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Fourth course Basics in human genetic diagnostics –
A course for CLGs* in education
*Clinical Laboratory Geneticists, Figueira da Foz, Portugal

09.-13. September 2019


~1800 studies included
Created by Dr. Thomas Liehr (PhD)
professional affiliation: Institute of Human Genetics, 07740 Jena, Germany;
e-mail: or:
last update: 15.01.2018


How to cite this database:
Liehr T. 201
X. Basics and literature on multicolor fluorescence in situ hybridization application.
[accessed XX/XX/XXXX]


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  introduction purpose of this page / probe-sets / first mFISH experiment  
  reviews technique and applications  
    pitfalls about fluorochromes  
  mFISH using wcps clinical cytogenetics  
    translocations cryptic rearr. marker chrs. PGD  
    tumor cytogenetics  
    leukemia lymphomas solid tumors cell lines  
    mutagenesis radiobiology linkage/mapping interphase /3D  
    immunohisto Zoo-FISH FISH-midi alternatives  
  FISH-banding available approaches  
    arm specific DAPI-banding barcode (CBC) Rx-FISH  
    SCAN MCB/ mband mouse mcb    
  CEP & SC-probes available approaches  
    subtel "multi-FISH" prenatal & PGD clin. CG  
    tumor CG mapping alphoid probes pod-FISH  
  additional mFISH available approaches  
    "four"-color CGH other "specials"  
  mFISH in animal mFISH in mice  
    mouse wcp mouse CBC mouse mcb other animal  
  mFISH in plant    
  some mFISH probes are available at....  


links helpful for diagnostics:





Multicolor-FISH (mFISH) is defined (on this page) as the simultaneous use of at least three different ligands or fluorochromes for the specific labeling of DNA (excluding the counterstain).


The purpose is to give a complete overview on the available mFISH literature.

When you as a reader become aware of lacking literature please send an e-mail to:

Dr. Thomas Liehr:

and the article will be listed as soon as possible.


Also the following probe-sets are available on request (Dr. Thomas Liehr:
at cost price and/or in connection with cooperation /common projects:

  • cenM-FISH (Nietzel et al., 2001, Hum Genet)

  • subcenM-FISH (Starke et al., 2003; Hum Genet)

  • regionspecific microdissection derived probes (pcp) for human and mouse - whole genome coverage!

The first successful mFISH experiment

It was performed by Nederlof and coworkers, 1989.
Three differently labeled nucleic acid sequences were visualized
simultaneously in blue (amino methyl coumarin acetic acid = AMCA), red
(tetramethylrhodamine isothiocyanate = TRITC) and green (fluorescein isothiocyanate = FITC).


  • Nederlof PM, Robinson D, Abuknesha R, Wiegant J, Hopman AH, Tanke HJ, Raap AK (1989) Three-color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences. Cytometry 10: 20-27.

Reviews on m-FISH

(including microscopy and image processing for mFISH)

