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Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to simulate histology-like appearance.

ABSTRACT: Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Mosaicing may offer a means to perform rapid histology at the bedside. A possible barrier toward clinical acceptance is that the mosaics are based on a single mode of grayscale contrast and appear black and white, whereas histology is based on two stains (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple and pink. Toward addressing this barrier, we report advances in digital staining: fluorescence mosaics that show only nuclei, are digitally stained purple and overlaid on reflectance mosaics, which show only cellular cytoplasm and dermis, and are digitally stained pink. With digital staining, the appearance of confocal mosaics mimics the appearance of histology. Using multispectral analysis and color matching functions, red, green, and blue (RGB) components of hematoxylin and eosin stains in tissue were determined. The resulting RGB components were then applied in a linear algorithm to transform fluorescence and reflectance contrast in confocal mosaics to the absorbance contrast seen in pathology. Optimization of staining with acridine orange showed improved quality of digitally stained mosaics, with good correlation to the corresponding histology.

SUBMITTER: Bini J

PROVIDER: S-EPMC3154052 | biostudies-other | 2011 Jul

REPOSITORIES: biostudies-other

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Project description:BackgroundEx vivo confocal microscopy (CM) works under two modes, fluorescence and reflectance, allowing the visualization of different structures. Fluorescence CM (FCM) requires a contrast agent and has been used for the analysis of basal cell carcinomas (BCCs) during Mohs surgery. Conversely, reflectance CM (RCM) is mostly used for in vivo diagnosis of equivocal skin tumours. Recently, a new, faster ex vivo confocal microscope has been developed which simultaneously uses both lasers (fusion mode).ObjectivesTo describe the BCC features identified on reflectance, fluorescence and fusion modes using this novel device. To determine the best mode to identify characteristic BCC features. To develop a new staining protocol to improve the visualization of BCC under the different modes.MethodsFrom September 2016 to June 2017, we prospectively included consecutive BCCs which were excised using Mohs surgery in our department. The lesions were evaluated using ex vivo CM after routine Mohs surgery. The specimens were first stained with acridine orange and then stained using both acetic acid and acridine orange.ResultsWe included 78 BCCs (35 infiltrative, 25 nodular, 12 micronodular, 6 superficial). Most features were better visualized with the fusion mode using the double staining. We also identified new CM ex vivo features, dendritic and plump cells, which have not been reported previously.ConclusionsOur results suggest that nuclei characteristics are better visualized in FCM but cytoplasm and surrounding stroma are better visualized in RCM. Thus, the simultaneous evaluation of reflectance and fluorescence seems to be beneficial due to its complementary effect. What's already known about this topic? Ex vivo fluorescent confocal microscopy (FCM) is an imaging technique that allows histopathological analysis of fresh tissue. FCM is faster - at least one-third of the time - than conventional methods. FCM has a sensitivity of 88% and a specificity of 99% in detecting basal cell carcinomas (BCCs). What does this study add? Reflectance and fluorescence modes can be used simultaneously in a new ex vivo CM device. Each mode complements the other, resulting in an increase in the detection of BCC features in fusion mode. A combined staining using acetic acid and acridine orange enhances the visualization of tumour and stroma without damaging the tissue for further histopathological analysis.

Project description:ObjectiveTo characterize the tissue and cellular changes found in trachomatous scarring (TS) and inflammation using in vivo confocal microscopy (IVCM).DesignTwo complimentary case-control studies.ParticipantsThe first study included 363 cases with TS (without trichiasis), of whom 328 had IVCM assessment, and 363 control subjects, of whom 319 had IVCM assessment. The second study included 34 cases with trachomatous trichiasis (TT), of whom 28 had IVCM assessment, and 33 control subjects, of whom 26 had IVCM assessment.MethodsAll participants were examined with ×2.5 loupes. The IVCM examination of the upper tarsal conjunctiva was carried out with a Heidelberg Retina Tomograph 3 with the Rostock Cornea Module (Heidelberg Engineering GmbH, Dossenheim, Germany).Main outcome measuresThe IVCM images were graded in a masked manner using a previously published grading system evaluating the inflammatory infiltrate density; the presence or absence of dendritiform cells (DCs), tissue edema, and papillae; and the level of subepithelial connective tissue organization.ResultsSubjects with clinical scarring had a characteristic appearance on IVCM of well-defined bands and sheets of scar tissue visible. Similar changes were also seen in some clinically normal subjects consistent with subclinical scarring. Scarred subjects had more DCs and an elevated inflammatory infiltrate, even after adjusting for other factors, including the level of clinical inflammation. Cellular activity was usually seen only in or just below the epithelium, rarely being seen deeper than 30 ?m from the surface. The presence of tissue edema was strongly associated with the level of clinical inflammation.ConclusionsIn vivo confocal microscopy can be quantitatively used to study inflammatory and scarring changes in the conjunctiva. Dendritic cells seem to be closely associated with the scarring process in trachoma and are likely to be an important target in antifibrotic therapies or the development of a chlamydial vaccine. The increased number of inflammatory cells seen in scarred subjects is consistent with the immunopathologic nature of the disease. The localization of cellular activity close to the conjunctival surface supports the view that the epithelium plays a central role in the pathogenesis of trachoma.Financial disclosure(s)The author(s) have no proprietary or commercial interest in any materials discussed in this article.

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Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to simulate histology-like appearance.

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