DE102007010860B4 - Method of identifying a particle with two or more monodisperse embedded fluorophores - Google Patents

Abstract

Method for identifying a particle made of a polymeric material that contains two or more fluorophores embedded in the material with absorption and fluorescence maxima in the UV / VIS / NIR spectral range, the fluorophores being selected such that (i) the absorption maxima of the fluorophores are spaced from each other by at least 5 nm; (ii) the fluorescence maxima of the fluorophores are each spaced apart by at least 10 nm; and (iii) the absorption maxima and fluorescence maxima of the fluorophores are each spaced apart by at least 5 nm in a sample, comprising the steps of: (i) irradiating particles preferably present in monolayer with one or more excitation wavelengths in the overlap region of the absorption spectra of the fluorophores and im Range of absorption maxima of the fluorophores (+/- 10% of the absorbance); (ii) Detecting the intensities of the fluorescence at predetermined measurement points, with at least one measurement point being specified for each fluorophore contained in the particle, which lies in a range of at least 5% of the intensity of the fluorescence maximum of the respective fluorophore and with the measurement points being at least 5 nm apart are spaced; (iii) determining an intensity ratio of the intensities detected at the measurement points; and (iv) comparing the determined intensity ratio with a reference value for identifying the particle, characterized in that the absorption spectra of the fluorophores have a common excitation wave range, in this range an extinction of at least 0.001 and a quantum yield of at least 0.01 applies to each fluorophore and the irradiation in step (i) is carried out using a single excitation wavelength which lies in the overlap region.

Description

The invention relates to a method for the identification of particles of a polymeric material containing two or more monophosphorus embedded in the material fluorophores with absorption and fluorescence maxima in the UV / VIS / NIR spectral range.

Technological background and state of the art

Monodisperse particles (usually microparticles) made of polymeric materials are increasingly being used in medical diagnostics, as carriers for immobilized enzymes, as markers in environmental analysis and in numerous microbiological investigation methods in which, for example, target compounds are bound to the surface of the microparticles be identified spectroscopically. Polymer particles of uniform shape and size can be obtained by precipitation, suspension or emulsion polymerization.

It is state of the art to add fluorophores to such particles, ie fluorescent chromophores whose fluorescence (emitted fluorescent light) can be used to identify the particles. As a rule, the particle contains only a single fluorophore, so that either the position of the fluorescence maximum or the absolute intensity of the fluorescence must be detected for identification. The latter identification via the absolute intensity has already been realized with particles containing two fluorophores. The intensities of the dyes are determined separately.

If several fluorophores are present in one particle, however, there is a general possibility that photophysical processes such as radiationless deactivation or bimolecular processes (sensitization, quenching), as well as possibly also primary photochemical processes under spin reversal, influence the fluorescence. Aggregation of fluorophores in the particle causes intermolecular interactions. It is to be expected with a concentration-dependent changed spectroscopic behavior. In addition, the measured fluorescence intensity is dependent on the absolute total amount of the fluorophores in the particle and thus also on the respective size of the particles. If a number n of different particles are present in a sample to be examined, they must be distinguishable by fluorescence spectroscopy; Accordingly, a number n of different and spectroscopically distinguishable fluorophores is to be used in a conventional procedure or, for a given fluorophore, particles having graded concentrations of the fluorophore are to be prepared. It is obviously disadvantageous that the number of fluorophores suitable for the purpose, which are sufficiently accessible and which are economically justifiable in their costs, is limited. It is also to be considered that an exchange of a component (ie a fluorophore or a component of the polymer material) of the particle may require a modification of the entire system, either by a necessary adjustment of the production route or the replacement of other components which would otherwise be the spectroscopic Investigation could cause disruptive interaction. Finally, the identification method for samples with a variety of different particles, each with different fluorophores is complicated: There are appropriate excitation wavelengths for the different fluorophores to specify and the detection of fluorescence is to be optimized simultaneously for the variety of different fluorophores. The assignment of particles containing the same dye at different levels of concentration alone, by detecting the absolute intensity, can be detrimental for several reasons. Even small variations in the particle size or inhomogeneities in the dye distribution lead to ambiguous results or to a very limited number of distinguishable populations.