Technique and applications

  • Anderson R (2010) Multiplex fluorescence in situ hybridization (M-FISH). Methods Mol Biol 659:83-97.
  • Bayani J, Squire J (2001) Advances in the detection of chromosomal aberrations using spectral karyotyping. Clin Genet 59: 65-73.
  • Bayani J, Squire J (2002) Spectral Karyotyping. In: Molecular Cytogenetics - Protocols and Applications. Ed. Fan Y-S. pp85-104.
  • Cao H, Deng HW, Li M, Wang YP (2012) Classification of multicolor fluorescence in situ hybridization (M-FISH) images with sparse representation. IEEE Trans Nanobioscience 11: 111-118.
  • Carpenter NJ (2001) Molecular cytogenetics.Semin Pediatr Neurol 8: 135-146.
  • Castleman KR (1998) Concepts in imaging and microscopy: color image processing for microscopy. Biol. Bull 194: 100-107.
  • Castleman KR, Eils R, Morrison L, Piper J, Saracoglu K, Schulze MA, Speicher MR (2000) Classification accuracy in multiple color fluorescence imaging microscopy. Cytometry 41: 139-147.
  • Chang SS, Mark HFL (1997) Emerging molecular cytogenetic techniques. Cytobios 90 7-22.
  • Cigudosa JC, Calasanz MJ, Garcia Miranda JL (1999) [Multicolor spectral karyotyping (SKY) and its application to the cytogenetic diagnosis of multiple myeloma], Sangre (Barc).44:301-304. Spanish
  • Fauth C, Speicher MR (2001) Classifying by colors: FISH-based genome analysis. Cytogenet Cell Genet 93:1-10.
  • Garini Y, Macville M, du Manoir S, Buckwald RA, Lavi M, Katzir N, Wine D, Bar-Am I, Schröck E, Ried T (1996) Spectral Karyotyping. Bioimaging 4: 65-72.
  • Garini Y, Gil A, Bar-Am I, Cabib D, Katzir N (1999) Signal to noise analysis of multiple color fluorescencve imaging microscopy. Cytometry 35: 211-226.
  • Ioannou D, Meershoek EJ, Thornhill AR, Ellis M, Griffin DK (2011) Multicolour interphase cytogenetics: 24 chromosome probes, 6 colours, 4 layers. Mol Cell Probes 25: 199-205.
  • Imataka G, Arisaka O (2012) Chromosome analysis using spectral karyotyping (SKY). Cell Biochem Biophys 62:13-17.
  • Jaffray JY, Giollant M, Perissel B, Vago P (2002) [From "monocolor" karyotype to "multicolor" karyotype: applications of M-Fish in hematology and oncology] Bull Cancer 89: 174-80. French.
  • Jalal SM, Lew ME (2002) Multicolor FISH. In: Molecular Cytogenetics - Protocols and Applications. Ed. Fan Y-S. pp 105-120.
  • Kahn E, Hotmar J, Frouin F, Di Paola M, Bazin JP, Di Paola R, Bernheim A (1996) Spectral and dynamic confocal fluorescence characterization of cytogenetic preparations. Anal Cell Pathol 12: 45-56.
  • Karvelis PS, Fotiadis DI, Georgiou I, Sakaloglou P (2009) Enhancement of the classification of multichannel chromosome images using support vector machines. Conf Proc IEEE Eng Med Biol Soc 2009:3601-4.
  • Knutsen T, Veldman T, Padilla-Nash H, Schröck E, Liynage M Ried T (1997) Spectral kyrotyping: chromosomes in color. Appl Cytogenet 23: 26-32.
  • Knutsen T, Ried T (2000) SKY: a comprehensive diagnostic and research tool. J. Assoc. Genetic Technologists 26: 3-15.
  • Koehler MR, Steinlein C, Schröck E, Yu V, Nanni M, Schmid M, Venkitaraman A, Calabrese G, Palka G, Bar-Am I, Garini Y (1999) Spectral BioImaging in modern cytogenetics and poathology. ECA Newsletter 4: 3-8.
  • Langer S, Kraus J, Jentsch I, Speicher MR (2004) Multicolor chromosome painting in diagnostic and research applications. Chromosome Res. 2004;12:15-23.
  • Le Beau MM (1996) One FISH, two FISH, red FISH, blue FISH. Nat Genet. 12: 341-344.
  • Lee C, Lemyre E, Miron PM, Morton CC (2001) Multicolor fluorescence in situ hybridization in clinical cytogenetic diagnostics. Curr Opin Pediatr 13: 550-555.
  • Li J, Lin D, Cao H, Wang YP (2013) An improved sparse representation model with structural information for Multicolour Fluorescence In-Situ Hybridization (M-FISH) image classification. BMC Syst Biol 7 Suppl 4: S5.
  • Li J, Lin D, Wang YP (2017) Segmentation of multicolor fluorescence in situ hybridization images using an improved fuzzy C-means clustering algorithm by incorporating both spatial and spectral information. J Med Imaging (Bellingham) 4: 044001.
  • Lichter P (1997) Multicolor FISHing: what’s the catch? TIGs 13: 475-479.
  • Liehr T, Heller A, Starke H, Claussen U (2002) Fluorescence in situ hybridization (FISH) banding methods - applications in research and diagnostic. Expert Review of Molecular Diagnostics 2:217-225.
  • Liehr T, Claussen U (2002) Review: Multicolor-FISH approaches for the characterization of human chromosomes in clinical genetics and tumor cytogenetics. Current Genomics 3: 213-235.
  • Liehr T, Claussen U (2002) Current developments in human molecular cytogenetic techniques. Curr Mol Med 2: 283-297.
