A series of new fluorescent labeling reagents based on sulfoindocyanine dyes has been developed. We describe the synthesis and properties of these reagents. They contain succinimidyl ester reactive groups and can be readily conjugated to antibodies, avidin, DNA, lipids, polymers, and other amino-group-containing materials. The labeling reagents are water soluble, pH insensitive, and show much reduced dye aggregation under labeling conditions. One of the reagents, Cy3, can be excited with the 488-, 514- and 532-nm laser lines and is optimally excited with the 546-nm mercury arc line. Another, Cy5, can be excited with the 633-nm HeNe and 647-nm Kr laser lines available with many flow cytometers and confocal laser-scanning microscopes. New laser diodes emitting near 650 nm should also be excellent excitation sources for Cy5.

A synthetic method for shifting the absorption and emission wavelengths of cyanine dye labels by 15-30 nm to the red has been developed. This step significantly increases the potential for preparing fluorescent probes for multiparameter analysis in cytometry and diagnostics. The new sulfobenzindocyanine dyes contain succinimidyl esters as reactive groups and can be readily conjugated to antibodies, avidin, modified DNA, and other amino group-containing materials. The labeling reagents are water soluble, and their fluorescence is not sensitive to pH. One of the reagents, Cy3.205, can be optimally excited with the 568 nm Kr laser line and is useful for confocal microscopy and flow cytometry. Another dye, Cy5.205, can be excited with the 633 He-Ne or 647 nm Kr laser lines available with many flow cytometers and laser-scanning microscopes. New laser diodes emitting near 660-690 nm should also be excellent excitation sources for Cy5.205.

Ten carboxymethylindocyanine dyes which form the basis of a new series of fluorescent probes have been synthesized and converted into succinimidyl active esters for fluorescent labeling of proteins or other amino-containing substances. Fluorescence emission maxima for members of the series range from 575 to 780 nm. Hydrophilic, water-soluble reagents have been obtained which yield labeled antibodies with little tendency to form precipitates. The fluorescence intensities achieved are higher than those produced by labeling with the cyanine isothiocyanates described previously (Mujumdar et al.: Cytornetry 1011-19, 1989). The utility of these reagents has been demonstrated in antibody labeling for two-color immunofluorescent imaging of internal structures in a mammalian cell and for two-color flow-cytometry experiments. The use of values of chromophore-equivalent weight (W/Ceq), calculated from quantitative absorption data on dye samples, is proposed as an aid in formulating labeling procedures.Key terms: Fluorescent biolabeling probes, fluorescent cyanine dyes, succinimidyl ester biolabeling probes, twocolor immunofluorescent imaging, longwavelength fluorescent probes, flow cytometry, chromophore equivalent weight Earlier papers from this laboratory (4,111 have described the preparation of two series of cyanine and merocyanine dyes which are useful as fluorescent labeling reagents for biological investigations. Functional groups utilized in these reagents for covalent linking to biomolecules have been the iodoacetamido group, for sulfhydryl tagging, and the isothiocyanato moiety, for attachment to primary or secondary amino groups. The incorporation of carboxylic acid groups into the basic cyanine structure was expected to permit fluorescent labeling through the use of derived active esters. The synthesis of (2,3,3-trimethyl-3-H-indol-5-yl)-acetic acid and derived intermediates suitable for the construction of highly fluorescent carboxyl-containing indocyanine dyes has been described recently (12).Here we describe the preparation of the dyes themselves, the procedures used for converting the carboxylic acid groups into active ester functions (esters of N-hydroxysuccinimide) which are reactive toward proteins, and procedures for attaching these dyes to various proteins. Advantages which characterize reagents in this group include ease of preparation, higher activity toward protein substrates in aqueous media under mild conditions, and the high extinction coefficients and quantum yields required for intense fluorescence. As with the other dyes treated in this series of papers, the fluorescence emission maxima are to be found from the long-wavelength portion of the visible range into the near infrared, well separated from the shorterwavelength autofluorescence which is characteristic of many biological specimens and which presents a difficulty in working with such fluorophores as fluorescein.

Two homologous indocyanine dyes, Cy3.18 and Cy5.18, can be used as a ratio pair for fluorometric determination of solvent viscosity. Succinimidyl ester derivatives of these dyes can be attached to inert carrier macromolecules, such as Ficoll 70, for measurement of intracellular or intravesicular solvent viscosity. When the viscosity of the solvent was varied by various methods, the fluorescence intensity ratio (Cy3/Cy5) in a mixture of Cy3.18-Ficoll 70 (Cy3F70) and Cy5.18-Ficoll 70 (Cy5F70) in solution was found to be solely a function of solvent viscosity and was insensitive to other solvent parameters such as dielectric constant, temperature, and the ability of the solvent to form hydrogen bonds. Most important, it was insensitive to the presence of large macromolecules, such as proteins, which increase the shear viscosity but have little effect on solvent viscosity. Following microinjection into the cytoplasm of living tissue culture cells, no binding of Cy3F70 or Cy5F70 to intracellular components was detected by fluorescence recovery after photobleaching. Fluorescence intensity ratio imaging of Cy3F70 and Cy5F70 in non-motile interphase CV1 and PtK1 cells showed that the solvent viscosity of cytoplasm was not significantly different from water and showed no spatial variation.

Cyanine and merocyanine dyes are introduced as new fluorescent reagents for covalently labeling proteins and other biomolecules. These dyes, which contain iodoacetamide functional groups, have high extinction coefficients and moderate quantum yields. A major advantage of these polymethine dyes is the easy manipulation of their spectral properties during synthesis. Cyanines containing reactive functional groups can be made with absorption maxima ranging from < 500 nm to > 750 nm. This property opens additional regions of the spectrum for experiments involving the simultaneous multicolor analysis of different fluorescent probes. The cyanines, which are relatively insensitive to solvent property changes, are complemented by the merocyanines, which are keen indicators of solvent polarity.