Imaging method using a dye dimer, dye dimer, and kit for use in an imaging method

Abstract

The invention relates to an imaging method using a dye dimer, wherein the dye dimer comprises a single-stranded DNA (nucleotide strand) formed by a sequence of nucleotides, and a molecule of a dye that can be excited to emit fluorescence radiation is attached to the mutually opposite terminal regions of the nucleotide strand. The dye dimer can be weakly excited to emit fluorescence radiation in a first conformation and can be highly excited to emit fluorescence radiation in a second conformation. Structures (antigens) of a sample to be imaged are bound to antibodies which are provided with first binding molecules, these first binding molecules being compatible with at least one section of the nucleotide strand of the dye dimer. The dye dimer binds to a plurality of the first binding molecules, a dye dimer bound to a first binding molecule being present in the second conformation. The sample is illuminated with excitation radiation suitable for exciting an emission of fluorescence radiation from the dye dimer and shaped to form a light sheet, dye dimers in the second conformation being excited to emit fluorescence radiation. This fluorescence radiation is detected and evaluated for imaging purposes. The invention is characterised in that the excitation radiation is designed as a lattice light sheet and is directed into the sample. The invention also relates to a dye dimer and to the provision and use thereof.

Description

[0001] Imaging technique using a dye dimer, dye dimer and kit for use in an imaging technique

[0002] The invention relates to an imaging method using a dye dimer according to the preamble of the independent claim. The invention further relates to a dye dimer and a kit.

[0003] For high-resolution imaging, especially in living cells and tissues (sample), as well as for investigations of processes in living cells, a number of imaging techniques are known which are based on the marking of structures of interest with markers and their excitation to emit detection radiation.

[0004] In recent years, the method known as DNA-PAINT (DNA-point accumulation for imaging in nanoscale topography) has become established and further developed (Chung, KKH et al, 2022, Fluorogenic DNA-PAINT for faster, low-background super-resolution imaging, Nature Methods 19: 554–559). In this method, for example, a structure (antigen) of the sample to be examined is coated with a specifically binding antibody. The antibody is equipped with a single-stranded sequence of deoxyribonucleic acid (DNA), which is referred to as the "docking strand" (hereinafter also: binding molecule).

[0005] To image antibodies bound to an antigen, a number of dye molecules are added to the medium containing the sample to be imaged. These molecules contain a dye, specifically a fluorophore, which is bound to a short segment of single-stranded DNA (nucleotide strand, "imager strand"). The nucleotide sequence of the nucleotide strand is compatible with the binding molecule, meaning the dye molecule can bind to it. It is advantageous if the dye molecules are designed to exhibit strong fluorescence emission only when the dye molecule is attached to the binding molecule, i.e., when the imager strand and docking strand are fused together.

[0006] Recent studies show that unwanted background radiation can be significantly reduced by attaching a dye to each end of the nucleotide strand of the dye molecule (TDI, "two-dye imager"; Kessler, LF et al. 2023, Self-quenched fluorophore dimers for DNA-Paint and STED microscopy, Angew. Chem. Int. Ed. 62, e202307538). Such dye molecules, i.e., the combination of a nucleotide strand and two dyes, are henceforth referred to as dye dimers. The dyes are chosen so that they largely prevent each other from emitting fluorescence radiation when the dyes are in close proximity (self-quenching). In the state not coupled to a binding molecule, the dye dimer has a compact form. This state (hereinafter also referred to as the first conformation) is also called the "H-dimer".

[0007] When the dye dimer binds to the binding molecule, i.e., when the image strand and docking strand align, the nucleotide strand is stretched and the dyes are so far apart that the self-quenching effect is no longer relevant. Therefore, in this second conformation, the dyes can be excited, for example, by suitable excitation radiation to emit fluorescence with a higher intensity than in the first conformation. 2024P01041WG 09.10.2024

[0008] The desired high spatial resolution for investigating interactions between cells and cell components can be achieved if only a narrow region of the sample in the direction of the detection axis of a detection optic (z-direction) is illuminated with excitation radiation and fluorescence radiation is excited only in the limited illuminated area (Kessler, LF et al. 2023, Self-quenched fluorophore dimers for DNA-Paint and STED microscopy, Angew. Chem. Int. Ed. 62, e202307538).

