Fluorescent viability assay using cyclic-substituted unsymmetrical cyanine dyes

A method of analyzing cell viability, comprising:

• a) combining a sample of cells with an aqueous dye solution comprising an effective amount of a fluorescent Dye I of the formula:
• ##STR21## where t=1-4 and each R.sup.1, which may be the same or different, is H, C.sub.1-6 alkyl;
• trifluoromethyl;
• halogen;
• or --OR.sup.8, --SR.sup.8 or --(NR.sup.8 R.sup.9) where R.sup.8 and R.sup.9, which can be the same or different, are independently H;
• C.sub.1-6 alkyl;
• or 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings having a total of 3-16 ring atoms wherein the heteroatoms are O, N or S;
• or R.sup.8 and R.sup.9 taken in combination are --(CH.sub.2).sub.2 --L--(CH.sub.2).sub.2 -- where L=a single bond or --CH.sub.2 --, --O--, or --NR.sup.10, where R.sup.10 is H or C.sub.1-6 alkyl;
• R.sup.2 is C.sub.1-6 alkyl;
• Z.sup.- is a biologically compatible counterion;
• X is O;
• S;
• Se;
• or NR.sup.15, where R.sup.15 is H or C.sub.1-6 alkyl;
• or CR.sup.16 R.sup.17, where R.sup.16 and R.sup.17, which may be the same or different, are independently H or C.sub.1-6 alkyl, or the carbons of R.sup.16 and R.sup.17 taken in combination complete a five or six membered saturated ring;
• n=0, 1, or 2;
• Q has the formula Q1 or Q2 ##STR22## wherein Y is --CR.sup.3 .dbd.CR.sup.4 --;
• p and m=0 or 1, such that p+m=1;
• R.sup.5 is a C.sub.1.varies.6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 polyalkenyl, C.sub.1-6 alkynyl or C.sub.1-6 polyalkynyl group;
• or R.sup.5 is an OMEGA;
• R.sup.3, R.sup.4, R.sup.6 and R.sup.7, which may be the same or different, are independently H;
• or a C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 polyalkenyl, C.sub.1-6 alkynyl or C.sub.1-6 polyalkynyl group;
• or halogen;
• or --OR.sup.8, --SR.sup.8, --(NR.sup.8 R.sup.9), as defined previously;
• or --OSO.sub.2 R.sup.19 where R.sup.19 is C.sub.1-6 alkyl, or C.sub.1-6 perfluoroalkyl, or aryl;
• or an OMEGA;
• or R.sup.6 and R.sup.7, taken in combination are --(CH.sub.2).sub.v -- where v=3 or 4, or R.sup.6 and R.sup.7 form a fused aromatic ring according to formula Q2;
• R.sup.11, R.sup.12, R.sup.13, and R.sup.14, which may be the same or different, are independently H;
• or a C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 polyalkenyl, C.sub.1-6 alkynyl or C.sub.1-6 polyalkynyl group;
• or a halogen;
• or an OMEGA;
• or --OH, --OR.sup.8, --SR.sup.8, or --(NR.sup.8 R.sup.9);
• OMEGA is a saturated or unsaturated, substituted or unsubstituted, cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms wherein the heteroatoms are O, N or S, that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, alkoxy or carboxyalkyl, having 1-6 carbons, and that is attached as R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.13, or R.sup.14 by a single bond;
• such that at least one of R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.13, and R.sup.14 is an OMEGA, and, where more than one of R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.13, and R.sup.14 is an OMEGA, each OMEGA is optionally the same or different;
• and an effective amount of a fluorescent Dye II that selectively stains only viable cells, where Dye II and Dye I together have a detectable fluorescent response in combination with cells in the sample that is different from a fluorescent response of Dye I alone, resulting in a sample of stained cells;
• b) illuminating the sample of stained cells at a suitable absorption wavelength that results in one or more illuminated cells;
• and
• c) observing the illuminated cells with means for detecting a fluorescent response resulting from illumination, where non-viable cells show the fluorescent response of Dye I alone.

