Method for photobleaching stained cells
The invention is directed to a method for detecting a target moiety in a sample of biological specimens by: a) providing at least one conjugate with the general formula (I) X n —P—Y m , b) contacting the sample of biological specimens with at least one conjugate, thereby labeling the target moiety recognized by the antigen recognizing moiety Y with the conjugate c) exciting the labelled target moieties with light having a wavelength within the absorbance spectrum of fluorescent moiety X d) detecting the labelled target moieties by detecting the fluorescence radiation and e) degrading the fluorescent moiety X of the labelled target moieties by irradiating the conjugate with light having a wavelength within the absorbance spectrum of fluorescent moiety X Use of the method in fluorescence microscopy, flow cytometer, spectrofluorometry, cell separation, pathology or histology.
1. A method for detecting a target moiety in a sample of biological specimens comprising:
a) providing at least one conjugate with the general formula III:
wherein Z is at least one fluorescent moiety X and at least one antigen recognizing moiety Y; and p is an integer ranging from 27 through 112;
b) contacting the sample of biological specimens with the at least one conjugate, thereby labelling the target moiety recognized by the at least one antigen recognizing moiety Y with the conjugate;
c) exciting the labelled target moieties with light having a wavelength within the absorbance spectrum of the at least one fluorescent moiety X;
d) detecting the labelled target moieties by detecting the fluorescence radiation emitted by the at least one fluorescent moiety X; and
e) degrading the at least one fluorescent moiety X of the labelled target moieties by irradiating the conjugate with light having a wavelength within the absorbance spectrum of the at least one fluorescent moiety X for a time sufficient to deliver enough energy to reduce the fluorescence radiation emitted by the at least one fluorescent moiety X at least by 75% of the initial fluorescence radiation.
2. The method according to claim 1 , wherein the at least one fluorescent moiety X is selected from the group consisting of xanthene dyes, rhodamine dyes, coumarine dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyl oxazole dyes and pyrromethene dyes.
3. The method according to claim 1 , wherein the at least one fluorescent moiety X is substituted with one more water solubility imparting substituents selected from the group consisting of sulfonates, phosphonates, phosphates, sulfonamides, polyethers and carbonates.
4. The method according to claim 1 , wherein the at least one antigen recognizing moiety Y is at least one biomolecule selected from group consisting of an immunoglobulin, antibody, fragmented antibody, Fab, Fab′, F(ab′) 2 , sdAb, scFv, and di-scFv, each of naturally or recombinant origin.
5. The method according to claim 1 , wherein the at least one antigen recognizing moiety Y is at least one biomolecule selected from the group consisting of peptide/MHC-complexes, receptors for cell adhesion or costimulatory molecules, receptor ligands, antigens, hapten binders, avidin, streptavidin, neutravidin, aptamers, primers and ligase substrates.
6. The method according to claim 1 , wherein the at least one antigen recognizing moiety Y is an antibody, an fragmented antibody, an fragmented antibody derivative, peptide/MHC-complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules or artificial engineered binding molecules.