Differentiating intratumoral melanocytes from Langerhans cells in nonmelanocytic pigmented skin tumorsin vivoby label-free third-harmonic generation microscopy

Abstract

Abstract. 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. © The Authors.Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Slide-free clinical imaging of melanin with absolute quantities using label-free third-harmonic-generation enhancement-ratio microscopy

“…THG microscopy, with the advantages of non-invasiveness, label-free, bleach-free, high penetration, and sub-femtoliter 3D resolution, has been applied for slide-free morphological and molecular visualizations of 3D sub-cellular structures inside living animals and humans [22], [26][27][28][29]. Under a tight focus, the high THG nonlinearity provides the 3D auto-sectioning capability without the need of a confocal pinhole [30].…”

In vivo third-harmonic generation microscopy study on vitiligo patients

“…Melanin is known to provide strong third-harmonic generation (THG) contrast in human skin. [7][8][9] A recent study 10 has further indicated the capability of noninvasive label-free THGM to differentiate melanocytes from Langerhans cells, which is not feasible in other in vivo labelfree clinical-imaging modalities. This characteristic makes THGM ideal for the assessment of the vitiligo therapy and, with its high three-dimensional (3-D) resolution, to facilitate the understanding of the biologic mechanisms of repigmentation in vitiligo.…”

Presence of intralesional melanocytes as a histopathological feature of actinic keratosis based on in vivo harmonic generation microscopy in Asians

“…They proposed that the presence of melanocytes might be explained by chronic sun‐damaged skin with mottled hyperpigmentation, often associated with AK and LCs was shown histologically to belong to the inflammatory infiltrates in skin lesions 12,33,34 . However, unlike RCM imaging, we previously showed that intratumoral THG‐bright dendritic cell‐like signals were enhanced by melanin and endogenously derived from melanocytes rather than LCs 28 . The kappa coefficient, sensitivity, and specificity for the consistency between THG‐bright dendritic cell‐like signals and HMB‐45 melanocyte staining were 0.807, 84.6%, and 95%, respectively 28 …”

“…However, unlike RCM imaging, we previously showed that intratumoral THG‐bright dendritic cell‐like signals were enhanced by melanin and endogenously derived from melanocytes rather than LCs 28 . The kappa coefficient, sensitivity, and specificity for the consistency between THG‐bright dendritic cell‐like signals and HMB‐45 melanocyte staining were 0.807, 84.6%, and 95%, respectively 28 …”

“…Our previous study showed that THG microscopic imaging did not enhance LCs. 28 Therefore, the THG-bright dendritic cells in AKs were melanocytes, which might increase in number or become active with dendrite elongation due to the influence of the AK microenvironment.…”