Methods for photobleaching stained cells

Abstract

The present invention relates to a method for photobleaching stained cells, in particular, a method for detecting or identifying a target moiety or a target cell from a cell sample by labeling the target moiety or the target cell with a conjugate having a detection moiety and an antigen recognition moiety, wherein after detecting the target moiety, the detection moiety is degraded by irradiation with light, thereby enabling subsequent labeling and detection; and use of the method in fluorescence microscopy, flow cytometry, fluorescence spectrophotometry, cell separation, pathology or histology.

Description

Technical Field

[0001] The present invention relates to a method for detecting or identifying a target moiety or a target cell from a cell sample by labeling the target moiety or the target cell with a conjugate having a detection moiety and an antigen recognition moiety, wherein after detecting the target moiety, the detection moiety is degraded by irradiation with light, thereby enabling subsequent labeling and detection. Background Art

[0002] Fluorescent dyes conjugated to one or more antibodies are commonly used in immunofluorescence analysis. Over the past two decades, a large number of variations have been developed in antibodies, fluorescent dyes, flow cytometers, flow sorters, and fluorescence microscopes to enable specific detection and isolation of target cells.

[0003] It is known to use fluorescent dyes to separate or detect target cells, which can be removed or destroyed after detection. For example, US7776562 discloses a reversible fluorescent labeling method based on indirect, non-covalent labeling of target cells with reversible peptide / MHC-polymers or Fab-Streptamer.

[0004] In order to reduce the fluorescence radiation after detection, GB2372256 discloses a method of quenching fluorescence radiation by providing a conjugate comprising a plurality of fluorescent dyes connected to an antibody via a linker. High density of fluorescent dyes will quench the fluorescent signal. In addition, GB2372256 describes enzymatic degradation of the linker to release the fluorescent dye from the conjugate. The released fluorescent dye does not undergo self-quenching, resulting in a stronger fluorescent signal, i.e., better resolution.

[0005] Elimination of the fluorescent signal is essential for immunofluorescence techniques based on sequentially stained samples. These techniques have been shown to offer a higher multiplexing potential compared to standard procedures using simultaneous labeling and detection. However, these techniques are based on oxidative destruction of the conjugated fluorescent moieties by chemical bleaching procedures (US 7741045 B2, EP 0810 428 B1 or DE10143757) or, in the case of methods based on photobleaching, on slower bleaching rates than the method proposed here.

[0006] The main purpose of the prior art is to provide dyes or conjugates comprising said dyes, which emit as strong a fluorescence radiation as possible, i.e., have a maximum quantum yield. In order to provide a reliable and reproducible signal, dyes are designed to be as stable as possible. Although these properties are advantageous for cell detection and cell separation (such as FACS processes), they prevent cells from being repeatedly dyed and detected. Summary of the invention

[0007] It is therefore an object of the present invention to provide an enhanced method for specific labeling and detection of a target in or on a sample of a biological specimen and subsequent degradation of the detection moiety to enable further labeling and detection cycles.

[0008] Surprisingly, it was found that the conjugate consists of an antigen binding portion connected to a detection portion via a spacer, the detection portion having both a higher quantum yield (and therefore higher brightness) and reduced photostability, if the spacer is a branched PEG oligomer or polymer provided with multiple oligomer PEG side chains (i.e., a certain degree of branching), and the detection portion is covalently linked to the branched PEG, then the detection portion can be easily photodegraded. Without being bound by this theory, it is believed that the branched PEG oligomer or polymer "wraps" the detection portion covalently linked thereto, resulting in a closer contact and a higher quantum yield, with photoinduced oxidation to destroy the detection portion.

[0009] The object of the present invention is therefore a method for detecting a target moiety in a sample of a biological specimen, by:

[0010] a) providing at least one compound having the general formula (I): X n – P – Y m A conjugate of the invention, wherein X is a fluorescent moiety, Y is an antigen recognition moiety, n and m are integers between 1 and 100, and P is a spacer group comprising polyethylene glycol according to formula I.