[0009] However, the solutions known from the prior art require long measurement times to image a volume of the sample. In addition, the unwanted background radiation must be kept within an acceptable range by using only small amounts of dye molecules.

[0010] The invention is based on the objective of proposing a method that reduces the disadvantages of the prior art and, in particular, reduces the excitation of unwanted background signals, thereby increasing the signal-to-background ratio. Improved dye dimers and, optionally, the combined use of different dye dimers are also proposed.

[0011] The problem is solved by an imaging method according to the main claim. Dye dimers, as well as their provision and application, are the subject of the dependent claims. Advantageous embodiments are specified in the dependent claims.

[0012] The imaging technique is performed using a dye dimer. The dye dimer comprises a single strand (nucleotide strand) formed by a sequence of nucleotides. A molecule of a dye capable of emitting fluorescence is bound to each of the opposite terminal regions of the nucleotide strand. The dye dimer can adopt at least two conformations. In a first conformation, the dye dimer is barely excitable to emit fluorescence. Upon excitation, the dye dimer exhibits only relatively low fluorescence emission. In this conformation, the dye dimer may, for example, undergo self-quenching of the two dye molecules and be structured as an H-dimer. In a second conformation, the dye dimer is excitable to emit fluorescence and emits with a significantly higher fluorescence intensity than in the first conformation.Under otherwise identical conditions, the fluorescence intensities in the second conformation are at least five times higher, particularly at least eight times higher, and advantageously at least ten times higher, than in the first conformation. In the second conformation, the nucleotide strand can be stretched, so that the two dyes are as far apart as possible and no or no relevant self-quenching occurs. The second conformation is particularly present when the dye dimer is bound to a binding molecule.

[0013] In this process, structures (antigens) of the sample to be imaged are linked to antibodies. These antibodies are equipped with first binding molecules, also known as "docking strands." These first binding molecules are compatible with at least one segment of the nucleotide strand of the dye dimer, meaning that the dye dimer and the binding molecule can bind to each other. Sufficient compatibility between the nucleotide strand and the binding molecule is sufficient to enable binding. This binding is preferably reversible, so that only a fraction of the dye dimers present are in the second conformation and can be detected at any given time. Unbound dye dimers in the first conformation diffuse too rapidly and exhibit insufficient fluorescence intensity to be localized. However, they contribute to the background signal.

[0014] The sample to be imaged is illuminated with excitation radiation suitable for inducing fluorescence emission from the dye dimer. The excitation radiation is shaped into a planar shape by optical elements of an illumination optic, such that it propagates essentially in a plane obliquely or perpendicular to the optical axis of the illumination optic, and the sample is illuminated by a corresponding light sheet. The dye dimers present in the area illuminated by the light sheet and residing in the second conformation are excited to emit fluorescence radiation. The emitted fluorescence radiation is detected with two-dimensional resolution and evaluated for imaging.

[0015] A characteristic feature of the invention is that the excitation radiation is designed as a lattice light sheet and directed into the sample.

[0016] State-of-the-art light sheets are generated by compressing illumination radiation in a direction perpendicular to its propagation direction using optical lenses, such as cylindrical lenses (static light sheet). Alternatively, the illumination radiation, shaped into a thin beam, can be rapidly moved back and forth in a plane, for example, using a deflection device (scanner) (dynamic light sheet). Both approaches can also be combined. In contrast to the state of the art, a lattice light sheet, such as the Lattice Light Sheet 7 from Carl Zeiss Microscopy GmbH, shapes the illumination radiation into a defined grating or network. A spatial light modulator (SLM) can be used for this purpose. The shaped illumination radiation can then be moved in a plane using a scanner.Furthermore, the illumination radiation can be provided in the form of non-diffractive rays, for example as Bessel rays or Sinc3 rays. Due to the effect of this special form of illumination radiation, a very thin light sheet is produced, which also exhibits hardly any or no unwanted side lobes.

