Fast Green FCF Improves Depiction of Extracellular Matrix in Ex Vivo Fluorescence Confocal Microscopy

Rapid microscopic analysis of tissue is an essential diagnostic tool in oncological surgery. The gold standard for intraoperative histological tissue evaluation is frozen sections. However, frozen sections are prone to a variety of artefacts and require skilled staff and specialized lab equipment. A potential method for rapid intraoperative tissue evaluation that does not require fixation, freezing, or sectioning of the tissue is ex vivo fluorescence confocal microscopy (FCM). The visualization of the structurally important extracellular matrix (ECM) in conventional ex vivo FCM lags behind the standards of conventional histology. The objective of this study was to find a stain that would improve the depiction of the ECM to resemble FFPE H&E sections as closely as possible. Eleven different tissue stains were tested on 122 tissue samples submitted to the Department of Pathology at the Medical University of Vienna. This study was conducted on the RS-G4 Upright (Caliber I.D. Rochester, NY, USA, distributed in Europe by MAVIG GmbH, Munich, Germany). Fast Green FCF (FGFCF) in combination with acridine orange as a nuclear stain improved the visibility of the structural details of the ECM. Morphological details in FCM were equivalent or even superior to frozen sections in most analyzed categories. The addition of FGFCF to the conventional staining protocol improves the assessment of the ECM and analysis of fibrosis. The rapid staining protocol is compatible with an application in intraoperative microscopy.