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Some facts

Origin of probes: Whole chromosome painting probes can be derived from flow sorted chromosomes [Speicher et al., 1996; for flow sorting see e.g. Gray et al. 1990 or Carter, 1994] or by chromosome microdissection [Senger et al., 1998; Thalhammer et al., 2004].

COT1-blocking: Normally repetitive sequences have to be blocked in the probe DNA by COT1-DNA. However, also an approach was published [Bolzer et al., 1999] describing the generation of probes depleted of repetitive sequences. By that it is possible to refrain from expensive blocking agent. Dugan et al. 2005 shows another way to use less COT1 DNA

Number of fluorochromes: Five [Speicher et al., 1996, Schröck et al., 1996, Senger et al., 1998, Padilla-Nash et al., 2006], six [Tanke et al., 1998] or seven [Azofeifa et al., 2000, Saracoglu et al., 2001] different fluorescence dyes are used to achieve the required 24 specific color combinations. The principle of combinatorial labeling as described by others before [Nederlof et al., 1990; Dauwerse et al., 1992, Ried et al., 1992; Wiegant et al., 1992] is used.

Image analysis: As in any case at least one of the used fluorochromes has its emission maximum in the near-infrared spectrum, i.e. is invisible for the human eye, a CCD-camera based image acquisition and a computer based image analysis are required. Automatization is only limited possible [Lee et al., 2001].
Image acquisition can be done based on two different principles: (i) split spectra are acquired via a set of specific filter sets as suggested by Speicher and coworkers (Multiplex-FISH = M-FISH [Speicher et al., 1996]) or (ii) complete emission spectra are acquired by an interferometer-based spectral imaging system as recommended by Schröck and coworkers (spectral karyotyping = SKY [Schröck et al., 1996]). A comparison between both approaches has been done e.g. by Rens et al. (2001). For the classification accuracy of M-FISH see as well Castleman and coworkers (2000) and Rooms et al. (2005), Wang and Castleman (2005) and Wang (2008).

New approaches for image analysing: Blind Spectral Unmixing of M-FISH Images by Non-negative Matrix Factorization [Munoz-Barrutia et al., 2007]; watershed based segmentation method for multispectral chromosome images classification [Karvelis et al., 2006, 2008 and 2009], Maximum-likelihood techniques for joint segmentation-classification [Schwartzkopf et al., 2005]; Wavelet-based compression of M-FISH images [Hua et al., 2005]; Feature normalization via expectation maximization and unsupervised nonparametric classification [Choi et al., 2008]; Color compensation [Choi et al., 2009]; Modeling of clonal expansion from M-FISH experiments [Stolte et al., 2008]; Supervised parametric and non-parametric classification [Sampat et al., 2005]

Combinatorial labeling is most frequently applied for mFISH.

Ratio labeling: The required 24 color combinations can also be achieved using the principle of the combinatorial labeling plus the principle of ratio-labeling [Dauwerse et al. 1992; Nederlof et al., 1992; Morrison et al., 1997] which has been suggested by Tanke and coworkers (COBRA-FISH: COmbined Binary RAtio labelling-FISH [Tanke et al., 1999]). In that approach only 4 fluorochromes are necessary to come to 24 color-combinations or pseudocolors and more than 96 could be easily obtained, according to the authors [Tanke et al., 1999; Szuhai et al., 2000].

Color-changing karyotyping: Another approach to be mentioned in that connection is the recently published mFISH technique named color-changing karyotyping (CCK) [Henegariu et al., 1999]. Using CCK up to 41 different targets can be discriminated by three fluorochromes only. Similar is the approach of Wu et al. (2006) coloring 2 times 12 chromosomes sequentially or Yang et al., 2008.

Counterstaining: Alternative to DAPI is presented by Christian et al. (1998).

Literature to this part:

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  • Choi H, Bovik AC, Castleman KR (2008) Feature normalization via expectation maximization and unsupervised nonparametric classification for M-FISH chromosome images. IEEE Trans Med Imaging 27:1107-1119.
  • Choi H, Castleman KR, Bovik AC (2009) Color compensation of multicolor fish images. IEEE Trans Med Imaging 28:129-136.
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