detection with these optical methods. Results indicate that OCT allowed differentiating caries from sound enamel, hypocalcification, and stain, with calculus deposits recognizable on OCT images. ANOVA and post-hoc unequal N HSD analyses to compare the mean Raman depolarization ratios from the various groups showed that the mean values were statistically significant at P < .05, except for several comparison pairs. With the current PRS analysis method, the mean depolarization ratios of stained enamel and caries are not significantly different due to the sloping background in the stained enamel spectra. Overall, calculus and hypocalcification are not confounding factors affecting WSL detection using OCT and PRS. Stain does not influence WSL detection with OCT. Improved PRS analysis methods are needed to differentiate carious from stained enamel.

Multimodal nonlinear optical imaging of atherosclerotic plaque development in myocardial infarction-prone rabbits.

Label-free imaging of bulk arterial tissue is demonstrated using a multimodal nonlinear optical microscope based on a photonic crystal fiber and a single femtosecond oscillator operating at 800 nm. Colocalized imaging of extracellular elastin fibers, fibrillar collagen, and lipid-rich structures within aortic tissue obtained from atherosclerosis-prone myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits is demonstrated through two-photon excited fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering, respectively. These images are shown to differentiate healthy arterial wall, early atherosclerotic lesions, and advanced plaques. Clear pathological changes are observed in the extracellular matrix of the arterial wall and correlated with progression of atherosclerotic disease as represented by the age of the WHHLMI rabbits.

An update on novel non-invasive approaches for periodontal diagnosis.

For decades there has been an ongoing search for clinically acceptable methods for the accurate, non-invasive diagnosis and prognosis of periodontitis. There are several well-known inherent drawbacks with current clinical procedures. The purpose of this review is to summarize some of the newly emerging diagnostic approaches, namely, infrared spectroscopy, optical coherence tomography (OCT), and ultrasound. The history and attractive features of these new approaches are briefly illustrated, and the interesting and significant inventions related to dental applications are discussed. The particularly attractive aspects for the dental community are that some of these methods are totally non-invasive, do not impose any discomforts to the patients during the procedure, and require no tissue to be extracted. For instance, multiple inflammatory indices withdrawn from near infrared spectra have the potential to identify early signs of inflammation leading to tissue breakdown. Morphologically, some other non-invasive imaging modalities, such as OCT and ultrasound, could be employed to accurately measure probing depths and assess the status of periodontal attachment, the front-line of disease progression. Given that these methods reflect a completely different assessment of periodontal inflammation, if clinically validated, these methods could either replace traditional clinical examinations for the diagnosis of periodontitis or at least serve as attractive complementary diagnostic tools. However, the potential of these techniques should be interpreted more cautiously given the multifactorial character of periodontal disease. In addition to these novel tools in the field of periodontal inflammatory diseases, other alternative modalities like microbiologic and genetic approaches are only briefly mentioned in this review because they have been thoroughly discussed in other comprehensive reviews.

Differentiating atherosclerotic plaque burden in arterial tissues using femtosecond CARS-based multimodal nonlinear optical imaging.

A femtosecond CARS-based nonlinear optical microscope was used to simultaneously image extracellular structural proteins and lipid-rich structures within intact aortic tissue obtained from myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits (WHHLMI). Clear differences in the NLO microscopic images were observed between healthy arterial tissue and regions dominated by atherosclerotic lesions. In the current ex-vivo study, we present a single parameter based on intensity changes derived from multi-channel NLO image to classify plaque burden within the vessel. Using this parameter we were able to differentiate between healthy regions of the vessel and regions with plaque, as well as distinguish plaques relative to the age of the WHHLMI rabbit.

Signal attenuation and box-counting fractal analysis of optical coherence tomography images of arterial tissue.

The sensitivity of optical coherence tomography images to sample morphology is tested by two methods. The first method estimates the attenuation of the OCT signal from various regions of the probed tissue. The second method uses a box-counting algorithm to calculate the fractal dimensions in the regions of interest identified in the images. Although both the attenuation coefficient as well as the fractal dimension correlate very well with the anatomical features of the probed samples; the attenuation method provides a better sensitivity. Two types of samples are used in this study: segments of arteries collected from atherosclerosis-prone Watanabe rabbits (WHHL-MI) and healthy segments of porcine coronary arteries.