[0017] The thin sheet of light thus generated can be moved through the sample by creating a relative movement between the light sheet and the sample. This can be achieved by using a deflection device for the light sheet and / or by moving the sample using a movable sample stage. A three-dimensional image of the sample can be generated from a multitude of acquired two-dimensional images sequentially in the z-direction (z-stack).

[0018] Using the method according to the invention, a high spatial resolution can therefore be achieved with a combination of dye dimers and the use of a lattice light sheet, while simultaneously achieving an improved signal-to-noise ratio (SNR) compared to the prior art. The method can be described as LLS-TDI-DNA-PAINT. The high fluorescence intensity of the dye dimers excited in the second conformation advantageously supports the accuracy of the localization of the individual detected signals.

[0019] In one embodiment of the process according to the invention, the dye ATTO Oxa14 (Fig. 1) is used as the dye of the dye dimer. This oxazine dye, which is a derivative of the dye MR121 (CAS 185213-55-2) with a side group of the form R2 = CH2COOH bonded to a nitrogen atom (Fig. 1), is well-established and readily available. The dye absorbs and emits in aqueous solution at 660 nm and 680 nm, respectively (similar to: Marme, N. et al., 2005, Aggregation behavior of the red-absorbing oxazine derivative MR 121 : A new method for determination of pure dimer spectra, Chemical Physics Letters 408: 221-225).

[0020] The use of ATTO 520 (e.g., ATTO-TEC GmbH, Siegen, Germany; Fig. 2) as the dye in the dye dimer is possible in a further embodiment of the process according to the invention. ATTO 520 is based on the known rhodamine G6. Its absorption wavelength is 517 nm, and its emission wavelength is 538 nm.

[0021] It is also possible to use the dye AF 647, whose absorption wavelength is 647 nm and whose emission wavelength is 667 nm.

[0022] As described above, the dye dimers can bind to antibodies via their nucleotide strand. These antibodies are modified with corresponding binding molecules. The antibodies can bind to specific antigens. In one method configuration, the B-lymphocyte antigen CD20, hereinafter referred to simply as CD20, is chosen as the structure of the sample acting as the antigen.

[0023] CD20 is a transmembrane protein of B lymphocytes (B cells). As an essential part of the adaptive immune system, B cells are capable of producing and releasing antibodies. It is possible to induce changes in B cells via CD20, even leading to their cell death (see below). Various pathways for the destruction of B cells have been postulated. These include the direct induction of cell death through interaction with CD20, phagocytosis, Fc receptor-mediated antibody-dependent cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) (Ghosh, A. et al (in press), Decoding the molecular interplay of endogeneous CD20 and therapeutic antibodies with fast volumetric nanoscopy, Science; Rouge, L. et al., 2020, Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab, Science 367: 1224-1230).

[0024] Several monoclonal antibodies (mAbs) are known that bind to the CD20 antigen and can be used in the method according to the invention. The antibodies rituximab (RTX, CAS 174722-31-7) and 2H7 (OCR, ocrelizumab; CAS 637334-45-3) bind to almost the same epitope of CD20. The monoclonal antibodies ofatumumab (OFA; CAS 679818-59-8) and obinutuzumab (OBZ; CAS 949142-50-1) can also be used.

[0025] The monoclonal antibodies rituximab (RTX), ocrelizumab (OCR), and ofatumumab (OFA) are classified as class I (Rouge, L. et al., 2020, Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab, Science 367: 1224-1230). These monoclonal antibodies are intended to induce cross-linking of CD20 and trigger the CDC pathway for the destruction of B cells.

[0026] In contrast, the monoclonal antibody obinutuzumab OBZ is classified as class II. Current literature suggests that it does not induce cross-linking and that the mechanism of action of ADCC is more relevant than that of CDC.

