Morphology and distribution of melanocytes are critical imaging information for the diagnosis of melanocytic lesions. However, how to image intratumoral melanocytes noninvasively in pigmented skin tumors is seldom investigated. Third-harmonic generation (THG) is shown to be enhanced by melanin, whereas high accuracy has been demonstrated using THG microscopy for in vivo differential diagnosis of nonmelanocytic pigmented skin tumors. It is thus desirable to investigate if label-free THG microscopy was capable to in vivo identify intratumoral melanocytes. In this study, histopathological correlations of label-free THG images with the immunohistochemical images stained with human melanoma black (HMB)-45 and cluster of differentiation 1a (CD1a) were made. The correlation results indicated that the intratumoral THG-bright dendritic-cell-like signals were endogenously derived from melanocytes rather than Langerhans cells (LCs). The consistency between THG-bright dendritic-cell-like signals and HMB-45 melanocyte staining showed a kappa coefficient of 0.807, 84.6% sensitivity, and 95% specificity. In contrast, a kappa coefficient of −0.37, 21.7% sensitivity, and 30% specificity were noted between the THG-bright dendritic-cell-like signals and CD1a staining for LCs. Our study indicates the capability of noninvasive label-free THG microscopy to differentiate intratumoral melanocytes from LCs, which is not feasible in previous in vivo label-free clinical-imaging modalities.
In vivo harmonic generation microscopy (HGM) has been applied successfully in healthy human skin and can achieve a submicron resolution, similar to histopathologic examination, even at a penetration depth up to 270 μm. This study aims to investigate the clinical applicability of HGM imaging for differential diagnosis of nonmelanoma pigmented skin lesions. A total of 42 pigmented skin tumors, including pigmented basal cell carcinoma, melanocytic nevus, and seborrheic keratosis were evaluated by HGM ex vivo or in vivo. Based on the standard histopathologic characteristics, we established the corresponding HGM imaging criteria for each pigmented tumor. Diagnostic performance of HGM for differentiating nonmelanoma pigmented skin tumors was evaluated through the observers’ direct general assessment (overall evaluation) or the presence of two imaging criteria with the highest sensitivity and specificity (major criteria evaluation). Our results show that, based on the direct general assessment, the sensitivity is 92% [95% confidence interval (CI): 67 to 97%] and the specificity is 96% (95% CI: 83 to 99%); by major criteria evaluation, 94% sensitivity (95% CI: 70 to 99%) and 100% specificity (95% CI: 87 to 100%) are achieved. Our study indicates that HGM serves as a promising histopathological examination tool for noninvasive differential diagnostics of nonmelanoma pigmented skin tumors.
Third-harmonic generation (THG) microscopy has been reported to provide intrinsic contrast in elastic fibers, cytoplasmic membrane, nucleus, actin filaments, lipid bodies, hemoglobin, and melanin in human skin. For advanced molecular imaging, exogenous contrast agents are developed for a higher structural or molecular specificity. We demonstrate the potential of the commonly adopted tattoo dye as a THG contrast agent for in vivo optical biopsy of human skin. Spectroscopy and microscopy experiments were performed on cultured cells with tattoo dyes, in tattooed mouse skin, and in tattooed human skin to demonstrate the THG enhancement effect. Compared with other absorbing dyes or nanoparticles used as exogenous THG contrast agents, tattoo dyes are widely adopted in human skin so that future clinical biocompatibility evaluation is relatively achievable. Combined with the demonstrated THG enhancement effect, tattoo dyes show their promise for future clinical imaging applications.
