Journal:ChromaWizard: An open-source image analysis software for multicolor fluorescence in situ hybridization analysis

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Full article title ChromaWizard: An open-source image analysis software for multicolor fluorescence in situ hybridization analysis
Journal Cytometry Part A
Author(s) Auer, Norbert; Hrdina, Astrid; Hiremath Chaitra; Vcelar, Sabine; Baumann, Martina; Borth, Nicole; Jadhav, Vaibhav
Author affiliation(s) Austrian Centre for Industrial Biotechnology
Primary contact Email: nicole dot borth at boku dot ac dot at
Year published 2018
Volume and issue 93(7)
Page(s) 749–54
DOI 10.1002/cyto.a.23505
ISSN 1552-4930
Distribution license Creative Commons Attribution-NonCommercial 4.0 International
Website https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.23505
Download https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.23505 (PDF)

Abstract

Multicolor image analysis finds its applications in a broad range of biological studies. Specifically, multiplex fluorescence in situ hybridization (M‐FISH) for chromosome painting facilitates the analysis of individual chromosomes in complex metaphase spreads and is widely used to detect both numerical and structural aberrations. While this is well established for human and mouse karyotypes, for which species sophisticated software and analysis tools are available, other organisms and species are less well served. Commercially available software is proprietary and not easily adaptable to other karyotypes. Therefore, a publicly available open-source software that combines flexibility and customizable functionalities is needed. Here we present such a tool, called “ChromaWizard,” which is based on popular scientific image analysis libraries (OpenCV, scikit‐image, and NumPy). We demonstrate its functionality on the example of primary Chinese hamster (Cricetulus griseus) fibroblasts metaphase spreads and on Chinese hamster ovary cell lines, known for their large number of chromosomal rearrangements. The application can be easily adapted to any kind of available labeling kits and is independent of the organism and instrumentation used. It allows direct inspection of the original hybridization signals and enables either manual or automatic assignment of colors, making it a functional and versatile tool that can also be used for other multicolor applications.

Keywords: fluorescence in situ hybridization (FISH), chromosome painting, open-source image analysis software

Introduction

fluorescence in situ hybridization (FISH) has greatly facilitated the characterization of gene position, chromosomes, and genome organization.[1] FISH involves the generation and hybridization of loci-specific fluorescence‐labeled nucleic acid probes on to metaphase chromosomes or onto interphase nuclei.[2] The use of this method made gene mapping possible at much higher resolution compared with other physical mapping technologies.[3] FISH was further developed into chromosome painting to simultaneously and unequivocally distinguish chromosomes and their rearrangements in complex karyotypes. Using chromosome-specific probes and combining multiple fluorophores allows the visualization of all chromosomes with individual and unique color combinations.[4] Chromosome painting has proven to be very important not only in diagnostics and in cancer research, but also in biological research for numerical and structural aberrations, as analysis can be done at the entire genome level.[5][6] There have been considerable technical improvements for analysis of human and murine samples. However, even though chromosome painting probes become available for a larger number of non‐model species from commercial or specialized labs[7], this method is still not widely applied due to the absence of open‐source software for analysis.[8]

Specialized cytogenetics labs are equipped with the necessary infrastructure, including highly sophisticated software and instruments specifically set up for the purpose of karyotype analysis and chromosome painting, including examples such as Isis (by Metasystems), CytoVision (by Leica Biosystems), and Hyperspectral (by Applied Spectral Imaging). However, if this method needs to be established and used as a side project in a lab not thus equipped, this might turn into a challenge; while imaging facilities are typically accessible, software packages come at a cost that may not be budgeted for given the restricted budgets of most core facilities.