[0027] The high resolution and the SNR, which is also increased compared to the prior art, allow at least two different dye dimers to be used simultaneously with the method according to the invention and a two-color DNA paint to be carried out using a lattice light sheet (two-color-LLS-TDI-DNA-PAINT).

[0028] Instead of just one antibody, a second antibody is used, which is equipped with a second set of binding molecules. In this way, a first dye dimer can bind to the first set of binding molecules, while a second dye dimer can bind to the second set of binding molecules. The second dye dimer differs from the first dye dimer 2024P01041WG 09.10.2024 in its excitation wavelength, its emission wavelength, and / or its compatibility with the existing binding molecules. It is possible to use different antibodies and equip them with different binding molecules. It is also conceivable to use the same antibodies but couple them with different binding molecules, so that different dye dimers can be bound as a result.

[0029] It has been found that in such a two-color DNA-PAI NT variant, it is advantageous to use ATTO Oxa14 as the dye of the first dye dimer and ATTO 520 as the dye of the second dye dimer. The two dyes, ATTO Oxa14 and ATTO 520, are sufficiently far apart in their excitation and emission wavelengths to reliably separate signals from the detection radiation. However, considering their spectra and their behavior upon addition of an excess of binding molecules (see below), both dyes behave very similarly (Figs. 3 and 4), making simultaneous use quite feasible.

[0030] The advantages of a two-color LLS-TDI-DNA-PAI NT application are further emphasized by another experiment conducted by the inventors. Raji cells were labeled with 5 pg / mL of either anti-CD20 RTX-AF647 or 2H7-AF647 and anti-CD45 HI30-CF568 (562 / 584 nm). After incubation with RTX / 2H7, the cells were fixed. Cell samples were scanned using a Lattice Light Sheet 7 from Carl Zeiss Microscopy GmbH. The results showed that RTX accumulated in certain areas of the cells, particularly in cell protrusions. Furthermore, the RTX signal was predominantly located on one side of the cell, indicating cell polarization, which confirms the results of other investigations by the inventors.

[0031] Furthermore, Raji cells were labeled against actin with 2H7-AF647 and with SPY-555 (555 / 580 nm), and the living cells were observed and imaged using LLS-TDI-DNA-PAINT.

[0032] The inventive method has also led to further insights, which are presented in detail in the currently in-press publication Ghosh, A. et al, Decoding the molecular interplay of endogeneous CD20 and therapeutic antibodies with fast volumetric nanoscopy, Science, and are only briefly outlined below.

[0033] It was thus demonstrated that, contrary to previous assumptions, therapeutic antibodies of both type I and type II crosslink CD20 molecules on the cell membrane of B cells. The efficiency of this crosslinking varies considerably from antibody to antibody. However, it always occurs, for example, when the cells are incubated with the antibodies at room temperature or at 37°C. Furthermore, the efficiency of crosslinking increases significantly with the concentration of the added antibodies. Precise determination of the number of therapeutic antibodies binding to the cell membrane of B cells was made possible.

[0034] It was also possible to show using the method according to the invention that CD20 is highly concentrated on the microvilli of the cells (cell extensions) and that the antibodies mainly bind there.

[0035] The method according to the invention allows the molecular distribution of CD20 / antibody signals to be mapped three-dimensionally across the entire cell. Compared to the prior art, this also makes it possible to capture their distribution on the upper surface of the cell with molecular precision.

[0036] It was also found that type I antibodies strongly polarize B cells and stabilize the microvilli, meaning the microvilli are stable, very long, and all point in one direction. Type II 2024P01041WO 09.10.2024

[0037] Antibodies, on the other hand, do not polarize B cells, although they also cross-link CD20 molecules. The length of the microvilli stabilized by the antibodies depends strongly on the concentration and type of antibody. This finding can be used to classify antibodies.