DE 699 07 630 T2 describes microparticles with multiple fluorescence signals. The particles may contain a plurality of dyes, the emission spectra having maxima separated by more than 10 nm. US 2005/0260676 A1 . US 6,632,526 B1 and DE 33 31 017 A1 Further examples of fluorescent particles with two or more fluorophores can be found. DE 600 27 576 T2 describes an identification method for fluorophore-containing particles having a different size or different concentrations of the fluorophore.

The invention has for its object to accelerate the particle identification of particles with two or more fluorophores.

Inventive solution

• (i) die Absorptionsmaxima der Fluorophore um jeweils mindestens 5 nm voneinander beabstandet sind;
• (ii) die Fluoreszenzmaxima der Fluorophore um jeweils mindestens 10 nm voneinander beabstandet sind; und
• (iii) die Absorptionsmaxima und Fluoreszenzmaxima der Fluorophore um jeweils mindestens 5 nm voneinander beabstandet sind.

The invention relates to a method for the identification of a particle of a polymeric material containing two or more monodisperse embedded in the material fluorophores with absorption and fluorescence maxima in the UV / VIS / NIR spectral range. The fluorophores are selected such that

• (i) the absorption maxima of the fluorophores are at least 5 nm apart;
• (ii) the fluorescence maxima of the fluorophores are at least 10 nm apart; and
• (iii) the absorption maxima and fluorescence maxima of the fluorophores are each at least 5 nm apart.

With the aid of a particle system of two (possibly more) fluorophores, a multiplicity of particles which can be differentiated by means of fluorescence spectroscopy can be made available if the selection criteria mentioned are observed. Because the wavelengths of the fluorescence maxima of the fluorophores are at least 10 nm apart from each other, the wavelengths of the absorption maxima are at least 5 nm apart, and the fluorescence and absorption maxima of all fluorophores differ by at least 5 nm with regard to their position, a ratio of the fluorescence intensities of the fluorescence intensities can be individual fluorophores are detected. From this ratio can be - as explained in more detail below - Identification of particles. A sample to be examined may therefore contain different types of particles which differ in each case in the ratio of the detected fluorescence intensities and thus relative proportions of the fluorophores, but otherwise have an identical structure. Of course, particles with different fluorophores can be used as well. The distributed in the polymeric material fluorophores are distributed monodisperse, so are in the ideal case as a homogeneous solution of the fluorophores in the polymeric matrix, or if it is a dispersion, embedded as a dye of substantially the same size in the polymer matrix. The absorption spectra of the fluorophores have an overlap range, in which range for each fluorophore an extinction greater than 0.001 and a quantum yield greater than zero. As a result, the identification method can be initiated by excitation with only one wavelength-explained in more detail below. Compared to conventional methods, a significantly reduced number of individual particle specimens of a population is needed to identify this population.

Preferably, the absorption maxima of the fluorophores are spaced apart by 10 to 100 nm. Independently or in addition, the fluorescence maxima of the fluorophores are spaced apart by 20 to 100 nm.

The absorption and fluorescence maxima are in the UV / VIS / NIR spectral range, that is to say in the wavelength range from 250 to 2500 nm. The absorption maxima are preferably in the wavelength range from 350 to 850 nm. Independently or additionally, the fluorescence maxima are preferably in the wavelength range of 400 up to 900 nm.