KEYWORDS: Skin, Liquid crystals, In vivo imaging, Second-harmonic generation, Microscopy, Lymphatic system, FDA class II medical device development, Video, Molecules, Nonlinear optics
Atopic dermatitis (AD) is an allergic inflammatory disease of skin. It remains unclear that CD207 of Langerhans cells (LCs) plays a central role in the development of allergic sensitization. There is little data on LCs within the microenviroment in vivo. We used a murine model of epicutaneous (EC) ovalbumin (OVA) sensitization inducing an inflammatory skin resembling AD to explore the role of CD207 in the pathogenesis of AD. Cr:forsterite laser-based multimodality nonlinear microscopy was applied for in situ imaging. Peritoneal injections of Alexa Fluor 647-rat anti-mouse CD207 into mice were performed to specifically trace the LCs. Peritoneal injections of OVA-Alexa Fluor 647 conjugate into mice were performed to specifically trace the OVA. We found that combining Alexa Fluor fluorescent probes with multimodality nonlinear microscopy permitted the unequivocal in situ imaging of CD207-expressing LCs. The relevant time-course, expressional, and functional studies reveal that CD207 of LCs plays an essential role during the induction of EC sensitization. We establish and validate that Cr:forsterite laser-based multimodality nonlinear microscopy is applicable for the specific detection of labeled mAb-bound LCs and labeled antigen. We suggest that CD207-expressing LCs initiate the allergic response through the CD207 mediated epicutaneous sensitization associated with the development of AD.
Atrial fibrillation (AF) is the most common irregular heart rhythm and the mortality rate for patients with AF is approximately twice the mortality rate for patients with normal sinus rhythm (NSR). Some research has indicated that myocardial fibrosis plays an important role in predisposing patients to AF. Therefore, realizing the relationship between myocardial collagen fibrosis and AF is significant. Second-harmonic generation (SHG) is an optically nonlinear coherent process to image the collagen network. We perform SHG microscopic imaging of the collagen fibers in the human atrial myocardium. Utilizing the SHG images, we can identify the differences in morphology and the arrangement of collagen fibers between NSR and AF tissues. We also quantify the arrangement of the collagen fibers using Fourier transform images and calculating the values of angle entropy. We indicate that SHG imaging, a nondestructive and reproducible method to analyze the arrangement of collagen fibers, can provide explicit information about the relationship between myocardial fibrosis and AF.
Oral cancer ranked number four in both cancer incident and mortality in Taiwanese male population. Early disease
diagnosis and staging is essential for its clinical success. However, most patients were diagnosed in their late disease
stage as ideal prescreening procedures are yet to be developed especially when dealing with a large surface of
precancerous lesions. Therefore, how to detect and confirm the diagnosis of these early stage lesions are of significant
clinical value. Harmonic generation process naturally occurred in biological molecules and requires no energy deposition
to the target molecule. Thus harmonic generation microscopy (HGM) could potentially serve as a noninvasive tool for
screening of human oral mucosal diseases. The in vivo optical biopsy of human oral cavity with HGM could be achieved
with high spatial resolution to resolve dynamic physiological process in the oral mucosal tissue with equal or superior
quality but devoid of complicated physical biopsy procedures. The second harmonic generation (SHG) provide
significant image contrast for biomolecules with repetitive structures such as the collagen fibers in the lamina propria
and the mitotic spindles in dividing cells. The cell morphology in the epithelial layer, blood vessels and blood cells flow
through the capillaries can be revealed by third harmonic generation (THG) signals. Tissue transparent technology was
used to increase the optical penetration of the tissue. In conclusion, this report demonstrates the first in vivo optical
virtual biopsy of human oral mucosa using HGM and revealed a promising future for its clinical application for
noninvasive in vivo diseases diagnosis.
Myocardial fibrosis, a common sequela of cardiac hypertrophy, has been shown to be associated with arrhythmias in
experimental models. Some research has indicated that myocardial fibrosis plays an important role in predisposing
patients to atrial fibrillation. Second harmonic generation (SHG) is an optically nonlinear coherent process to image the
collagen network. In this presentation, we observe the SHG images of the collagen matrix in atrial myocardium and we
analyzed of collagen fibers arrangement by using Fourier-transform analysis. Moreover, comparing the SHG images of
the collagen fibers in atrial myocardium between normal sinus rhythm (NSR) and atrial fibrillation (AF), our result
indicated that it is possible to realize the relation between myocardial fibrosis and AF.
Cartilage matrix is damaged in diseased states such as osteoarthritis, while adult articular cartilage does not have the
capacity to repair structural damage. A least invasive mean to diagnose these diseased states of human articular cartilage
with a high spatial resolution is thus highly desired. In this paper, we present our harmonic generation microscopic
studies on the human articular cartilage samples. Without any staining, third and second harmonic generation can
provide strong contrast in chondrocytes and collagen matrix, respectively. Our study indicates the high capability of
harmonic generation microscopy for future articular cartilage disease diagnosis.
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