Fluorescence ratio measurements of double‐labeled probes for multiple in situ hybridization by digital imaging microscopy

Abstract

To expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposed.For this purpose, chromosome-specific repetitive probes were double-labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different doublehaptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), and mercury (Hg) were used. The fluorescence intensity ratio (FITCI TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%.To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double-labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin-11-dUTP and digoxigenin-11-dUTP) in the nick-translation reaction.Measurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variation.The results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double-labeled probes is feasible. o 1992 Wiley-Liss, Inc.

Chromosomal bar codes produced by multicolor fluorescence in situ hybridization with multiple YAC clones and whole chromosome painting probes

“…As a result, it can be anticipated that multiple color FISH should become a routine procedure in cytogenetic laboratories. While microphotographs showing 'real color pictures' (25) can be taken directly on color slides as demonstrated by Dauwersee et al (20), approaches for the quantitative evaluation of multicolor FISH by digital fluorescence microscopy and image analysis have also been developed (22,26) and will become indispensable for the automated evaluation of CBCs (see below). A number of Alu-PCR amplified YACs used for the present experiments yielded chromosomal bars on target chromosome bands of both homologs and both chromatids in all evaluated metaphase spreads.…”

Statistical methods in interphase cytogenetics: An experimental approach

“…For further improvement of the sensitivities of ISH applications, it will be necessary to employ advanced microscopic and optical instrumentation in combination with (semi)automated image analysis (21,22,29). This is further underlined by the possible influences of small interobserver variations, and the inadequacy of photographing random microscopic fields for quantitative interphase cytogenetics.…”

COBRA: combined binary ratio labeling of nucleic-acid probes for multi-color fluorescence in situ hybridization karyotyping

“…Several strategies were therefore designed to use combinations of fluorochromes. Examples are combinatorial labeling 1 and ratio labeling 2 . In combinatorial labeling, the achievable number of targets with n available fluorochromes is the number of permutations: 2 n -1.…”