The polymeric material should be insoluble in water and optionally swellable, so that the use of the particles in aqueous media or in vivo is possible. The polymeric material may include a polymer made from one or more monomers selected from the group consisting of substituted or unsubstituted acrylates, vinyls, and allylene. In addition, in particular polysilicates and melamine resins can be used. The polymeric material is preferably selected from the group comprising polysilicates, polyadducts (especially polyurethanes, polyureas, polyisocyanates and polydiols), polycondensates (especially polyester / polycarbonates, polyamides, polyimides, polysulfones, polyethersulfones and melamine resins), polyacrylates (especially polymethylmethacrylate, polymethylacrylate, polyhydroxyethylmethacrylate , Polyethyleneglycol monomethacrylates of various chain lengths, poly-N-isopropylacrylamide, polydiethylacrylamide, polyhexylmethacrylate, poly-tert-butylmethacrylate and polyacrylonitrile), polyolefins (especially polystyrene, polypropylene, polyethylene, aliphatic polyvinyls, aliphatic polyallylene), their copolymers or blends. The abovementioned materials permit the production of particles of defined size and are therefore particularly suitable as a polymeric matrix for the fluorescence spectroscopic examination of the particles since they show no or negligible interference with the chromophore system and the photophysical processes, in particular with the fluorescence of the fluorophores. The polymeric material particularly preferably consists of polymethyl methacrylate (PMMA) or polystyrene (PS). In the case of particle preparation, preference is also given to adding suspension stabilizers, such as, for example, the substances PVP K-30 and aerosol OT listed in the exemplary embodiment.

A particle diameter is preferably in the range of 10 nm to 2000 .mu.m, in particular 1 .mu.m to 50 .mu.m.

As fluorophores organic or organometallic compounds, but also from a semiconductor material or metal oxides or metal sulfides existing quantum dots can be used. Preferably, the particle contains one or more fluorophores selected from the group of polyenes, azo compounds, carboximides / nitro compounds / quinacridones, quinoids, oxazines, indigoids, diphenylmethanes / triphenylmethane polymethines, porphyrins / phthalocyanines, metal complexes, in particular the noble metals, rare earths or Transition metals, conjugated betaines, and multiple chromophores. The particle particularly preferably contains a combination of coumarin and rhodamine derivatives as fluorophores.

A test kit based on the aforementioned particles contains two or more batches of particles, the batches each containing the same fluorophores but with a different concentration ratio. Of course, different test kits can be combined with different fluorophores or fluorophore combinations. The particles of different batches can therefore be the same size and composed of the same components; they differ from each other in the concentration ratio of the fluorophores. The particles of the different batches can be distinguished with the aid of the identification method according to the invention in a manner which will be explained in more detail. In other words, in a sample to be measured, a large number of particles of the same size can be present, all of which also contain the same fluorophores. Due to the special selection and determination of the concentration ratios, regardless of the total amount of fluorophore, however, it is possible to assign individual particles to the known batches.

The particles can be prepared according to the method described below. The method provides that the particles are formed by polymerization of a solution or molecular or colloidally disperse dispersion of the fluorophores in a monomer / monomer mixture. In other words, the fluorophores are initially dissolved in the monomer or - in the preparation of a copolymer - in the monomer mixture. Unless they dissolve sufficiently, they are preferably molecularly disperse (particle size <1 nm) or at least colloidally disperse (particle size 1 nm to 1 μm). This ensures that the fluorophores in the particle to be produced are distributed monodisperse, that is, interactions which could influence the emission properties are avoided.

Preferably, the polymerization is carried out under free-radical conditions, in particular, a thermally induced free radical polymerization takes place at 20 to 90 ° C. On the one hand, the abovementioned conditions make it possible to use a large number of known fluorophores which are stable under the test conditions mentioned, and, on the other hand, make it possible to resort to known preparation processes for monodisperse microparticles. For the polymerization, preference is given to using one or more of the monomers selected from the group consisting of substituted or unsubstituted acrylates, vinyls and allylene. Also conceivable is ionic polymerization.

It is further preferred if one or more surface-active substances are added to the solution / dispersion.

Finally, it is preferred if the solution / dispersion polyvinylpyrrolidone (PVP) and / or a small amount of other polymers such as polyethylene glycol (PEG) is added.