[0011] (R7CH2CHR5O)-(CR1R2CR3R4O) o -(CH2CHR6R8) (I)

[0012] wherein R1 to R8 are independently H or a branched or linear oligomer of ethylene glycol having at least 1 and a maximum of 20,000 repeating units or a covalent bond to Y or X, provided that at least two of R1 to R8 are covalent bonds to Y or X

[0013] and o is an integer from 1 to 500;

[0014] b) contacting a sample of the biological specimen with at least one conjugate, thereby labeling the target portion recognized by the antigen recognition portion Y with the conjugate;

[0015] c) exciting the target portion of the label with light of a wavelength within the absorption spectrum of the fluorescent moiety X;

[0016] d) detecting the labeled target moiety by detecting fluorescent radiation emitted by the fluorescent moiety; and

[0017] e) degrading the fluorescent moiety X of the labeled target moiety by irradiating the conjugate with light of a wavelength within the absorption spectrum of the fluorescent moiety X for a time sufficient to deliver sufficient energy to reduce the fluorescent radiation emitted by the fluorescent moiety by at least 75% of the initial fluorescent radiation.

[0018] Irradiation of the conjugate can be performed by a white broadband light source, a single wavelength laser, or an LED with a wavelength within the absorption spectrum of the fluorescent moiety.

[0019] Due to the spacer P, the fluorescent moiety X degrades significantly faster than in the absence of the spacer P, which results in an accelerated and complete quenching of the emission signal. This enables repeated staining and destaining procedures, wherein the same or different conjugates provided with the same or different fluorescent moieties X and / or antigen recognition moieties Y can be sequentially applied to the same cell in order to stain different targets or the same target with the same or different dyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shows a single exponential decay fit f(x)=y0 * e -k*y , τ = 1 / k and t 1 / 2 =τ * ln(2), the xanthene dye is schematically decomposed by light.

[0021] Figure 2 The photodegradation curves for a series of dyes belonging to different chemical classes according to the nature of their chromophore (i.e., coumarins, xanthenes, rhodamines, cyanines) are shown, showing that all dye classes are susceptible to this light-induced degradation.

[0022] Figure 3 The photodegradation curves of cyanine dyes under four different conditions are shown. DETAILED DESCRIPTION

[0023] In the method of the present invention, the conjugate is irradiated with light of a wavelength within the absorption spectrum of the fluorescent moiety X so as to reduce the fluorescence radiation emitted by the fluorescent moiety so that any residual fluorescence radiation from the first staining cycle does not interfere with subsequent staining and detection cycles. Generally, a reduction of at least 75% of the initial fluorescence radiation is considered sufficient, but in order to achieve higher detection quality, i.e., to reduce background radiation not originating from the intended staining step, it is preferred to reduce the fluorescence radiation by at least 85%, more preferably by at least 95%, and most preferably by at least 99%. Although a 100% reduction is best, there is a trade-off between the quality of quenching and the duration of the entire process.

[0024] In an alternative definition, the degradation of the fluorescent moiety X of the labeled target moiety is carried out by irradiating the conjugate with light of a wavelength within the absorption spectrum of the fluorescent moiety X for a period of time sufficient to transfer sufficient energy to reduce the half-life of the fluorescent radiation emitted by the fluorescent moiety. The k value from the single exponential decay fit analysis of the fluorescent moiety covalently conjugated to the spacer P should give a degradation rate of at least 1,02 times and at most 10.000.000 times compared to the k obtained for the same fluorescent moiety not conjugated to the spacer P.

[0025] The fluorescent moiety X and the antigen recognition moiety Y can be covalently or quasi-covalently bound to the spacer P. The term "covalently or quasi-covalently" means that the bond between X and P is ≤ 10 -9 The dissociation constant of M.

[0026] The method of the invention may be performed in one or more sequences of steps a) to e). After each sequence, the fluorescent moiety is degraded by irradiation with light.

[0027] The terms "quenching" and "bleaching" are used interchangeably herein and should be understood to mean a decrease in fluorescence intensity from a labeled biological sample due to alteration of the fluorophore by radiation. For example, "quenching" or "bleaching" of a fluorescent moiety X can be achieved by radiation-induced oxidation and / or by cleavage of the fluorescent moiety X from the spacer P and removal of unbound fluorescent moieties from the labeled target by washing.

