Method of analysing a cell or other biological material containing a nucleic acid

A composition suitable for use as a fluorescent dye for analyzing cells or other biological materials, comprising a mixture of:

• a fluorescent, cell-impermeant, compound of Formula (I)
• or a derivative in which the group NR' is quaternarized, wherein
• A is a C2-8 alkylene group;
• R1 selected from hydrogen, C1-4 alkyl, and C3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups, and R2, R3, and R4 are independently selected from C1-4 alkyl, and C3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups,
• or R1 is selected from hydrogen, C1-4 alkyl, and C3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups, R4 is selected from C1-4 alkyl, and C3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups, and R2 and R3 together with the nitrogen atom to which R2 and R3 are attached form a heterocyclic ring, wherein the heterocyclic ring consists of a C24 alkylene group together with the nitrogen atom;
• X1, X2, and X3 are independently selected from hydrogen, hydroxyl, NR1-A-NR2R3R4+(Zm−) 1/m, halogeno amino, C1-4 alkoxy, and C2-8 alkanoyloxy;
• and
• (Zm−) 1/m is an anion of charge m, where m is 1, 2 or 3;
• together with
• at least a second fluorochrome or light-emitting compound, wherein the compound is cell permeant and is of Formula (II)
• or an N-oxide thereof;
• wherein
• A is a C2-8 alkylene group;
• R1, R2, and R3 are independently selected from hydrogen, C1-4 alkyl, and C3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups,
• or R1 is selected from hydrogen, C1-4 alkyl, and C3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups, and R2 and R3 together with the nitrogen atom to which R2 and R3 are attached form a heterocyclic ring, wherein the heterocyclic ring consists of a C2-6 alkylene group together with the nitrogen atom;
• and
• X1, X2, and X3 are independently selected from hydrogen, hydroxyl, NR1-A-NR2R3, halogeno amino, C1-4 alkoxy, and C2-8 alkanoyloxy;
• said composition further comprising a first nucleic acid and a second nucleic acid,
• wherein the compound of Formula (I) forms a first fluorescent complex including the first nucleic acid, and the compound of Formula (II) forms a second fluorescent complex including the second nucleic acid, and
• the first fluorescent complex is formed in a non-intact cell and the second fluorescent complex is formed in an intact cell.