• (i) Bestrahlen von vorzugsweise in Monoschicht vorliegenden Partikeln mit einer oder mehreren Anregungswellenlängen im Überlappungsbereich der Absorptionsspektren der Fluorophore bzw. im Bereich der Absorptionsmaxima der Fluorophore (+/–10% der Extinktion);
• (ii) Erfassen der Intensitäten der Fluoreszenz an vorgegebenen Messpunkten, wobei für jeden im Partikel enthaltenen Fluorophor wenigstens ein Messpunkt vorgegeben wird, der in einem Bereich von mindestens 5% der Intensität des Fluoreszenzmaximums des jeweiligen Fluorophors liegt und wobei die Messpunkte um mindestens 5 nm voneinander beabstandet sind;
• (iii) Bestimmen eines Intensitätsverhältnisses der an den Messpunkten erfassten Intensitäten; und
• (iv) Vergleichen des bestimmten Intensitätsverhältnisses mit einem Referenzwert zur Identifikation des Partikels.

The identification method according to the invention of particles of the aforementioned embodiment in a sample. includes the steps:

• (i) irradiating particles which are preferably present in monolayer and having one or more excitation wavelengths in the overlap region of the absorption spectra of the fluorophores or in the region of the absorption maxima of the fluorophores (+/- 10% of the extinction);
• (Ii) detecting the intensities of the fluorescence at predetermined measuring points, wherein for each fluorophore contained in the particle at least one measuring point is set, which lies in a range of at least 5% of the intensity of the fluorescence maximum of the respective fluorophore and wherein the measuring points by at least 5 nm from each other are spaced apart;
• (iii) determining an intensity ratio of the intensities detected at the measurement points; and
• (iv) comparing the determined intensity ratio with a reference value for identification of the particle.

Accordingly, according to the identification method according to the invention, initially in step (i) particles which are ideally present in a monolayer are irradiated, specifically in a wavelength range in which the fluorophores are excited to emit. The particles can only be identified if they can be reached directly with the excitation light. Not directly accessible particles, the identification u. U. disturb and represent in each case an unused material use. It is therefore desirable to arrange the particles such that the largest possible number of particles present in a sample can be directly irradiated. In addition, the aim is for many particles to be directly excited and identified in as short a time as possible, ie, if possible, several particles at the same time. These criteria lead to the optimal distribution of the particles in a monolayer and not, as in conventional methods (eg flow cytometer), in the singulation of the particles.

To create a monolayer, particles in solution are distributed in a microtiter plate. The bottom of the wells of the microtiter plate is prepared so that the particles adhere to it. Non-adherent particles are removed again and can be used for evaluation at another time.

A sufficiently accurate identification result can also be achieved if a second particle layer lies over the interstices of the monolayer. In that case, both the lower and upper particles are identifiable. Finally, it is also possible to assign the particles in solution, so distributed randomly. However, it is disadvantageous in that the detection of moving particles is more complex and time-consuming and superimposed particles u. U. can not be clearly identified and therefore not available for evaluation.

Because the absorbance spectra of the fluorophores have an overlap region in which for each fluorophore an absorbance is greater than 0.001 and a quantum yield greater than zero, irradiation in step (i) is accomplished using a single excitation wavelength that is in the overlap region.

In the subsequent step (ii), the intensities of the fluorescence are detected at predetermined measuring points. These measurement points are at the fluorescence maximum or in a wavelength range at which at least 5%, preferably at least 50%, particularly preferably at least 90%, of the intensity of the fluorescence maximum are present for each individual fluorophore. In other words, if the sample contains two fluorophores, at least two measurement points should be specified; one for the first and one for the second fluorophore. Of course, to increase the measurement accuracy, a plurality of measurement points in the wavelength range mentioned can be assigned to the respective fluorophore, but this is generally not necessary. If the above-mentioned conditions for the position of the absorption and fluorescence maxima are met, the intensities at the measuring points can be clearly detected.