[0028] Therefore, the quenching system used in the method of the present invention comprises at least one light source.

[0029] Therefore, the quenching device used in the present invention can be provided with more than one light source of quenching radiation emitting different wavelengths. For example, the quenching device can be provided with 1-5 kinds of light sources, and these light sources have a combined emission spectrum within the range of 350-850nm, preferably 450-650nm. The emission of the light source can be optically combined to irradiate the sample simultaneously or successively. For example, the quenching device can be provided with three kinds of light sources emitting within the range of 450-500nm (blue), 520-560nm (green) and 630-650nm (red). In another embodiment, only a light source emitting light within the range of 350-850nm, preferably 450-650nm is provided. The advantage of a single light source is that the sample is exposed to the radiation necessary for only quenching (eliminating) fluorescent dyes, thereby avoiding the sample from being unnecessarily exposed to radiation with other wavelengths. The radiation of a single light source can be combined by suitable devices such as mirrors or optical waveguides such as optical fibers.

[0030] After and / or before each sequence, washing steps may be performed to remove unwanted material, such as unbound conjugate moieties and / or unbound fluorescent moieties X, from the sample.

[0031] The quenching process can be further enhanced by adding an oxidant. The oxidant can be, for example, O2, H2O2, peroxide or DMSO. The added oxidant can generate reactive oxygen species, which should be present in a concentration of 0.1 to 5 ppm, preferably 2 to 5 ppm, calculated as O2.

[0032] Target part

[0033] The target moiety detected by the method of the present invention can be on any biological sample, such as tissue sections, cell aggregates, suspended cells or adherent cells. The cell can be alive or dead. Preferably, the target moiety is an antigen expressed intracellularly or extracellularly on a biological sample, such as a whole animal, an organ, a tissue section, a cell aggregate or an invertebrate (e.g., Caenorhabditis elegans, Drosophila melanogaster), a vertebrate (e.g., zebrafish (Danio rerio), African clawed frog (Xenopus laevis)) and a single cell of a mammal (e.g., mouse (Mus musculus), Homo sapiens (Homo sapiens)).

[0034] Spacer P

[0035] The spacer P comprises polyethylene glycol according to the general formula I, which can be based on ethylene glycol grafted on polyhydroxy compounds, polyamino compounds and polythiol compounds. These compounds or their precursors are commercially available from Nanocs Inc., Sigma-Aldrich or NOF Corporation.

[0036] Preferred precursors are polyols such as pentaerythritol having four hydroxyl groups as attachment points for 3 to 4 polyether residues via ether bonds, dipentaerythritol having six hydroxyl groups as attachment points for 3 to 6 polyether branches via ether bonds, tripentaerythritol having eight hydroxyl groups as attachment points for 3 to 8 polyether branches via ether bonds, or hexaglycerol.

[0037]

[0038] The polyol may be substituted with ether monomer units which may be homopolymeric or copolymeric, ie alternating or block copolymers. The polyether residues may be linear or branched.

[0039] In order to achieve the covalent bond of the spacer group P with Y and X, the polyethylene glycol of formula I can be provided with at least two reactive groups known in the field of fluorescent conjugates, such as acetal, anhydride, acyl chloride, acyl azide, acyl halide, acyl nitrile, acyl phosphate, acyl amide, aldehyde, alkoxy, alkyl azide, alkyl sulfonate, alkyne, allyl, amide, anhydride, aryl halide, azido nitrophenyl, aziridine, borate, carbamate, carbodiimide, carbonate, carboxylate, carboxylic acid, diazaalkane, dichlorotriazine, diene, disulfide, enamine, epoxide, fluorobenzene, fluorophenyl ester, glyoxal, haloacetamide, haloplatinum Acid ester, halothiazine, hemiacetal, hemiketal, highly strained cycloalkane, highly strained cycloolefin, highly strained cycloalkyne, hydrazide, hydrazine, hydroxyl, hydroxylamine, imidazole, imidate, imine, iodoacetamide, isocyanate, isothiocyanate, ketal, ketone, lactone, maleimide, monochlorotriazine, N-hydroxysuccinimide ester, nitrile, nitro, oxime, pentafluorophenyl ester, peroxide group, phosphoramidite, primary amine, secondary amine, silyl halide, mercapto, sulfide, sulfodichlorophenyl ester, sulfonate, sulfone, sulfonyl halide, tertiary amine, tetrafluorophenyl ester, thioamide, thioester, thioether, thiocarboxylic acid, active ester, olefin or conjugated olefin group.

