Quantification of intraepidermal nerve fiber density using three‐dimensional microscopy

Three‐dimensional microscopy provides more extended depth of penetration compared with conventional light microscopy and is known to be useful in clinical evaluation of thick biological specimens. Skin nerve biopsy together with the quantification of intraepidermal nerve fibers in multiple thick sections has been widely adopted for evaluating peripheral neuropathies. The aim of the present study was to evaluate the effectivity of three‐dimensional microscopy in reducing the required time and inter‐rater discrepancies, especially in the case of personnel not familiar with the quantification methods. A total of six cryo‐sectioned specimens were analyzed for the study and the skin samples were collected from one patient with postherpetic neuralgia who voluntarily participated in the study. Two investigators, a physician and non‐physician assessed the intraepidermal nerve fiber densities and required analysis time using three different methods including direct visualization of tissue slides, and analysis with two‐ and three‐dimensional images. Three‐dimensional microscopy could produce images that enabled reliable evaluation of intraepidermal nerve fibers; the accuracy of analysis was statistically comparable between the physician and non‐physician (p > .05). Three‐dimensional microscopy also enabled the non‐physician to proceed meaningfully faster evaluation compared with the direct visualization method (p = .03). Three‐dimensional microscopy could be one of the useful methods to improve accuracy and convenience of the analysis of intraepidermal nerve fibers especially appropriate for unaccustomed physician or non‐physician. Research Highlights Three‐dimensional microscopy is capable of producing images with more extended depth of penetration compared with conventional light microscopy and has been known to be suitable for clinical evaluation of thick biological specimens. Cutaneous nerve biopsy and the quantification of nerve fibers in thick sections has been widely adopted for evaluating peripheral neuropathies. Three‐dimensional microscopy could be especially appropriate for unaccustomed physician or non‐physician to improve accuracy and convenience of the analysis of intraepidermal nerve fibers. Three dimensional images of cutaneous nerve fibers produced by 3D microscopy (50‐μm‐thick section, ×400). The image presents detailed course of intraepidermal nerve fibers which contribute to clear evaluation of investigators