  • Liehr T, Starke H, Weise A, Lehrer H, Claussen U (2004) Multicolor FISH probe sets and their applications. Histol Histopathol 19:229-237.
  • Liehr T, Weise A, Hamid AB, Fan X, Klein E, Aust N, Othman MAK, Mrasek K, Kosyakova N (2013) Multicolor fluorescence in situ hybridization methods in nowadays clinical diagnostics. Exp Rev Mol Diag 13, 3: 251-255
  • Luke S, Shepelsky M (1998) FISH: recent advances and diagnostic aspects. Cell Vision 5: 49-53.
  • Mackinnon RN, Chudoba I (2011) The use of M-FISH and M-BAND to define chromosome abnormalities. Methods Mol Biol 730:203-218.
  • MacLeod RA, Drexler HG (2013) Classical and molecular cytogenetic analysis. Methods Mol Biol 946:39-60.
  • Macville M, Veldman T, Padilla-Nash H, Wangsa D, O'Brien P, Schrock E, Ried T (1997) Spectral karyotyping, a 24-colour FISH technique for the identification of chromosomal rearrangements. Histochem Cell Biol 108: 299-305.
  • Marx J (1996) New methods for expanding the chromosomal paint kit. Science 271: 430.
  • Price CM (1993) Fluorescence in situ hybridization - state of the art. Blood Reviews 7: 127-134.
  • Raimondi SC (2000) Fluorescence in situ hybridization: molecular probes for diagnosis of pediatric neoplastic diseases. Cancer Invest. 18: 135-147.
  • Rao VK (1998) Course in advanced molecular cytogenetics. Pathology 30: 428-429.
  • Ried T, Liyanage M, du Manoir S, Heselmeyer K, Auer G, Macville M, Schröck E (1997) Tumor cytogenetics revisited: comparative genomic hybridization and spectral karyotyping. J Mol Med 75: 801-814.
  • Ried T, Schröck E, Ning Y, Wienberg J (1998) Chromosome painting: a useful art. Hum Mol Genet 7:1619-1626.
  • Rothmann C, Bar-Am I, Malik Z (1998) Spectral imaging for quantitative histology and cytogenetics. Histol Histopathol 13: 921-926.
  • Schwartz S (1999) Molecular cytogenetics: Show me the color. Genetics in Medicine 1: 178-180.
  • Schröck E, Veldman T, Padilla-Nash H, Ning Y, Spurbeck J, Jalal S, Schaffer JG, Papenhausen P, Kozma C, Phelan MC, Kijeldsen E, Schonberg SA, Biesecker L, du Manoir S, Ried T (1997) Spectral karyotyping refines cytogenetic diagnostics of constitutional chromosomal abnormalities. Hum Genet 101: 255-262.
  • Schröck E, Garini Y, Köhler M, Ried T (1999) Spectral karyotyping in clinical and tumor cytogenetics. In: Diagnostic Cytogenetics, Springer-labmanual, Springer, Berlin, pp 416-438, ISBN: 3-540-64602-7.
  • Schröck E, Padilla-Nash H (2000) Spectral karyotyping and multicolor fluorescence in situ hybridization reveal new tumor-specific chromosomal aberrations. Semin Hematol 37: 334-347.
  • Shuster M, Bockmuhl U, Gollin SM (1997) Early experiences with SKY: a primer for the practicing cytogenetic technologist. Appl Cytogenetics 23: 33-37.
  • Speel EJM (1999) Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: Looking inside cells with a spectrum of colors. Histochem Cell Biol 112: 89-113.
  • Speicher MR, Ward DC (1996) The coloring of cytogenetics. Nat Med 2:1046-1048.
  • Speicher MR, Eils R (1998) Multiplex-FISH (M-FISH) Ein neues zytogenetisches Verfahren in der zytogenetischen Diagnostik. BIOspektrum 2, 84-86.
  • Speicher MR (1999) Chromosome analysis by multiplex-FISH (M-FISH). In: Diagnostic Cytogenetics, Springer-labmanual, Springer, Berlin, pp 439-455, ISBN: 3-540-64602-7.
  • Speicher MR (2001) Multiplex-FISH (M-FISH) in clinical cytogenetics and research applications. ECA Newsletter 7: 3-7.
  • Stanley RJ, Caldwell CW, Keller J, Gader P (1997) The effects of image manipulation on automated karyotyping.
  • Stocker G, Fischer M, Rieder D, Bindea G, Kainz S, Oberstolz M, McNally JG, Trajanoski Z (2009) iLAP: a workflow-driven software for experimental protocol development, data acquisition and analysis. BMC Bioinformatics 10:390.
  • Stumm M, Tönnies H, Wieacker PF (1999) Molecular cytogenetic techniques for the diagnosis of chromosomal abnormalities in childhood disease. Europ J Pediat 158: 531-536.
  • Tönnies H (2005) Modern molecular cytogenetic techniques in genetic diagnostics. Mol Diag:139-157.
  • Toretsky JA, Helman LJ (1997) Cytogenetics and experimental models. Curr Opin Oncol 9: 342-247.
  • Wan TS, Ma ES (2012) Molecular cytogenetics: an indispensable tool for cancer diagnosis. Chang Gung Med J 35: 96-110.
  • Weise A, Mrasek K, Ewers E, Mkrtchyan H, Kosyakova N, Liehr T (2009) Diagnostic applications of fluorescence in situ hybridization. Expert Opin Med Diagn 3:453-460.
  • Wilcox B (1997) Genetic technologies: the brave new world? Family Futures 1: 4-5.
  • Williams ES, Cornforth MN, Goodwin EH, Bailey SM (2011) CO-FISH, COD-FISH, ReD-FISH, SKY-FISH. Methods Mol Biol 735:113-124.