[0040] wherein Z = at least one fluorescent moiety X and at least one antigen recognition moiety Y; p is an integer from 1 to 500.

[0041] Fluorescent Part X

[0042] Suitable fluorescent moieties X are those known in the art of immunofluorescence techniques (e.g., flow cytometry or fluorescence microscopy). In these embodiments of the invention, the target moiety labeled with the conjugate is detected by exciting the fluorescent moiety X and detecting the resulting emission (photoluminescence). In this embodiment, the fluorescent moiety X is a preferred fluorescent moiety.

[0043] Useful fluorescent moieties X can be protein-based (such as phycobiliproteins), polymeric (such as polyfluorenes), small organic molecule dyes (such as xanthenes, such as fluorescein, or rhodamine, cyanine, oxazine, coumarin, acridine, oxadiazole, pyrene, pyrromethene, pyridyloxazole) or metal organic complexes, such as Ru, Eu, Pt complexes. In addition to single molecule entities, clusters of fluorescent proteins or small organic molecule dyes, and nanoparticles (such as quantum dots, upconversion nanoparticles, gold nanoparticles, dyed polymer nanoparticles) can also be used as fluorescent moieties.

[0044] In another embodiment of the invention, the target labeled with the conjugate is not detected by radiation emission, but by absorption of UV, visible light or NIR radiation. Suitable light absorbing detection moieties are light absorbing dyes without fluorescence emission, such as small organic molecule quencher dyes such as N-aryl rhodamines, azo dyes and stilbenes.

[0045] In another embodiment, the light absorbing fluorescent moiety X can be irradiated by a pulsed laser to generate a photoacoustic signal.

[0046] In one variation of the invention, the fluorophore X is substituted with one or more substituents conferring water solubility selected from the group consisting of sulfonates, phosphonates, phosphates, polyethers, sulfonamides and carbonates. The use of fluorescent dyes with sulfonate substituents is particularly advantageous, such as dyes of the Alexa Fluor family provided by Thermo Fisher Scientific Inc. For rhodamine dyes or cyanine dyes, the degree of sulfonate substitution per fluorophore may be 2 or more. The use of sulfonated dyes results in even brighter conjugates of the fluorophores polymerized on the polyether scaffold compared to unsulfonated dyes, such as Figure 2 Shown: T helper cells stained with a CD4 conjugate of Alexa Fluor 488 polymerized on branched PEG (PEG-AF488) were almost twice as bright (average MFI 98) as T helper cells stained with a CD4 conjugate of fluorescein polymerized on branched PEG (PEG-FAM) (average MFI 55).

[0047] Suitable commercially available fluorescent moieties may be selected from the product line "Vio" from Miltenyi Biotec GmbH, or FITC, or Promofluor, or the Alexa dyes and / or Bodipy dyes from Thermofisher, or cyanines from Lumiprobe.

[0048] Antigen recognition part Y

[0049] The term "antigen recognition portion Y" refers to any type of antibody, fragmented antibody or fragmented antibody derivative directed to a target portion expressed on a biological sample, such as an antigen expressed intracellularly or extracellularly on a cell. The term relates to completely intact antibodies, fragmented antibodies or fragmented antibody derivatives, such as Fab, Fab', F(ab')2, sdAb, scFv, di-scFv, nanobodies. Such fragmented antibody derivatives can be synthesized by recombinant procedures, including covalent and non-covalent conjugates comprising such molecules. Other examples of antigen recognition portions are peptide / MHC-complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules, artificially engineered binding molecules, such as peptides or aptamers targeting, for example, cell surface molecules.

