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

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 double-haptenized 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 (FITC/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.