[0038] The invention also allows for the examination of large volumes of a sample. Due to the high fluorescence intensity, low noise, and high localization precision, unlike prior art methods, the illumination does not need to be limited to a small area of ​​the sample, for example, using TIRF. Since previous methods, such as those using TIRF technology, require the concentration of the dye molecules to be kept very low, such imaging takes several hours.

[0039] The higher increase in fluorescence intensity during the transient binding of the dye dimers to the binding molecules allows the inventive method to also be used for applications without TIRF excitation. High-resolution images of entire cells, not just the lower (basal) membrane, can be generated. A 3D scan of an entire cell takes only 4 hours. A measurement time of 10 to 20 minutes is sufficient per image plane. Furthermore, the measurements can be performed with an imager concentration 10 to 50 times higher than is known from the prior art.

[0040] The method according to the invention can therefore be used to obtain high-resolution images of a cell and to investigate interactions between an antibody, an antigen and a cell.

[0041] Furthermore, the effectiveness of an antibody after it binds to an antigen can be assessed by determining the degree of cross-linking of addressed tumor-associated receptors and / or an induced polarization of the cell.

[0042] The invention thus opens up new avenues for testing novel therapeutic antibodies and for a better understanding of their mode of action.

[0043] It has been shown during the work on the inventive method that an excess of bonding molecules by a factor of at least 10 3 , advantageous but of at least 10 4 , leading to an approximately 6 to 9-fold increase in the fluorescence intensity of the bound dye dimers (Fig. 3 and 4).

[0044] The newly developed dye dimer, using ATTO 520 as the dye, is described in more detail below. As described above, the dye dimer comprises a single strand of DNA (nucleotide strand) formed by a sequence of nucleotides. One molecule of the dye ATTO 520 is bound to each of the opposite terminal regions of the nucleotide strand. In its first conformation, the dye dimer is barely excitable to emit fluorescence. In this first conformation, the effect of self-quenching is particularly noticeable. In contrast, in a second conformation, the dye dimer is excitable to emit fluorescence of higher intensity (see above).

[0045] In one embodiment of the dye dimer according to the invention, the nucleotide strand has a length of ten nucleotides with the sequence 5'-GTA ATG AAG A-3' (also designated P3). A compatible docking strand has the sequence methyltetrazine-5-TTT CTT CATTA-3' (Schnitzbauer, J. et al., 2017, Super-resolution microscopy with DNA-PAINT, Nature Protocols 12: 1198-1228). In another embodiment, the dye dimer according to the invention can have a nucleotide strand with seven nucleotides and the sequence 5'-TGT GTG T-3' (also designated R4). A compatible binding molecule may possess the sequence Methyltetrazine-5'-ACACACACACACACACACA (Reinhardt, SCM et al., 2023, Ängström-resoLution fluorescence microscopy, Nature 617: 711-716; Narayanasamy, KK et al., 2021, Visualizing synaptic multi-protein patterns of neuronal tissue with DNA-assisted singlemolecule Localization microscopy, Frontiers in Synaptic Neuroscience 13: 671288, doi: 10.3389 / fnsyn.2021 .671288).

[0046] The dye dimers and / or the antibodies, in particular their modified form with the corresponding binding molecules Methyltetrazine-5 -TTT CTT CATTA-3 ' or Methyltetrazine-5 '-ACACACACACACACACA, may be provided to a user in the form of a kit.

[0047] A kit for use in a TDI-DNA-PAINT procedure, particularly for the characterization of CD20, comprises a quantity of a first dye dimer, each containing two molecules of the dye ATTO Oxa 14 or ATTO 520. Rituximab (RTX), ocrelizumab (ORC), ofatumumab (OFA), or obinutuzumab (OBZ) is present as an antibody specific for the antigen CD20. The antibody is equipped with a binding molecule that is compatible with at least one segment of the nucleotide strand of the dye dimer and allows reversible binding of the nucleotide strand of the dye dimer to the binding molecule. The kit may also contain additional components such as buffers, enzymes, and excipients.

