Masking of the background fluorescence and luminescence in the optical analysis of biomedical assays
In a process for the quantitative optical analysis of biological cells labelled with a fluorescent dye, the sensitivity of analytical detection can be considerably improved if a masking dye, which absorbs the excitation light for the fluorescent dye and/or its emission light is added to the solution surrounding the biological cells and/or if a separating layer permeable to the solution and absorbing and/or reflecting the excitation light or the emission light is applied to a layer of the biological cells at the bottom of a reaction vessel. This process can also be used for improving the sensitivity in the quantitative optical analysis of a luminescent biological cell layer. Analogously, these process principles can also be used in receptor studies for the masking of the interfering background radiation in the quantitative optical analysis of fluorescently or luminescently labelled reaction components.
1. A process for the quantitative optical analysis of biological cells labeled with a fluorescent dye, said process comprising the following steps:
a) providing a coherent layer of biological cells labeled with a fluorescent dye applied to a transparent support at the bottom of a reaction vessel and in contact with a solution comprising the fluorescent dye;
b) shining excitation energy through the transparent support to cause the biological cells labeled with the fluorescent dye to exhibit fluorescence;
c) adding a masking dye to the solution comprising the fluorescent dye, the masking dye absorbing at least one of:
i) the excitation energy of the fluorescent dye in the solution; and
ii) the emission light of the fluorescent dye in the solution; and
d) detecting and analyzing the fluorescence of the biological cells labeled with the fluorescent dye through the transparent support;
wherein steps b) and c) are carried out in any order.
2. The process according to claim 1 , wherein the masking dye is water-soluble and has no cytotoxic effects.
3. The process according to claim 1 , wherein the biological cells are labeled with a fluorescent dye which is a FURA-AM dye.
4. The process according to claim 3 , wherein the fluorescent dye is FURA2-AM.