Abstract

Volume.127 Number.12

Original article : Clinical science

3D Assessment of Sebaceous Carcinoma of the Eyelid Based on Digitized Hematoxylin Eosin Staining Images Using Transparency-enhancing Technology
Yuichi Asahina1,2, Munetoshi Hinata2, Asami Tanaka3, Makoto Aihara1, Hiroshi Onodera4, Tetsuo Ushiku2
1 Department of Ophthalmology, The University of Tokyo, Graduate School of Medicine and Faculty of Medicine
2 Department of Pathology, Graduate School of Medicine, The University of Tokyo
3 Photon Science Center of the University of Tokyo
4 Institute for Photon Science and Technology, The University of Tokyo

Purpose: Pathological diagnosis of surgical specimens is usually limited to evaluation of cross sections at 2 mm intervals, and it is difficult to adequately evaluate the three-dimensional distribution of lesions. In this study, we attempted to evaluate pathological specimens of sebaceous carcinoma of the human eyelid three-dimensionally using a tissue transparency reagent (iILUmination of Cleared organs to IDentify target molecules method: LUCID).
Subjects and methods: A right lower eyelid specimen obtained from a woman in her 80s who had undergone excisional surgery for sebaceous carcinoma was preserved after it was fixed with formalin and embedded in paraffin. The specimen was deparaffinized, labeled with 4', 6-diamidino-2-phenylindole (DAPI) and tomato lectin conjugated with DyLight 594 (TL), immersed in LUCID for at least 24 hours for transparency, and photographed under a two-photon microscope. The measurement wavelengths were 430-460 μm (blue, DAPI short wavelength), 520-580 μm (green, autofluorescence), and 610-630 μm (red, TL), and the three-dimensional structure of the eyelid was reconstructed from the images obtained. Next, the structure was observed after it had been converted to a digitized HE-stained image by converting blue to hematoxylin (H) and green to eosin (E) color using ImageJ.
Results: The three-dimensional structure of the specimen after immersion in LUCID was clearly observed, and the digitized HE-stained images enabled identification of the extent of cancer in any cross section and observation of nuclear fission. The images also helped understand the three-dimensional structure of sebaceous carcinoma as a series of congregating nodules of lesions, which appeared to be independent in two-dimensions. Preparation of HE stained sample was equally possible on the transparency-enhanced specimen, similar to normal specimens.
Conclusion: LUCID enables three-dimensional observation of images obtained from paraffin-embedded pathological specimens and may aid in understanding the three-dimensional structures of normal tissues and tumors in the eyes.
Nippon Ganka Gakkai Zasshi (J Jpn Ophthalmol Soc) 127：1110-1118, 2023.

Key words
Tissue transparency, Digitized hematoxylin eosin (HE)-stained image, Sebaceous carcinoma
Reprint requests to
Yuichi Asahina, M. D.　Department of Ophthalmology, The University of Tokyo, Graduate School of Medicine and Faculty of Medicine. 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
Received: 18 May 2023. Accepted in revised form: 19 July 2023.
Nihon Ganka Gakkai Zasshi 127: 1110-1118,2023