[0048] Another kit for use in a two-color TDI DNA-PAINT procedure provides quantities of a first dye dimer and a second dye dimer. The first dye dimer contains two molecules of the dye ATTO Oxa 14, while the second dye dimer contains two molecules of the dye ATTO 520.

[0049] Both kits can be used in a process where the excitation radiation is shaped and irradiated as a lattice light sheet.

[0050] Character description

[0051] Figure 1 shows the structural formulas of the dyes MR121 and ATTO Oxa14.

[0052] Figure 2 shows the structural formula of the dye ATTO 520.

[0053] The behavior of the dye dimers with a nucleotide strand of the form R4 and the dye ATTO 520 is plotted as relative intensity (normalized to 1) over a wavelength range of approximately 530 nm to 650 nm in Figure 3 (simplified from Ghosh, A. et al (in press), Decoding the molecular interplay of endogeneous CD20 and therapeutic antibodies with fast volumetric nanoscopy, Science). The curve with a dashed solid line shows the intensity profile without the addition of an excess of binding molecules. The solid line shows the profile with a 10 4 -fold excess of binding molecules.

[0054] The behavior of the dye dimers with a nucleotide strand of the form R4 and the dye ATTO Oxa14 is plotted as relative intensity (normalized to 1) over a wavelength range of approximately 650 nm to 750 nm in Figure 4 (reproduced from Ghosh, A. et al (in press), Decoding the molecular interplay of endogeneous CD20 and therapeutic antibodies with fast volumetric nanoscopy, Science). The curve with a dashed solid line shows the intensity profile without the addition of an excess of binding molecules. The solid line shows the profile with a 10 4 -fold excess of binding molecules.

Claims

Patent claims 1. Imaging method using a dye dimer, wherein the dye dimer comprises a single strand of DNA (nucleotide strand) formed by a sequence of nucleotides, and a molecule of a dye excitable to emit fluorescence radiation is bound to the opposite terminal regions of the nucleotide strand, wherein the dye dimer is: o slightly excitable to emit fluorescence radiation in a first conformation; and o highly excitable to emit fluorescence radiation in a second conformation, wherein the fluorescence intensity in the second conformation is at least 5 times, in particular at least 8 times, advantageously at least 10 times higher than in the first conformation;and in the process, structures (antigens) of a sample to be imaged are linked to antibodies that are equipped with first binding molecules, wherein these first binding molecules are compatible with at least one section of the nucleotide strand of the dye dimer; the dye dimer is added and binds to a number of the first binding molecules, wherein a dye dimer bound to a first binding molecule is in the second conformation; the sample is illuminated with excitation radiation suitable for exciting emission of fluorescence radiation from the dye dimer, wherein the excitation radiation is shaped into a light sheet; and wherein dye dimers in the second conformation are excited to emit fluorescence radiation; and emitted fluorescence radiation is detected as detection radiation and evaluated for imaging;characterized in that the excitation radiation is designed as a lattice light sheet and directed into the sample.

2. Method according to claim 1, characterized in that ATTO Oxa14 is used as the dye of the dye dimer.

3. Method according to claim 1, characterized in that ATTO 520 is used as the dye of the dye dimer.

4. Method according to one of the preceding claims, characterized in that the B-lymphocyte antigen CD20 is selected as the structure (antigen).

5. Method according to one of claims 2 to 4, characterized in that one of the monoclonal antibodies rituximab (CAS 174722-31-7) or 2H7 (ocrelizumab; CAS 637334-45-3) is used as an antibody binding to a structure of the sample.

6. Method according to one of claims 2 to 4, characterized in that the monoclonal antibody ofatumumab (OFA; CAS 679818-59-8) or obinutuzumab (OBZ; CAS 949142-50-1) is used as an antibody binding to a structure (antigen) of the sample.