In step (iii), the intensities previously detected at the measuring points are related to one another. The thus determined intensity ratio is compared in step (iv) with a stored reference value, thus allowing identification of the particle. That is, for the individual batches of particles with fluorophores of different concentration ratios, reference values are prepared in advance by measuring the intensities at the measuring points and setting them in relation to one another. The method according to the invention is characterized in that a clear identification of the particles of different batches is already possible on the basis of the intensity ratios. The apparatus for carrying out the spectroscopic examination as well as the identification method itself need only be set / optimized once to a very specific spectral system in order to identify a multiplicity of different particles. This is a great advantage of the method according to the invention.

As a further identification parameter, the fluorescence lifetime can be used. In other words, in step (ii), fluorescence lifetime may be additionally detected for selected fluorophores. This is particularly useful if an additional fluorophore (eg n-th dye) is used to expand the number of distinguishable particles and this fluorophore is not sufficiently different from the other fluorophores properties. Has absorption and fluorescence behavior.

It is also conceivable that the particles to be measured consist of batches of different diameters and in step (ii) additionally an absolute intensity of the fluorescence is detected. In step (iv), a particle diameter is then determined on the basis of the detected absolute intensity of the fluorescence by comparison with a reference value. Taking into account this parameter accordingly particles of the same fluorophores and the same concentration ratios of the fluorophores can be used, which differ in diameter. The difference in particle diameter requires a different one absolute total amount of the fluorophores, which in turn leads to differences in the absolute intensity of the fluorescence (at the measuring points or in a predetermined wavelength range). The latter difference then serves as the distinguishing criterion for the particles of different sizes.

At the same time, a different absolute total amount of the fluorophores at the same particle size produces a different intensity, which can be used in the described way for particle identification. In other words, the microparticles to be measured may have the same diameter but different absolute amounts of fluorophore, and in step (ii) an absolute intensity is additionally detected and assigned to the respective charge.

If particles of the same dye coding and the same absolute total chromophore substance amounts but different particle diameters are present in a sample, an optical object identification can take place in addition to the detection of the intensities of the fluorescence. In other words, if the particles to be measured consist of batches of different diameters and have the same absolute amounts of fluorophore, in addition to the detection of the absolute intensity of the fluorescence, an optical object identification for determining the particle surface or the particle diameter can be carried out.

The detected absolute intensity of fluorescence as area integral is a function of the particle size and the total amount of fluorophores contained. An identical detected intensity of the fluorescence of two particles can therefore result both from the actual equality of the objects as well as from differences in the particle size with the same absolute amount of dye and thus does not allow any clear conclusions about the particle diameter.

By evaluating the projection of the emitted light on an image surface, the particle surface or the diameter can be determined in such cases. The detection of the absolute intensity together with the determination of the particle diameter then makes a clear distinction possible.

An alternative detection of the particle size is the detection of the backscattered excitation light. Particle size recognition can be used as described to distinguish different populations as well as for corrective purposes. In other words, the particles to be measured may consist of batches of different diameters, and in step (ii) an intensity of the backscattered excitation light is additionally detected and in step (iv) a particle diameter is determined by comparison with a reference value on the basis of the detected intensity of the backscattered excitation light ,

In step (ii), a surface potential (eg ζ potential) is preferably additionally determined.

The two or more fluorophores of a particle have an overlapping absorption region and the irradiation in step (i) takes place using an excitation wavelength lying in the overlap region. At the same time, in the case of the abovementioned criteria, a single excitation source can be used to excite both fluorophores to fluoresce, so that the identification method can be accelerated. In any case, however, a very high identification accuracy (small deviation) is guaranteed. In the case of two present fluorophores, the excitation wavelength is preferably at the intersection of the absorption spectra or in the region around the point of intersection with a difference of up to 10% of the value of the extinction at the point of intersection. In the case of three or more fluorophores, all fluorophores must have a common excitation wavelength range, in which range for each fluorophore an extinction of at least 0.001, preferably 0.1 to 1, and a quantum yield of at least 0.01, preferably 0.5 to 1 , and the irradiation in step (i) is performed using a single excitation wavelength which lies in the overlap region.