[0050] The conjugate used in the method of the present invention can contain up to 100, preferably 1-20 antigen recognition moieties Y. The interaction of the antigen recognition moiety with the target antigen can have high affinity or low affinity. The binding interaction of a single low-affinity antigen recognition moiety is too low to provide a stable combination with the antigen. The low-affinity antigen recognition moiety can be polymerized to provide high affinity by being conjugated with an enzymatically degradable spacer P. When the spacer P is enzymatically cleaved, the low-affinity antigen recognition moiety will be monomerized, which results in the complete removal of the fluorescent moiety X, the spacer P and the antigen recognition moiety Y.

[0051] Preferably, the term "antigen recognition part Y" refers to an antibody against an antigen expressed intracellularly (such as IL2, FoxP3, CD154) or extracellularly (such as CD19, CD3, CD14, CD4, CD8, CD25, CD34, CD56 and CD133) in a biological sample (target cell).

[0052] The antigen recognition moiety Y, in particular an antibody, can be coupled to the spacer P via a side chain amino or sulfhydryl group. In some cases, the glycosidic side chains of the antibody can be oxidized with periodate to generate an aldehyde functional group.

[0053] The antigen recognition moiety Y can be covalently or non-covalently coupled to the spacer P. Methods for covalent or non-covalent conjugation are known to those skilled in the art and are the same as those described for the conjugation of the fluorescent moiety X.

[0054] The method of the present invention is particularly useful for detecting and / or isolating specific cell types from complex mixtures, and may include more than one sequential sequence of steps a)-e). The method may use a variety of combinations of conjugates. For example, the conjugate may comprise antibodies specific for two different epitopes, such as two different anti-CD34 antibodies. Different conjugates comprising different antibodies may be used to address different antigens, for example, anti-CD4 and anti-CD8 are used to distinguish between two different T cell populations, or anti-CD4 and anti-CD25 are used to determine different cell subsets such as regulatory T cells.

[0055] Conjugate Xn-P-Ym

[0056] Preferably, the conjugate used in the method of the invention is provided with a spacer group P having 100 to 1000 ethylene glycol repeating units.

[0057] For example, the conjugates used in the methods of the invention are compounds according to formula II and III, respectively, wherein Z=at least one fluorescent moiety X and at least one antigen recognition moiety Y; p is an integer from 2 to 500.

[0058]

[0059]

[0060] Cell Detection Methods

[0061] Detection conjugate X n – P – Y m The methods and apparatus for labeling a target identified by a fluorescent moiety X.

[0062] The target labeled with the conjugate is detected by exciting the fluorescent moiety X and analyzing the resulting fluorescent signal. The excitation wavelength is usually selected according to the absorption maximum of the fluorescent moiety X and is provided by a laser or LED source known in the art. If several different detection moieties X are used for multi-color / parameter detection, care should be taken to select fluorescent moieties with non-overlapping absorption spectra, at least non-overlapping absorption maxima. In the case of multiple fluorescent moieties as fluorescent moieties, the target can be detected, for example, under a fluorescence microscope, in a flow cytometer, a fluorescence spectrophotometer or a fluorescence scanner. The light emitted by chemiluminescence can be detected by similar instruments, and the excitation is omitted.

[0063] Purpose of the method

[0064] The methods of the invention can be used in research, diagnostics and cell therapy, such as in various applications in fluorescence microscopy, flow cytometry, fluorescence spectrophotometry, cell separation, pathology or histology.

[0065] In a first variant of the invention, biological samples such as cells are tested for enumeration purposes, ie to determine the amount of cells from the sample that have a certain set of antigens recognized by the antigen recognition portion of the conjugate.

[0066] In another variation, the biological sample detected by the conjugate in step c) is separated from the sample by optical means, electrostatic force, piezoelectric force, mechanical separation or acoustic means. For this purpose, the biological sample detected by the conjugate in step d) is separated from the sample into one or more populations simultaneously or sequentially according to their detection signals by optical means, electrostatic force, piezoelectric force, mechanical separation or acoustic means before performing step e).

