Evaluation of a confocal WSI scanner for FISH slide imaging and image analysis

Background: Technological advances contribute to the maturation of digital pathology in clinical and research applications. However, there are only few reports on fluorescence scanning especially on confocal fluorescence imaging technology in digital pathology, which has superior depth resolution compared to wide-field fluorescence imaging. Here, we explored the features of a confocal WSI scanner for typical diagnostic and research imaging applications of fluorescence in situ hybridization (FISH) assay. Methods: Multi-layer stacking (Z-stack) which stores all image information from each layer, and extended focus which stores the optimal image information from all scanned layers, featured in the Pannoramic Confocal scanner (3DHISTECH Ltd., Budapest, Hungary) were employed in digitizing 14 FISH slides (ALK, EGFR, and multi-gene). The slides were scanned with a 40× water immersion objective producing a final image with pixel resolution of 0.1625 µm/pixel. Z-stack and extended focus were used for N=6, 13, and 26 multiple layers scanning at 1, 0.4, and 0.2 µm depth intervals respectively. Single-layer scanning was also done for comparison. Scanning time and resultant file size were recorded. The CaseViewer from 3DHISTECH was used to visualize images and export the annotated regions, and the exported images were further analyzed in Imaris (Bitplane, Zurich, Switzerland) for 3-dimensional reconstruction, nuclear segmentation, and the quantification and co-localization analysis of dots inside nuclei. Quantification data from Imaris were imported into Excel for statistic analysis. Results: Confocal fluorescence scanning of FISH slides enabled sharper image than wide-field scanning, although it required longer scanning time and larger file storage. More intra-nuclear dots were quantified from multi-layer Z-stack images than single-layer images, and the Z-stack increased scanning time and image file size. Furthermore, there were a reduced in the number of dots and an increased in the number of co-localized dots in extended-focus images compared to Z-stack. Dots in multiple channels were quantified and analyzed automatically, which supports clinical diagnosis of gene amplification, deletion, and translocation. Three-dimensional reconstruction of Z-stack produced precise measurement of spatial distance, which supports molecular research. Conclusion: Confocal provides sharper image than wide-field for FISH slide scanning. Extended focus reduces file size and storage, but could cause inaccurate analysis due to misinterpretation of overlapping information. Z-stack scanning provides high volume image information for spatial analysis. We foresee confocal multi-layer scanning as a digital pathology application tool for FISH imaging in both clinical and research in future. Fluorescence in situ hybridization; Whole slide image; Confocal; Three-dimensional reconstruction; Image analysi