State‐of‐the‐art FISHing: Automated analysis of cytogenetic aberrations in interphase nuclei

Abstract

Interphase fluorescence in situ hybridization (i‐FISH) is a powerful tool for visualizing various molecular targets in non‐dividing cells. Manual scoring of i‐FISH signals is a labor intensive, time‐consuming, and error‐prone process liable to subjective interpretation. Automated evaluation of signal patterns provides the opportunity to overcome these difficulties. The first report on automated i‐FISH analysis has been published 20 years ago and since then several applications have been introduced in the fields of oncology, and prenatal and fertility screening. In this article, we provide an insight into the automated i‐FISH analysis including its course, brief history, clinical applications, and advantages and challenges. The lack of guidelines for describing new automated i‐FISH methods hampers the precise comparison of performance of various applications published, thus, we make a proposal for a panel of parameters essential to introduce and standardize new applications and reproduce previously described technologies. © 2012 International Society for Advancement of Cytometry

Immense random colocalization, revealed by automated high content image cytometry, seriously questions FISH as gold standard for detecting EML4‐ALK fusion

“…For reliable automated image analysis of FISH patterns, an initial training process was performed ahead of scanning, as described in our previous publications (see also Supporting Information Supplement I, which briefly describes above mentioned training process, as well as provides exact values of fundamental morphometric parameters used for automated image analysis.) Following training for optimal automated image analysis, as well as optimization of image capture and ‐processing, high content scanning was performed in two distinct phases.…”

frenchFISH: Poisson models for quantifying DNA copy-number from fluorescence in situ hybridisation of tissue sections

“…However, determining the absolute 6 number of copies from bulk sequencing data remains difficult because of normal cell 7 contamination and intra-tumour heterogeneity [5], and results are generally reported in 8 terms of loss or gain of DNA relative to an assumed diploid or median background. 9 Information from single locus methods is therefore often required to validate estimates of 10 absolute copy-number [6,7]. 11 Fluorescence in situ hybridisation (FISH) of interphase nuclei is the most widely 12 established technique for interrogating single locus copy number.…”

“…Standard analysis of FISH data relies on 15 time-consuming manual counting of spots in these images [9]. Automated systems to 16 quantify foci using nuclei recognition and spot counting algorithms (reviewed in [10]) aim 17 to make the analysis of FISH data less labour-intensive, faster, and more objective. However, 18 the accuracy of most systems is limited to identification of spots in intact and separated 19 nuclei [11].…”

“…Additionally, tissue sections are typically 3 µm 25 to 5 µm, which is smaller than the diameter of most tumour nuclei, and thus the majority of 26 nuclei are not captured completely in the volume of the section [12,13]. 27 To address these challenges, both manual and automated analysis have been improved 28 by using control probes that bind to a specific locus with known copy-number state n ctrl 29 [10]. Two commonly used approaches are: 30 1.…”

Simultaneous Dual-Color Fluorescence Microscope: A Characterization Study

“…Possibly due to manufacture costs of the high-precision optical and electronic components, a commercialized confocal microscope is much more expensive than a wide-field fluorescence microscope, and the former is therefore a less appealing solution for many laboratories. As a result, conventional wide-field fluorescence microscopes have been remaining as a popular and practical imaging tool for clinical FISH examination [11].…”