Background: Proper assessment of dermal collagen fibers by dermatologists and researchers is essential. Histologic evaluation methods have limitations. We present a simple method for measurement of collagen fibers in human skin using Masson's trichrome staining. Materials and Methods: Normal skin specimens from a cadaver were processed with Masson's trichrome, which can effectively stain collagen fibers blue with aniline dye. Optical photomicrographs of these slides were analyzed using ImageJ software. Color image processing, a histogram-based function of ImageJ for image segmentation, was performed with color moments thresholding technique. We selected blue areas by adjusting the blue channel to include specific values. The selected areas were highlighted and evaluated. We divided the image into layers of 0.09-mm 2 areas from the top to bottom of the dermis. Each area was cropped and evaluated. Results: Quantitative assessment yielded the quantitative size occupied by collagen fibers in an area of 0.09 mm 2. Calculation of the percentage in each area can be used to determine the density of collagen fibers. Conclusion: Measurements obtained with our method can be applied to research on dermal collagen fibers. We present a convenient quantitative assessment method for the dermal constituents in Masson's trichrome-stained slides.

Skin biopsy for AK diagnosis is usually performed on only a limited part of the whole lesion. Therefore, a clinical diagnosis is important. According to a study, there is no significant correlation between histopathological and clinical classification system. We examined the correlation between microscopic information and dermoscopic findings to deduce if dermoscopic information reflects histopathologic grade severity. Forty seven patients with histologically confirmed AK were enrolled and positive ratio of red pseudonetwork, rosette, red background and targetoid signs, white-to-yellow scale, white structureless area, and pigmentation from dermoscopic findings were investigated. Furthermore, viable epidermal thickness, vessel lumen dimensions, existence and thickness of ortho- and parakeratosis, degree of sola elastosis, flag sign existence, and Roewert-Huber classification were measured as histologic findings. Red background did not show a significant correlation with vascular dimension or viable epidermal thickness. When targetoid sign was present, vascular dimension was significantly larger but showed no correlation with viable epidermal thickness, parakeratosis or orthokeratosis. Solar elastosis level was significantly higher when white-to-yellow scale was present. According to Spearman's correlation analysis, ortho/parakeratotic thickness showed correlations with each other. The negative correlation between viable epidermal thickness and vascular dimension was also shown. Roewert-Huber histologic AK classification showed no correlation with any factors we checked. Factors considered to be characteristic features of AK in dermoscopy seemed unassociated with histologic AK classification and additional research is needed to determine degree of dysplasia of AK lesions using dermoscopy.

Background and PurposeCutaneous nerve biopsies based on two-dimensional analysis have been regarded as a creditable assessment tool for diagnosing peripheral neuropathies. However, advancements in methodological imaging are required for the analysis of intact structures of peripheral nerve fibers. A tissue-clearing and labeling technique facilitates three-dimensional imaging of internal structures in unsectioned, whole biological tissues without excessive time or labor costs. We sought to establish whether a tissue-clearing and labeling technique could be used for the diagnostic evaluation of peripheral neuropathies.MethodsFive healthy individuals and four patients with small-fiber neuropathy (SFN) and postherpetic neuralgia (PHN) were prospectively enrolled. The conventional methods of indirect immunofluorescence (IF) and bright-field immunohistochemistry (IHC) were adopted in addition to the tissue-clearing and labeling method called active clarity technique-pressure related efficient and stable transfer of macromolecules into organs (ACT-PRESTO) to quantify the intraepidermal nerve-fiber density (IENFD).ResultsThe mean IENFD values obtained by IF, bright-field IHC, and ACT-PRESTO in the healthy control group were 6.54, 6.44, and 90.19 fibers/mm2, respectively; the corresponding values in the patients with SFN were 1.99, 2.32, and 48.12 fibers/mm2, respectively, and 3.06, 2.87, and 47.21 fibers/mm2, respectively, in the patients with PHN.ConclusionsThis study has shown that a tissue-clearing method provided not only rapid and highly reproducible three-dimensional images of cutaneous nerve fibers but also yielded reliable quantitative IENFD data. Quantification of the IENFD using a tissue-clearing and labeling technique is a promising way to improve conventional cutaneous nerve biopsies.

Background: Several experimental methods for evaluating dermal structures exist; however, most of these are not used in dermatology clinics because of cost and functional limitations. Objective: To propose a simple, non-invasive method for dermal structure evaluation using a green light-emitting diode (LED) with cross-polarized light (CPL) imaging and compare the quality of the images taken using either green or white LED. Materials and Methods: Skin specimens were taken from fifteen cadavers. Images were captured using CPL photography with a green or white LED. The Commission International d'Eclairage L*a*b* (CIELAB) values were calculated for each image. The skin specimens were processed and stained with Masson's trichrome to visualize collagen fibers with major image scattering. The images were histologically analyzed, and correlational and regression analyses were performed to determine the relationship between the L* values and histologic measurements. Results: The L* values for the green images were positively correlated with collagen fiber density, reticular collagen bundle diameter, and dermal thickness. They were effective for estimating dermal properties. The L* values for the white images were positively correlated with reticular collagen bundle diameter and dermal thickness. Correlational coefficients for white images were lower than those for green images. In regression analysis, green images showed a higher coefficient of determination (R 2) for predicting reticular collagen bundle diameter than white images (0.1128 vs. 0.0827). Conclusion: Cross-polarized light imaging with a green LED is a simple, non-invasive method for evaluating dermal structures. The use of a green LED was also more effective for image analysis.

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