Confocal Fluorescence Microscopy Platform Suitable for Rapid Evaluation of Small Fragments of Tissue in Surgical Pathology Practice.

Abstract

Rapid advances in the fields of biophotonics, computer science, and instrumentation have allowed for high-resolution imaging of biologic tissues. To evaluate the quality of images from an optimized confocal fluorescence microscopy (CFM) platform for rapid evaluation of small fragments of tissue, compared with hematoxylin-eosin staining. Tissue fragments (up to 1.0 × 0.3 cm) were stained with 0.6 mM acridine orange for 60 seconds and imaged using a CFM platform at 488-nm and 785-nm wavelength. The imaged tissues were then fixed in formalin and processed to generate hematoxylin-eosin-stained tissue sections. The quality of CFM images was scored on a scale of 0 to 3 on the basis of the percentage of the CFM images with recognizable tissue architecture (0, 0%; 1, <20%; 2, 20%-50%; 3, >50%). The diagnoses made using CFM images were compared with those made using histopathologic analysis of the hematoxylin-eosin-stained tissue sections. We imaged 118 tissue fragments obtained from 40 breast, 23 lung, 39 kidney, and 16 liver surgical excision specimens. We acquired CFM images in 2 to 3 minutes; 95.8% (113 of 118) of images showed a quality score of 3, and 4.2% (5 of 118) had a score of 2. We achieved a sensitivity of 95.5%, specificity of 97.3%, positive predictive value of 95.5%, and negative predictive value of 97.3%. Our results demonstrate the suitability of the CFM platform for rapid and accurate evaluation of small tissue fragments in surgical pathology practice.