[0067] In another variation of the present invention, the position of the target part such as antigen on the biological sample identified by the antigen recognition part of the conjugate is determined. Such technology is called "multi-epitope ligand mapping", "chip-based cell counting method" or "Multiomyx", and is described in, for example, EP 0810428, EP1181525, EP 1136822 or EP1224472. In this technology, cells are fixed and contacted with antibodies coupled to fluorescent moieties. Antibodies are recognized by the corresponding antigens on the biological sample (for example, on the cell surface), and after removing unbound markers and exciting the fluorescent moieties, the position of the antigen is detected by the fluorescent emission of the fluorescent moieties. In some variations, antibodies coupled to the detectable parts of MALDI imaging or CyTOF can be used instead of antibodies coupled to the fluorescent moieties. Those skilled in the art know how to change the technology based on the fluorescent moieties to work on these detection parts.

[0068] The localization of the target moiety is achieved by a digital imaging device having sufficient resolution and sensitivity to the wavelength of the fluorescent radiation. The digital imaging device can be used with or without optical magnification (e.g., using a fluorescence microscope). The resulting image is stored on a suitable storage device such as a hard drive, e.g., in RAW, TIF, JPEG or HDF5 format.

[0069] To detect different antigens, different antibody-conjugates may be provided with the same or different fluorescent moieties or antigen recognition moieties Y. Since parallel detection of fluorescence emissions with different wavelengths is limited, the antibody-fluorophore-conjugates are used sequentially either individually or in small groups (2-10).

[0070] In yet another variant of the method according to the invention, the biological specimen of the sample (in particular suspended cells) is immobilized by entrapment in a microcavity or by adhesion.

[0071] Generally, the method of the invention can be performed in several variations. For example, prior to detecting a target moiety labeled with a conjugate, the conjugate not recognized by the target moiety can be removed, for example by washing with a buffer.

[0072] In a modification of the present invention, at least two conjugates are provided simultaneously or in a subsequent staining sequence, wherein each antigen recognition part Y recognizes different antigens. In an alternative modification, at least two conjugates can be provided to the sample simultaneously or in a subsequent staining sequence, wherein each conjugate comprises different enzymatically degradable spacers P, and the spacers P are cut by different enzymes. In both cases, the target portion of the labeling can be detected simultaneously or sequentially. Sequential detection can include enzymatically degrading at least one spacer molecule P or enzymatically degrading spacer molecule P subsequently, optionally removing (washing) unbound portions in the middle. Example

[0073] To illustrate the general kinetics involved in photodegradation, Figure 1 A schematic curve is shown for the photodegradation of a xanthene dye by light, which is fitted with a single exponential decay curve such as f(x)=y0*e-k*x, where tau=1 / k and t 1 / 2 = tau * ln(2) is the half-life.

[0074] In order to measure the kinetics of photodegradation, organic fluorophores (i.e., coumarin, xanthene, rhodamine, cyanine, etc.) are dissolved in DMSO and diluted in PBS or directly dissolved in PBS so that the concentration is adjusted to obtain absorbance with its respective maximum value of about 0.3 AU, and the path length is 1 cm. In this way, all solutions are standardized and comparable by their absorbance. The solution is then placed in a 3-window fluorescent quartz cuvette with a low headspace and an airtight top to avoid evaporation and sample concentration. The sample in the cuvette is then radiated for a fixed amount of time and both absorbance and emission spectra are recorded. The intensity values ​​as their maximum values ​​are used to draw plots relative to the irradiation time, and then single exponential decay mathematical fitting is performed by suitable computer software to obtain a curve similar to Figure 1 The characteristic decay time (k) was used to calculate the half-life and other parameters.

[0075] Figure 2 Shown are the photodegradation decay curves for a series of dyes belonging to different chemical classes according to the nature of their chromophores (i.e., coumarins, xanthenes, rhodamines, cyanines) and their PEGylated forms, where each dye is covalently conjugated to a branched PEG.

