Fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection reagent is desirable. Dyes that are able to preferentially bind to a specific biological ingredient in a sample enable the researcher to determine the presence or quantity of that specific ingredient. In addition, specific cellular structures can be monitored with respect to their spatial and temporal distribution in diverse environments.
Mitochondria are the intracellular organelles that perform the aerobic mode of metabolic energy generation in eukaryotic cells. Their abundance varies as a function of cell type, cell-cycle stage and proliferative state. There is a need in biology to detect and observe mitochondria particularly in cells, as a specific application or in conjunction with additional labeling of other components under study. Due to the strong proton gradient across the mitochondrial membrane, a variety of substances that possess a cationic charge have been found to selectively localize within functioning mitochondria. Under the proper conditions, this property has been used to localize a variety of fluorescent dyes within mitochondria for use in imaging (Haugland, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS Set 30 (1992) (hereinafter "1992 HANDBOOK") incorporated by reference).
There are several "xanthylium" dyes that have proven useful for mitochondrial labeling. The fluorescent dyes tetramethylrosamine, rhodamine 123 and rhodamine 6G are readily sequestered in mitochondria, where localization of the dyes is a function of membrane potential. These mitochondrial stains have been used in conjunction with flow cytometry to sort and/or analyze cells. See, for example Rothe et al., JOURNAL OF IMMUNOLOGICAL METHODS 138, 133 (1991). These xanthylium stains for mitochondria, however, share common disadvantages. The xanthylium dyes are essentially fluorescent in any medium, resulting in problems with background fluorescence. Rhodamine 123, which is widely used because of its green fluorescence, is also the least photostable. Furthermore, the green fluorescent dye has significant spectral overlap with commonly used red fluorescent dyes, making multicolor assays more difficult to resolve.
In addition, the cationic xanthylium dyes are sequestered in the mitochondria in an equilibrium process, and transporting stained cells to fresh medium typically results in the loss of mitochondrial staining, as the stains are lost through leaching into the medium. Attempts to fix stained cells generally results in cell death, the loss of mitochondrial potential, and therefore the loss of mitochondrial staining. This is a drawback for researchers wishing to investigate mitochondrial function or viability in pathogenic species, who must choose between the very poor staining procedures available for fixed cells, or the additional hazards and costs associated with handling live pathogens.
These difficulties were partially overcome by the introduction of a family of xanthylium mitochondrial stains that are substituted by a reactive alkylating group, typically a halomethyl, that are well retained after fixation and permeabilization of the cell. The retention of staining also allows the use of other labeling techniques, including immunocytochemical staining of intracellular antigens and in situ hybridization in combination with mitochondrial staining, as many of these techniques require permeabilization of the cell membranes to allow the bulky labeling agents to enter the cellular space. These xanthylium dyes are described in U.S. Pat. No. 5,459,268 to Haugland et al. (1995) and U.S. Pat. No. 5,686,261 to Zhang et al. (1997) (incorporated by reference). Like the xanthylium dyes described above, these dyes are essentially fluorescent in aqueous medium. The green fluorescent dyes of this family also share the disadvantageous spectral overlap with commonly used red dyes.
The cyanine dyes of the present invention, with particular advantages in the staining of mitochondria, have advantageous spectral properties. Whereas xanthylium dyes and most known mitochondrial stains are intrinsically quite fluorescent, the dyes of the invention are very weakly fluorescent in aqueous solution but are enhanced by incorporation into a lipophilic environment (FIG. 4), which improves the quality of staining lipophilic compartments by improving the signal to noise ratio. The fluorescence emission of the subject dyes can be tuned from the blue-green to the far red region of the visible spectrum, and the green fluorescent dyes have less spectral overlap with commonly used red fluorophores. Additionally, the dyes of the invention are substantially more photostable than the xanthylium dyes previously used to stain mitochondria (FIG. 3). Dyes of the present invention with lower alkyl substituents are well retained in mitochondria, allowing cells under study to be washed and placed in fresh media without significant loss of staining. Where further retention is required, dye embodiments of the present invention possessing a haloalkyl substituent generally yield mitochondrial staining that is more resistant to fixation and permeabilization.
The symmetrical lipophilic carbocyanine dye DiOC.sub.7 (3) (3,3'-diheptyloxacarbocyanine iodide) has been reported to be useful as a stain for mitochondria of plant cells, e.g. Liu, et al., PLANT PHYSIOL. 84, 1385 (1987). Mitochondria stained with this carbocyanine dye, however, are more susceptible to dye removal by solvents and do not retain staining if the membrane potential is destroyed. DiO derivatives similar to DiOC.sub.7 (3) do not selectively stain the mitochondria of mammalian cells (e.g. DiOC.sub.6 (3) and DiOC.sub.5 (3), which are known to stain the endoplasmic reticulum). Another symmetrical cyanine dye that has been used to stain mitochondria is JC-1 (5,5', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine iodide). The dye tends to form so-called-J-aggregates within mitochondria that have a red fluorescence emission, and the fluorescence of the dye changes reversibly from green to greenish orange as the mitochondrial membrane becomes more polarized (U.S. Pat. No. 5,169,788 to Chen, Steele & Smiley, issued Dec. 8, 1992). This characteristic of JC-1, while advantageous for some applications, can result in non-specific red fluorescent staining in the cytoplasm and can obscure other dyes in multicolor applications. The dyes of the present invention do not form these aggregates, and the wavelength of fluorescence emission is essentially constant throughout the staining process. The dyes of the invention are less susceptible to removal by solvents after staining and will even stain mitochondria after they have been treated with solvents that fix and permeabilize cell membranes.
Other retention methods have been utilized to make cyanine dyes fixable. Schneider et al. (HISTOCHEMISTRY 101, 135 (1994)) incorporate a diazirine ring into the lipophilic chain of a carbocyanine dye. Similarly, carbocyanine dyes having the photoaffinity label 4-azido-2-nitrophenyl as a substituent are described by Hahn et al. (J. HISTOCHEM. CYTOCHEM. 41, 631 (1993)). However, the use of the above photoaffinity labels requires the additional step of photolysis before the subject cells are fixed. The photolysis also requires additional specialized equipment and exposes the sample to UV irradiation and heat that may damage the cells or tissues under examination.