Only to a limited extent can laboratories turn to free (or open‐source) tools such as ImageJ[9] for general applications, as they are technically challenging and require programming skills to write macros to customize the user application, which again limits their use. Therefore, we introduce a stand‐alone, simple open‐source program called ChromaWizard for analysis and visualization of multicolor fluorescence images that addresses the needs of custom non‐model organism samples. This software integrates image processing, multicolor integration, chromosome separation, and visualization with false color assignments. It processes images for any number of color channels/probes with flexible configuration. One of the important features of ChromaWizard is the use of a GUI to access full functionality so that the user can refine results. The software works with standard image formats (TIFF, PNG, and JPEG) taken either by epifluorescence or confocal microscopes. This open‐source tool is filling a gap for users lacking the ability or time to write macro programs, while being freely accessible for advanced users for further customization to enable specific or enhanced imaging applications. It can be connected with other image processing tools to improve the quality of final images as suggested.[10][11]

Acknowledgements

The authors acknowledge support from the Austrian BMWFW, BMVIT, SFG, Standortagentur Tirol, Government of Lower Austria, and Business Agency Vienna through the Austrian FFG‐COMET‐ Funding Program.

References

  1. ↑ Fauth, C.; Speicher, M.R. (2001). "Classifying by colors: FISH-based genome analysis". Cytogenetics and Cell Genetics 93 (1–2): 1–10. doi:10.1159/000056937. PMID 11474168. 
  2. ↑ Volpi, E.V.; Bridger, J.M. (2008). "FISH glossary: An overview of the fluorescence in situ hybridization technique". Biotechniques 45 (4): 385–6. doi:10.2144/000112811. PMID 18855767. 
  3. ↑ Cui, C.; Shu, W.; Li, P. (2016). "Fluorescence in situ hybridization: Cell-based genetic diagnostic and research applications". Frontiers in Cell and Developmental Biology 4: 89. doi:10.3389/fcell.2016.00089. PMC PMC5011256. PMID 27656642. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5011256. 
  4. ↑ Liehr, T.; Starke, H.; Weise, A. et al. (2004). "Multicolor FISH probe sets and their applications". Histology and Histopathology 19 (1): 229–37. doi:10.14670/HH-19.229. PMID 14702191. 
  5. ↑ Schröck, E.; Veldman, T.; Padilla-Nash, H. et al. (1997). "Spectral karyotyping refines cytogenetic diagnostics of constitutional chromosomal abnormalities". Human Genetics 101 (3): 255–62. doi:10.1007/s004390050626. PMID 9439652. 
  6. ↑ Ried, T.; Schröck, E.; Ning, Y. et al. (1998). "Chromosome painting: A useful art". Human Molecular Genetics 7 (10): 1619–26. doi:10.1093/hmg/7.10.1619. PMID 9735383. 
  7. ↑ Liehr, T. (2018). "Basics and literature on multicolor fluorescence in situ hybridization application". fish-tl.com. http://fish-tl.com/mfish.html. 
  8. ↑ Liehr, T.; Starke, H.; Heller, A. et al. (2006). "Multicolor fluorescence in situ hybridization (FISH) applied to FISH-banding". Cytogenetic and Genome Research 114 (3–4): 240–4. doi:10.1159/000094207. PMID 16954660. 
  9. ↑ Schindelin, J.; Rueden, C.T.; Hiner, M.C. et al. (2015). "The ImageJ ecosystem: An open platform for biomedical image analysis". Molecular Reproduction & Development 82 (7–8): 518-529. doi:10.1002/mrd.22489. PMC PMC5428984. PMID 26153368. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428984. 
  10. ↑ Partipilo, G.; D'Addabbo, P.; Lacalandra, G.M. et al. (2011). "Refinement of Bos taurus sequence assembly based on BAC-FISH experiments". BMC Genomics 12: 639. doi:10.1186/1471-2164-12-639. PMC PMC3268123. PMID 22208360. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3268123. 
  11. ↑ Vcelar, S.; Jadhav, V.; Melcher, M. et al. (2018). "Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting". Biotechnology and Bioengineering 115 (1): 165-173. doi:10.1002/bit.26453. PMID 28921524. 

Notes

This presentation is faithful to the original, with only a few minor changes to presentation, spelling, and grammar. We also added PMCID and DOI when they were missing from the original reference.

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