Abstract
Fluorescence in situ hybridization (FISH) is a technique to visualize specific DNA/RNA sequences within the cell nuclei and provide the presence, location and structural integrity of genes on chromosomes. A confocal Whole Slide Imaging (WSI) scanner technology has superior depth resolution compared to wide-field fluorescence imaging. Confocal WSI has the ability to perform serial optical sections with specimen imaging, which is critical for 3D tissue reconstruction for volumetric spatial analysis. The standard clinical manual scoring for FISH is labor-intensive, time-consuming and subjective. Application of multi-gene FISH analysis alongside 3D imaging, significantly increase the level of complexity required for an accurate 3D analysis. Therefore, the purpose of this study is to establish automated 3D FISH scoring for z-stack images from confocal WSI scanner. The algorithm and the application we developed, SHIMARIS PAFQ, successfully employs 3D calculations for clear individual cell nuclei segmentation, gene signals detection and distribution of break-apart probes signal patterns, including standard break-apart, and variant patterns due to truncation, and deletion, etc. The analysis was accurate and precise when compared with ground truth clinical manual counting and scoring reported in ten lymphoma and solid tumors cases. The algorithm and the application we developed, SHIMARIS PAFQ, is objective and more efficient than the conventional procedure. It enables the automated counting of more nuclei, precisely detecting additional abnormal signal variations in nuclei patterns and analyzes gigabyte multi-layer stacking imaging data of tissue samples from patients. Currently, we are developing a deep learning algorithm for automated tumor area detection to be integrated with SHIMARIS PAFQ.
Highlights
Fluorescence in situ hybridization (FISH) is a technique employed fluorescently labeled probes to bind a target genome sequence and it is in research and clinical use (Gozzetti and Le Beau 2000; Kajtar et al 2006; Tanas et al 2010; Hu et al 2014)
Application description for 3D scoring of FISH using a confocal whole slide imaging (WSI) scanner The application we developed, SHIMARIS PAFQ (Fig. 4), includes several functions, such as 3D data uploading, 3D data deletion, viewing of counting and scoring results, selecting and removing an individual nuclei from the calculations, z-stack image zooms and translations view, export and view of clinical report, quit the software, as well as assistance through manual view
FISH diagnosis with two or more different fluorescence probes can be applied to one sample (Li et al 2014, 2015) and relies on the number or the local relationship of gene signals within an individual cell nuclei
Summary
Fluorescence in situ hybridization (FISH) is a technique employed fluorescently labeled probes to bind a target genome sequence and it is in research and clinical use (Gozzetti and Le Beau 2000; Kajtar et al 2006; Tanas et al 2010; Hu et al 2014). Applications of FISH assay together with imaging techniques, such as confocal and wide-field fluorescence are commonly in use. Confocal imaging technology increases optical resolution compared to traditional wide-field fluorescent imaging by means of adding a spatial pinhole placed at the focal plane of the lens to eliminate the out-of-focus light (Wright et al 1993). In wide-field fluorescence the entire specimen of interest is exposed to the light source and the specimen axial dimension should be less than the waveoptical depth to satisfy in-focus condition. This condition limits the portion of the tumor that can be scanned
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