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

  • Xiujun Fu Memorial Sloan Kettering Cancer Center
  • Jochen K. Lennerz Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
  • Maristela Onozato Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
  • Anthony Iafrate Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
  • Yukako Yagi Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA


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 analysis


[1] Hu L, Ru K, Zhang L, Huang Y, Zhu X, Liu H, Zetterberg A, Cheng T, Miao W. Fluorescence in situ hybridization (FISH): an increasingly demanded tool for biomarker research and personalized medicine. Biomark Res. 2014, 2(1):3.
[2] Gozzetti A, Le Beau MM. Fluorescence in situ hybridization: uses and limitations. Semin Hematol. 2000, 37(4): 320-33.
[3] Tanas MR, Rubin BP, Tubbs RR, Billings SD, Downs-Kelly E, Goldblum JR. Utilization of fluorescence in situ hybridization in the diagnosis of 230 mesenchymal neoplasms: an institutional experience. Arch Patho Lab Med. 2010, 134(12):1797-803.
[4] Kajtár B, Méhes G, Lörch T, Deák L, Kneifné M, Alpár D, Pajor L. Automated fluorescent in situ hybridization (FISH) analysis of t(9;22)(q34;q11) in interphase nuclei. Cytometry A. 2006, 69(6): 506-14.
[5] Kozubek M. Confocal and two-photon microscopy: Foundations, applications, and advances, FISH Imaging. New York: Wiley-Lise, 2001 (ISBN: 0-471-40920-0): 389-429.
[6] White JG. An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J Cell Biol. 1987, 105(1): 41-48.
[7] Cornish TC, Swapp RE, Kaplan KJ. Whole-slide imaging: routine pathologic diagnosis. Adv Anat Pathol. 2012, 19(3): 152-9.
[8] Brachtel E, Yagi Y. Digital imaging in pathology-current applications and challenges. J Biophotonics. 2012, 5(4): 327-35.
[9] Laurent C, Guérin M, Frenois FX, Thuries V, Jalabert L, Brousset P, Valmary-Dagano S. Whole-slide imaging is a robust alternative to traditional fluorescent microscopy for fluorescence in situ hybridization imaging using break-apart DNA probes. Hum Pathol. 2013, 44(8): 1544-55.
[10] Van der Logt EM, Kuperus DA, van Setten JW, van den Heuvel MC, Boers JE, Schuuring E, Kibbelaar RE. Fully automated fluorescent in situ hybridization (FISH) staining and digital analysis of HER2 in breast cancer: a validation study. PLoS One. 2015, 10(4): e0123201.
[11] Alpár D, Hermesz J, Pótó L, László R, Kereskai L, Jáksó P, Pajor G, Pajor L, Kajtár B. Automated FISH analysis using dual-fusion and break-apart probes on paraffin-embedded tissue sections. Cytometry A. 2008, 73(7): 651-7.
[12] Lee RE, McClintock DS, Laver NM, Yagi Y. Evaluation and optimization for liquid-based preparation cytology in whole slide imaging. J Pathol Inform. 2011, 2: 46.
[13] Misteli T. Beyond the sequence: cellular organization of genome function. Cell. 2007, 128(4):787-800.
[14] Bolland DJ, King MR, Reik W, Corcoran AE, Krueger C. Robust 3D DNA FISH using directly labeled probes. J Vis Exp. 2013, 15(78): e50587.
[15] Ren Z, Chen N, Lam EY. Extended focused imaging and depth map reconstruction in optical scanning holography. App Opt. 2016, 55(5): 1040-7.
[16] Li C, Bai J, Hao X, Zhang S, Hu Y, Zhang X, Yuan W, Hu L, Cheng T, Zetterberg A, Lee MH, Zhang J. Multi-gene fluorescence in situ hybridization to detect cell cycle gene copy number aberrations in young breast cancer patients. Cell Cycle. 2014, 13(8): 1299-305.
[17] Li J, Su W, Zhang S, Hu Y, Liu J, Zhang X, Bai J, Yuan W, Hu L, Cheng T, Zetterberg A, Lei Z, Zhang J. Epidermal growth factor receptor and AKT1 gene copy numbers by multi-gene fluorescence in situ hybridization impact on prognosis in breast cancer. Cancer Sci. 2015, 106(5): 642-9.
[18] Roix JJ, McQueen PG, Munson PJ, Parada LA, Misteli T. Spatial proximity of translocation-prone gene loci in human lymphomas. Nat Genet. 2003, 34(3): 287-91.
[19] Gué M, Messaoudi C, Sun JS, Boudier T. Smart 3D-FISH: automation of distance analysis in nuclei of interphase cells by image processing. Cytometry A. 2005, 67(1): 18-26.
[20] Hildenbrand G, Rapp A, Spöri U, Wagner C, Cremer C, Hausmann M. Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy. Biophys J. 2005, 88(6): 4312-8.
[21] Farahani N, Parwani A, Pantanowitz L. Whole slide imaging in pathology: advantages limitations and emerging perspectives. Pathology and Laboratory Medicine International. 2015, 7:23-33.
[22] Wright SJ, Centonze VE, Stricker SA, DeVries PJ, Paddock SW, Schatten G. Introduction to confocal microscopy and three-dimensional reconstruction. Methods Cell Biol. 1993, 38:1-45.
How to Cite
FU, Xiujun et al. Evaluation of a confocal WSI scanner for FISH slide imaging and image analysis. Diagnostic Pathology, [S.l.], v. 3, n. 1, aug. 2017. ISSN 2364-4893. Available at: <http://www.diagnosticpathology.eu/content/index.php/dpath/article/view/249>. Date accessed: 21 aug. 2017. doi: https://doi.org/10.17629/www.diagnosticpathology.eu-2017-3:249.