Fluorescence confocal microscopy for pathologists

Abstract

Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on surgical specimens other than the skin and to evaluate the diagnostic capability of this technology from pathologists' viewpoint.

Ex vivo confocal microscopy imaging to identify tumor tissue on freshly removed brain sample

“…For meningiomas, some diagnostic criteria such as typical meningothelial, fibroblastic or transitional architecture were identifiable. The features of brain metastasis on confocal microscopy are the same encountered on formalin-fixed tissue section: cohesive or discohesive large and pleomorphic cells [9]. Our work provides additional data showing that ex vivo confocal microscopy is capable of identifying diagnostic histopathologic hallmarks of brain tumors and for some of them histopathological subtype.…”

Imaging breast cancer morphology using probe-based confocal laser endomicroscopy: towards a real-time intraoperative imaging tool for cavity scanning

“…Previous studies using bench-top confocal reflectance [42] and rigid confocal fluorescence microscopes [43][44][45] have reported that histological architecture of breast core biopsies and excision specimens, respectively, could be visualized in real time. Whilst these microscopes may have a role for imaging resection margins of excised specimens, in situ imaging of cavity walls during surgery warrants significant miniaturization to enable insertion through a small 3-4 cm skin incision and for deployment against the lateral walls of the breast cavity.…”

Evaluation of breast tissue with confocal strip-mosaicking microscopy: a test approach emulating pathology-like examination

“…In dermatological settings, for example, confocal mosaicking technology is being implemented for the detection of residual basal cell carcinoma margins to guide Mohs surgery of skin. [20][21][22][23] Dobbs et al 7 conducted an initial study to evaluate the usefulness of confocal microscopy for breast specimens. In this study, the mosaics (composite confocal images) were compared to the hematoxylin and eosin (H&E)-stained sections, and single images of ROIs in size 750 × 750 μm 2 were extracted for evaluation by the reader (pathologist).…”