7. Method according to one of the preceding claims, characterized in that Structures of the sample are provided with further antibodies which are equipped with second binding molecules, a second dye dimer is added alongside the dye dimer, wherein the second binding molecules are compatible with at least one section of a nucleotide strand of the second dye dimer and the second dye dimer binds to a number of the second binding molecules, wherein a second dye dimer bound to a second binding molecule is in the second conformation and the second dye dimer differs from the first dye dimer with respect to its excitation wavelength, its emission wavelength and / or its compatibility with the existing binding molecules;The sample is illuminated with excitation radiation in the form of a lattice light sheet, suitable for exciting the emission of fluorescence radiation from the second dye dimer, whereby second dye dimers in the second conformation are excited to emit fluorescence radiation, and the emitted fluorescence radiation is detected as a detection radiation and evaluated for imaging.

8. Method according to claim 7, characterized in that one of the dye dimers comprises the dye ATTO Oxa14 and the other dye dimer comprises the dye ATTO 520.

9. Method according to one of the preceding claims, characterized in that the amount of bonding molecules is reduced by a factor of at least 10 3 , advantageous by a factor of at least 10 4 is higher than the concentration of the respective compatible dye dimers.

10. Use of the method according to claim 1 for high-resolution observation and investigation of interactions between an antibody, an antigen and a cell.

11. Use of the method according to claim 1 for evaluating the efficacy of an antibody after its binding to an antigen by determining a degree of cross-linking of addressed tumor-associated receptors and / or an induced polarization of the cell.

12. Dye dimer, particularly for use in a process according to claim 3 and any one of claims 4 to 11, comprising a single strand of DNA (nucleotide strand) formed by a sequence of nucleotides, and one molecule of a dye excitable to emit fluorescence radiation, bound to the opposite terminal regions of the nucleotide strand, wherein the dye dimer is slightly excitable to emit fluorescence radiation in a first conformation; and is highly excitable to emit fluorescence radiation in a second conformation, wherein the fluorescence intensity in the second conformation is at least 5 times, in particular at least 8 times, advantageously at least 10 times higher than in the first conformation, characterized in that the dye is ATTO 520.

13. Dye dimer according to claim 12, characterized by a nucleotide strand having a length of ten nucleotides, in particular with the sequence GTAATGAAGA-3' or a nucleotide strand having a length of seven nucleotides, in particular with the sequence: 5'- TGTGTGT-3'.

14. Kit for use in a TDI-DNA-PAINT method, in particular for characterizing CD20, comprising a quantity of a first dye dimer in which a single strand of DNA (nucleotide strand) formed by a sequence of nucleotides is present, and one molecule of a dye excitable to emit fluorescence radiation is bound to each of the opposite terminal regions of the nucleotide strand, wherein the dye dimer is slightly excitable to emit fluorescence radiation in a first conformation;and is readily excitable to emit fluorescence radiation in a second conformation, wherein the fluorescence intensity in the second conformation is at least 5 times higher, in particular at least 8 times higher, advantageously at least 10 times higher, than in the first conformation, characterized in that the dye dimer comprises two molecules of the dye ATTO Oxa 14 or ATTO 520, and includes an antibody specific for the antigen CD20, such as rituximab, ocrelizumab, ofatumumab (OFA) or obinutuzumab (OBZ), wherein the antibody is provided with a binding molecule that is compatible with at least one section of the nucleotide strand and allows reversible binding of the nucleotide strand of the dye dimer to the binding molecule.

15. Kit for use in a two-color TDI DNA-PAINT process comprising an amount each of a first dye dimer and a second dye dimer, each of which has a single strand of DNA (nucleotide strand) formed by a sequence of nucleotides, and a molecule of a dye excitable to emit fluorescence radiation bound to opposite terminal regions of the nucleotide strand, each dye dimer being slightly excitable to emit fluorescence radiation in a first conformation;and is excitable in a second conformation to emit fluorescence radiation, wherein the fluorescence intensity in the second conformation is at least 5 times higher, in particular at least 8 times higher, advantageously at least 10 times higher, than in the first conformation, characterized in that a first dye dimer comprises two molecules of the dye ATTO Oxa 14, and a second dye dimer comprises two molecules of the dye ATTO 520.

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