The excitation can therefore take place with a wavelength.

As light sources, lamps (gas discharge lamps, if necessary in combination with corresponding filters), lasers, LEDs can be used.

Brief description of the figures

The invention will be explained in more detail with reference to two embodiments and the accompanying figures. Show it:

1 to 3 Absorption and fluorescence spectra of the Coumarin 314 / Rhodamine 6G system at two different concentration ratios showing possible excitation with only one wavelength (abscissa: wavelength [nm]; ordinate: intensity); and

4 Fluorescence behavior in PMMA at 11 different mixing ratios of a system of fluorophores Coumarin 334 / rhodamine 6G, which was excited at two wavelengths (abscissa: concentration ratio of the fluorescent dyes, ordinate: intensity ratio of the detected intensities at 550 nm and 490 nm).

5 Particles of the dye system coumarin 334 / rhodamine 6G of different diameters and at different concentration and thus determined intensity ratios (abscissa: particle diameter [μm]; ordinate: intensity ratio of the determined intensities at 550 nm and 490 nm).

Detailed description

Exemplary Embodiment - Production of the Particles

In general, all methods which produce adjustable particles by reaction in size can be used; specifically for precipitation polymerization, emulsion polymerization and suspension polymerization. The production of particles will be explained in more detail on the basis of the precipitation polymerization.

Reagents used for the polymeric matrix: methanol 70 ml Methyl methacrylate (MMA) 10 ml Polyvinylpyrrolidone (PVP K-30) 5 g Sodium bis (2-ethylhexyl) sulfosuccinate (Aerosol OT) 320 mg Azobisisobutyronitrile (AIBN) 40 mg

PVP and aerosol OT were presented dissolved in methanol. To the solution was added a solution of the radical initiator AIBN in the monomer MMA. The mixture was then stirred at 65 ° C bath temperature for 24 h at about 100 rpm. The still warm suspension was poured into 300 ml of water, and the precipitate was filtered off with suction through a frit and washed with water.

Fluorophors used:

• Rhodamine 6G and coumarin 314 and 334, respectively

The fluorophores were added in the amounts specified in Tab. 1 of the solution of AIBN in MMA. Particle number. Rhodamine 6G [mg / g plastic] Coumarin 334 [mg / g plastic] determined intensity ratio 1 0,475 0,015 2748 2 0,375 0,075 15 3 0,250 0,150 4 4 0,125 0.225 1 5 0.063 0,270 0.5 6 0.031 0,356 0.18 7 0.028 0.422 0.17 8th 0.016 0,366 0.12 9 0,006 0.422 0.04 10 0,004 0.422 0.02 11 0,002 0.422 0.01 Tab. 1 - proportions of fluorophores

A total of 11 different concentration ratios of Coumarin 334 and Rhodamine 6G were prepared in said PMMA matrix (cf. 4 ).

The microparticles obtained by the process have an average diameter of about 9 microns.

identification method

According to the above-described process dye mixtures were gem. Tab. 1 introduced into the material. The resulting particles were mounted on glass slides and analyzed in the fluorescence spectrophotometer. The device used (Shimadzu RF-5301 PC) is equipped with a standard xenon lamp that covers the entire UV / VIS spectrum.

Each specimen was exposed to light of wavelengths 470 nm and 525 nm and the values of the intensity of the emitted fluorescence radiation were determined at 490 nm and 550 nm, respectively. These intensity values were then set in relation to each other, the value obtained gives the identification parameter (cf. 4 ).

The 1 and 2 Exemplary excitation peaks and superimposed fluorescence spectra of the system coumarin 314 / rhodamine 6G can be seen. There are fluorescence spectra at two different concentration ratios, namely 1:15 ( 1 ) and 3: 1 ( 2 ), each shot at three different wavelengths.

