Directly labeled fluorescent DNA probes have been made by nick translation and PCR using dUTP attached to the fluorescent label, Cy3, with different length linkers. With preparation of probes by PCR we find that linker length affects the efficiency of incorporation of Cy3-dUTP, the yield of labeled probe, and the signal intensity of labeled probes hybridized to chromosome target sequences. For nick translation and PCR, both the level of incorporation and the hybridization fluorescence signal increased in parallel when the length of the linker arm is increased. Under optimal conditions, PCR yielded more densely labeled probes, however, the yield of PCR labeled probe decreased with greater linear density of labeling. By using a Cy3-modified dUTP with the longest linker under optimal conditions it was possible to label up to 28% of the possible substitution sites on the target DNA with reasonable yield by PCR and 18% by nick translation. A mechanism involving steric interactions between the polymerase, cyanine-labeled sites on template and extending chains and the modified dUTP substrate is proposed to explain the inverse correlation between the labeling efficiency and the yield of DNA probe synthesis by PCR.

Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps.

SUMMARYWe demonstrate that the CA talyzed R eporter D eposition method (CARD), utilizing the novel fluorescent reporter Cy3.29-tyramide, is successful in the F luorescent I n

A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections. Key terms: Image processing, quantitation, lymphocytesMulticolor fluorescence image analysis of cells present in tissue sections, smears, and imprints by microscopy offers a potentially powerful approach for correlating the locations of cells as well as their quantity in the two-or three-dimensional space of the sample. Intracellular and surface antigens, nucleic acid sequences from RNA and DNA, DNA content, and chromo/fluorogenic substrate precipitates are examples of parameters that could be related to sample morphology. In order for image cytometry to realize this potential, instrumentation with optimal light sources, filter sets, and detectors must be combined with software that permits identification of stained structures, accurate quantitation of the fluorescence or absorption signals, morphological analysis, and data reduction and presentation.While single-color fluorescence image cytometry of cells has been carried out previously in a number of laboratories (8,9,12,14,19), the optics, probes, hardware, and computers have recently advanced significantly, and now multicolor quantitative analysis offers enormous potential €or analysis of fixed and living cells and tissues. For example, the availability of high-resolution cooled CCD cameras with a broad spectral range, minimal spatial distortion, excellent linearity, and a wide dynamic range have made quantitative fluorescence analysis more simple than before (7,10,18). Laser scanning microscopes equipped with multiple excitation lines and detectors provide an alternative approach for multicolor immunofluorescence (2). Also, improvements in interference filters have made it possible to image multiple fluorophores that emit with narrow Stokes shifts in the visible region of the spectrum (6). Furthermore, additional fluorophores have become available at new wavelengths, such as CY5 (171, which emits in the deep red region of the spectrum where CCD cameras are very sensitive. And finally, imaging computers continue to be more powerful and cost effective year by year.Here we have developed software and methods for the four-color analysis of lymphocyte subsets on microscope slides. This work repre...

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