A method of analyzing cell viability, comprising:

• a) combining a sample of cells with an aqueous dye solution comprising a fluorescent Dye I of the formula:
• ##STR26## where t=1-4 and each R.sup.1, which may be the same or different, is H, C.sub.1-6 alkyl;
• trifluoromethyl;
• halogen;
• or --OR.sup.8, --SR.sup.8 or --(NR.sup.8 R.sup.9) where R.sup.8 and R.sup.9, which can be the same or different, are independently H;
• C.sub.1-6 alkyl;
• or 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings having a total of 3-16 ring atoms wherein the heteroatoms are O, N or S;
• or R.sup.8 and R.sup.9 taken in combination are --(CH.sub.2).sub.2 --L--(CH.sub.2).sub.2 -- where L=a single bond or --CH.sub.2 --, --O--, or --NR.sup.10, where R.sup.10 is H or C.sub.1-6 alkyl;
• R.sup.2 is C.sub.1-6 alkyl;
• Z.sup.- is a biologically compatible counterion;
• X is O;
• S;
• Se;
• or NR.sup.15, where R.sup.15 is H or C.sub.1-6 alkyl;
• or CR.sup.16 R.sup.17, where R.sup.16 and R.sup.17, which may be the same or different, are independently H or C.sub.1-6 alkyl, or the carbons of R.sup.16 and R.sup.17 taken in combination complete a five or six membered saturated ring;
• n=0, 1, or 2;
• Q has the formula Q1 or Q2 ##STR27## wherein Y is --CR.sup.3 .dbd.CR.sup.4 --;
• p and m=0 or 1, such that p+m=1;
• R.sup.5 is a C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 polyalkenyl, C.sub.1-6 alkynyl or C.sub.1-6 polyalkynyl group;
• or R.sup.5 is an OMEGA;
• R.sup.3, R.sup.4, R.sup.6 and R.sup.7, which may be the same or different, are independently H;
• or a C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 polyalkenyl, C.sub.1-6 alkynyl or C.sub.1-6 polyalkynyl group;
• or halogen;
• or --OR.sup.8, --SR.sup.8, --(NR.sup.8 R.sup.9), as defined previously;
• or --OSO.sub.2 R.sup.19 where R.sup.19 is C.sub.1-6 alkyl, or C.sub.1-6 perfluoroalkyl, or aryl;
• or an OMEGA;
• or R.sup.6 and R.sup.7, taken in combination are --(CH.sub.2).sub.v -- where v=3 or 4, or R.sup.6 and R.sup.7 form a fused aromatic ring according to formula Q2;
• R.sup.11, R.sup.12, R.sup.13, and R.sup.14, which may be the same or different, are independently H;
• or a C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 polyalkenyl, C.sub.1-6 alkynyl or C.sub.1-6 polyalkynyl group;
• or a halogen;
• or an OMEGA;
• or --OH, --OR.sup.8, --SR.sup.8, or --(NR.sup.8 R.sup.9);
• OMEGA is a saturated or unsaturated, substituted or unsubstituted, cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms wherein the heteroatoms are O, N or S, that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, alkoxy or carboxyalkyl, having 1-6 carbons, and that is attached as R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.13, or R.sup.14 by a single bond;
• such that at least one of R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.13, and R.sup.14 is an OMEGA, and, where more than one of R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.13, and R.sup.14 is an OMEGA, each OMEGA is optionally the same or different;
• and a fluorescent Dye II that selectively stains only non-viable cells, such that Dye II and Dye I together have a detectable fluorescent response in combination with cells in the sample that is different from a fluorescent response of Dye I alone, and where Dye I and Dye II are each present in an amount sufficient to give a detectable fluorescent response, resulting in a sample of stained cells;
• b) illuminating the sample of stained cells at a suitable absorption wavelength that results in one or more illuminated cells;
• and
• c) observing the illuminated cells with means for detecting a fluorescent response resulting from illumination, where viable cells show the fluorescent response of Dye I alone.