The peak at 449 nm results from the irradiation at this wavelength and corresponds to the absorption maximum of Coumarin 314, 525 nm corresponds to the absorption maximum of rhodamine 6G. In the case of different concentration ratios present in a mixture, therefore, the total fluorescence intensity (integral) of one or the other fluorophore and also the intensity at selected measurement points dominates, as additionally in 3 illustrated.

The peak at 470 nm results from the irradiation at the same wavelength, which represents the approximate maximum of the common excitation region. If the mixtures of the fluorophores are irradiated only at this one wavelength, sufficiently different fluorescence spectra result, which permit identification based on the ratio formation of intensities at two selected measurement points.

Intensity ratios of (i) 2.43 (1:15) and (ii) 0.30 (3: 1) were found for the fluorescence at the measurement points.

In the 4 are the intensity ratios at the different mixing ratios of the particle no. 1-11. The linear relationship of the two variables over a very wide range of concentrations can be seen. The ratio is therefore suitable as an identification parameter, possibly even without the deposit of concrete reference values, if the function is known.

5 shows particle populations that differ both in terms of the set concentration and thus determined intensity ratio as well as in terms of their particle diameter.

Claims (7)

A method of identifying a particle of a polymeric material containing two or more monodisperse fluorophores embedded in the material having absorption and fluorescence maxima in the UV / VIS / NIR spectral region, wherein the fluorophores are selected such that (i) the absorption maxima of the fluorophores each spaced at least 5 nm apart; (ii) the fluorescence maxima of the fluorophores are at least 10 nm apart; and (iii) the absorption maxima and fluorescence maxima of the fluorophores are each at least 5 nm apart in a sample comprising the steps: (i) irradiating particles which are preferably present in monolayer and having one or more excitation wavelengths in the overlap region of the absorption spectra of the fluorophores or in the region of the absorption maxima of the fluorophores (+/- 10% of the extinction); (Ii) detecting the intensities of the fluorescence at predetermined measuring points, wherein for each fluorophore contained in the particle at least one measuring point is set, which lies in a range of at least 5% of the intensity of the fluorescence maximum of the respective fluorophore and wherein the measuring points by at least 5 nm from each other are spaced apart; (iii) determining an intensity ratio of the intensities detected at the measurement points; and (iv) comparing the determined intensity ratio with a reference value for identifying the particle, characterized in that the absorption spectra of the fluorophores have a common excitation wavelength range, wherein in this range for each fluorophore an extinction of at least 0.001 and a quantum efficiency of at least 0.01 applies and the irradiation in step (i) is performed using a single excitation wavelength that lies in the overlap region. Identification method according to claim 1, wherein in step (ii) for selected fluorophores additionally a fluorescence lifetime is detected. Identification method according to claim 1, in which the particles to be measured consist of batches of different diameters and in step (ii) additionally an absolute intensity of the fluorescence is detected and in step (iv) on the basis of the detected absolute intensity of the fluorescence a particle diameter by comparison with a reference value is determined. Identification method according to claim 1, in which the particles to be measured have the same diameter but different absolute amounts of fluorophore and in step (ii) additionally an absolute intensity is detected and assigned to the respective charge. Identification method according to claim 1, wherein the particles to be measured from batches of different diameters and in step (ii) additionally an intensity of the backscattered excitation light is detected and in step (iv) based on the detected intensity of the backscattered excitation light, a particle diameter by comparison with a reference value is determined. Identification method according to claim 1, in which the particles to be measured consist of batches of different diameters and have the same absolute amounts of fluorophore and in which, in addition to the detection of the absolute intensity of the fluorescence, an optical object identification for determining the particle surface or the particle diameter is carried out. Identification method according to claim 1, wherein in step (ii) additionally a surface potential (eg ζ-potential) is determined.

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