Assessment of Extramammary Paget Disease by Two-Photon Microscopy.

Radu Hristu,Remus R Glogojeanu,Lucian G Eftimie,George A Stanciu,Stefan G Stanciu,Pavel Gheorghita

doi:10.3389/fmed.2022.839786

Abstract

Two-photon microscopy techniques are non-linear optical imaging methods which are gaining momentum in the investigation of fixed tissue sections, fresh tissue or even for in vivo experiments. Two-photon excited fluorescence and second harmonic generation are two non-linear optical contrast mechanisms which can be simultaneously used for offering complementary information on the tissue architecture. While the former can originate from endogenous autofluorescence sources (e.g., NADH, FAD, elastin, keratin, lipofuscins, or melanin), or exogenous eosin, the latter is generated in fibrillar structures within living organisms (e.g., collagen and myosin). Here we test the ability of both these contrast mechanisms to highlight features of the extramammary Paget disease on fixed tissue sections prepared for standard histological examination using immunohistochemical markers and hematoxylin and eosin staining. We also demonstrate the label-free abilities of both imaging techniques to highlight histological features on unstained fixed tissue sections. The study demonstrated that two-photon microscopy can detect specific cellular features of the extramammary Paget disease in good correlation with histopathological results.

Highlights

Extensive efforts have been deployed over the past couple of decades for advancing the state-of-the-art in clinical diagnosis of malignant dermatological conditions
Bright-field microscopy and two-photon microscopy (TPM) images were acquired on different regions of interest (ROIs) on (i) hematoxylin and eosin (H&E)-stained sections, (ii) IHC-stained sections using six different markers, and (iii) unstained tissue sections, respectively
Images acquired with bright-field microscopy on H&Estained tissue sections (Figure 1), highlight several polygonal or round-oval shaped large glandular cells with pale abundant cytoplasm which may contain small vacuoles and a big nucleus often pushed to a narrow peripheral rim which infiltrate in the epidermis

Summary

Introduction

Extensive efforts have been deployed over the past couple of decades for advancing the state-of-the-art in clinical diagnosis of malignant dermatological conditions (e.g., non-melanoma skin cancers). It cannot be neglected though that such procedures exhibit important limitations, such as sampling error, slow speed, high costs, or interpretative variability [1], to name just a few Such limitations result in a great need for alternative diagnosis methods that are faster and less invasive [2], while retaining the advantages of conventional histopathology, mainly those related to facile image interpretation. Among the most promising solutions that have been proposed to date for the non-invasive assessment of malignant epithelial pathologies are magnetic resonance imaging [3], optical coherence tomography [4], and more recently, reflectance confocal microscopy [5] and Assessment of EMPD by TPM non-linear optical (NLO) microscopy techniques [6] These latter exploit non-linear light-matter interactions [7] (e.g., two- or three-photon absorption, second or third harmonic generation) to achieve, in a label-free manner, high-contrast and easy to interpret images of tissues. Throughout this article we refer to the joint use of TPEF and SHG as two-photon microscopy (TPM)

Methods

Results

Conclusion