[0076] Figure 2 The data shown in show that: a) all dye classes are susceptible to photodegradation by light, and b) the rate of photodegradation is higher when conjugated to branched PEGs as defined in this application. Example

[0077] Photodegradation curves of cyanine dyes were obtained under four different conditions:

[0078] i) Not bound to the spacer and free in solution (control)

[0079] ii) Covalently linked to 20 kDa linear PEG (control)

[0080] iii) Free in a solution containing 5% non-covalently linked PEG 20kDA (control)

[0081] iv) covalently linked to a 20 kDa branched PEG (according to the present invention)

[0082] Figure 3 This indicates that both covalently conjugated but linear PEG and non-conjugated but branched PEG (5% PEG) of the same molecular weight have no effect on the degradation rate of the fluorophore.

[0083] Therefore, only the conjugates disclosed in the present application, ie, branched chain PEG covalently conjugated to a fluorescent moiety, have the accelerated light-induced photodegradation required for the method of the present invention.

Claims

1. A method for detecting a target moiety in a sample of a biological specimen, which is performed by: a) providing at least one conjugate, Wherein the conjugate is a compound according to formula II: Z is a fluorescent moiety X or an antigen recognition moiety Y, provided that at least one Z is a fluorescent moiety X and at least one Z is an antigen recognition moiety Y; and p is an integer from 2 to 500; or The conjugate is a compound according to the general formula III: Z is a fluorescent moiety X or an antigen recognition moiety Y, provided that at least one Z is a fluorescent moiety X and at least one Z is an antigen recognition moiety Y; and p is an integer from 2 to 500; b) contacting a sample of the biological specimen with said at least one conjugate, thereby labeling the target moiety recognized by the antigen recognition moiety Y with the conjugate; c) exciting the target portion of the label with light of a wavelength within the absorption spectrum of the fluorescent moiety X; d) detecting the labeled target moiety by detecting fluorescent radiation emitted by the fluorescent moiety; and e) degrading the fluorescent moiety X of the labeled target moiety by irradiating the conjugate with light of a wavelength within the absorption spectrum of the fluorescent moiety X for a time sufficient to deliver sufficient energy to reduce the fluorescent radiation emitted by the fluorescent moiety by at least 75% of the initial fluorescent radiation.

2. The method according to claim 1, characterized in that The fluorescent moiety X is selected from the group consisting of xanthene dyes, rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyloxazole dyes, and pyrromethene dyes.

3. The method according to claim 1 or 2, characterized in that The fluorescent moiety X is substituted with one or more substituents imparting water solubility selected from the group consisting of sulfonates, phosphonates, phosphates, sulfonamides, polyethers and carbonates.

4. The method according to claim 1 or 2, characterized in that The antigen recognition portion Y is at least one biological molecule selected from immunoglobulin, antibody, fragment antibody, Fab, Fab', F(ab')2, sdAb, scFv, di-scFv, each of which has a natural or recombinant origin.

5. The method according to claim 1 or 2, characterized in that The antigen recognition moiety Y is at least one biological molecule selected from the group consisting of peptide / MHC-complexes, receptors for cell adhesion or co-stimulatory molecules, receptor ligands, antigens, hapten binders, avidin, streptavidin, neutravidin, aptamers, primers and ligase substrates.

6. The method according to claim 1 or 2, characterized in that The antigen recognition part Y is an antibody, a fragment antibody, a fragment antibody derivative, a peptide / MHC-complex targeting a TCR molecule, a cell adhesion receptor molecule, a receptor for a co-stimulatory molecule, or an artificially engineered binding molecule.

7. The method according to claim 1 or 2, characterized in that Steps a) to d) are repeated by providing at least two of said conjugates, wherein each antigen recognition moiety Y recognizes a different antigen.

8. The method according to claim 1 or 2, characterized in that The fluorescent moiety X of the target moiety labeled in step e) is further degraded by adding an oxidizing agent.

9. Use of the method according to claim 1 or 2 in fluorescence microscopy, flow cytometry, fluorescence spectrophotometry, cell separation, pathology or histology.

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