Also see

Fluorescence in situ Hybridization (FISH) – Application Guide, T Liehr (Editor), 2nd Ed., Springer, Berlin, 2017, ISBN: 978-3662529577


  • Fauth C, Speicher MR (2001) Classifying by colors: FISH-based genome analysis. Cytogenet Cell Genet 93:1-10.
  • Lee C, Gisselsson D, Jin C, Nordgren A, Ferguson DO, Blennow E, Fletcher JA, Morton CC (2001) Limitations of chromosome classification by multicolor karyotyping. Am J Hum Genet 68: 1043-1047.
  • Rens W, Yang F, O'Brien PC, Solanky N, Ferguson-Smith MA (2001) A classification efficiency test of spectral karyotyping and multiplex fluorescence in situ hybridization: Identification of chromosome homologies between Homo sapiens and Hylobates leucogenys. Genes Chr Cancer 31: 65-74.
  • Strefford JC, Lillington DM, Young BD, Oliver RT (2001) The use of multicolor fluorescence technologies in the characterization of prostate carcinoma cell lines. a comparison of multiplex fluorescence in situ hybridization and spectral karyotyping data. Cancer Genet Cytogenet 124: 112-121.

Something about fluorochromes

  • Adarichev VA, Kalachikov SM, Kiseliova AV, Dymshits GM (1998). Molecular hybridization probes prepared with 4-aminooxybutylamine. Bioconjug Chem 9:671-675.
  • Castleman KR (1998) Concepts in imaging and microscopy: color image processing for microscopy. Biol. Bull 194: 100-107.
  • Dauwerse JG, Wiegant J, Raap AK, Breuning MH, van Ommen GJ (1992) Multiple colors by fluorescence in situ hybridization using ratio-labelled DNA probes create a molecular karyotype. Hum Mol Genet 1: 593-598.
  • Henegariu O, Bray-Ward P, Ward DC (2000) Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling. Nat Biotechnol 18:345-348.
  • Nederlof PM, Robinson D, Abuknesha R, Wiegant J, Hopman AH, Tanke HJ, Raap AK (1989) Three-color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences. Cytometry 10: 20-27:
  • Nederlof PM, van der Flier S, Wiegant J, Raap AK, Tanke HJ, Ploem JS, van der Ploeg M (1990) Multiple fluorescence in situ hybridization. Cytometry 11: 126-131.
  • Nederlof PM, van der Flier S, Vrolijk J, Tanke HJ, Raap AK (1992) Fluorescence ratio measurements of double-labeled probes for multiple in situ hybridization by digital imaging microscopy. Cytometry 13: 839-845.
  • Nimmakayalu M, Henegariu O, Ward DC, Bray-Ward P (2000) Simple method for preparation of fluor/hapten-labeled dUTP. Biotechniques 28:518-522.
  • Samiotaki M, Kwiatkowski M, Ylitalo N, Landegren U (1997) Seven-color time-resolved fluorescence hybridization analysis of human papilloma virus types. Anal Biochem 253: 156-61.
  • Schultz S, Smith DR, Mock JJ, Schultz DA (2000) Single-target molecule detection with nonbleaching multicolor optical immunolabels. Proc Natl Acad Sci U S A 97:996-1001.
  • Speel EJ, Ramaekers FC, Hopman AH (1997) Sensitive multicolor fluorescence in situ hybridization using catalyzed reporter deposition (CARD) amplification. J Histochem Cytochem 45:1439-1446.
  • Tanke HJ, De Haas RR, Sagner G, Ganser M, van Gijlswijk RP (1998) Use of platinum coproporphyrin and delayed luminescence imaging to extend the number of targets FISH karyotyping. Cytometry 33:453-459.
  • Vereb G, Jares-Erijman E, Selvin PR, Jovin TM (1998) Temporally and spectrally resolved imaging microscopy of lanthanide chelates. Biophys J 74:2210-2222.
  • Wiegant JC, van Gijlswijk RP, Heetebrij RJ, Bezrookove V, Raap AK, Tanke HJ (1999) ULS: a versatile method of labeling nucleic acids for FISH based on a monofunctional reaction of cisplatin derivatives with guanine moieties. Cytogenet Cell Genet 87:47-52.
  • Wiegant J, Wiesmeijer CC, Hoovers JM, Schuuring E, d'Azzo A, Vrolijk J, Tanke HJ, Raap AK (1993) Multiple and sensitive fluorescence in situ hybridization with rhodamine-, fluorescein-, and coumarin-labeled DNAs. Cytogenet Cell Genet 63:73-76.
  • Yurov YB, Soloviev IV, Vorsanova SG, Marcais B, Roizes G, Lewis R (1996) High resolution multicolor fluorescence in situ hybridization using cyanine and fluorescein dyes: rapid chromosome identification by directly fluorescently labeled alphoid DNA probes. Hum Genet 97:390-398.