Bispecific compound for DNA-paint and method

Abstract

The present invention relates to a bispecific compound capable of specifically binding to a target molecule, wherein the bispecific compound comprises: at least one target molecule binding moiety capable of binding to the target molecule, and at least one docking module capable of binding to at least one imager, wherein the at least one target molecule binding moiety corresponds to the target molecule. The present invention also relates to a method for detecting a target molecule, the method comprising: introducing into a sample chamber a sample containing the target molecule; introducing into the sample chamber a bispecific compound capable of binding the target molecule; allowing the bispecific compound to bind to the target molecule; and visualizing the target molecule bound to the bispecific compound, wherein the bispecific compound is according to any of the preceding bispecific compound claims, and wherein the method comprises visualizing the target molecule bound to the bispecific compound by means of points accumulation for imaging in nanoscale topography (PAINT) microscopy via at least one imager, wherein the step of visualizing the target molecule bound to the bispecific compound comprising at least one of: capturing at least one image data, and processing the at least one image data. Furthermore, the present invention also relates to a system for detecting a target molecule in a sample, the system comprising: a sample chamber configured to receive the sample and the bispecific compound; and a super-resolution microscope configured to perform points accumulation for imaging in nanoscale topography (PAINT) microscopy measurements, wherein the system is configured to perform the method of the present invention. The invention also relates to a kit for use in the method for detecting a target molecule, the kit comprising the bispecific compound, wherein the bispecific compound is at least one of: in a solution, and is freeze-dried.

Description

[0001] BISPECIFIC COMPOUND FOR DNA-PAINT AND METHOD

[0002] Field

[0003] The invention lies in the field of super-resolution microscopy and particularly in the field of DNA points accumulation for imaging in nanoscale topography (DNA-PAINT). The goal of the present invention is to provide a bispecific compound for DNA-PAINT, a method performed using such a bispecific compound and corresponding use of the method.

[0004] Introduction

[0005] Super-resolution microscopy methods have addressed some of the challenges faced by conventional microscopy techniques in achieving high spatial resolution, particularly in imaging biological structures at the nanoscale.

[0006] The field of super-resolution microscopy can be divided into two streams: one, which comprises deterministic nanoscale probing of a sample, as pioneered in STED microscopy, and another comprising stochastic localization of single emitters as used in various single molecule localization microscopy (SMLM) techniques.

[0007] DNA-PAINT is a super-resolution technique that breaks the optical diffraction limit by temporally separating signals from unresolvable targets, localizing them separately, and reconstructing the image from the localized data (single-molecule localization microscopy, SMLM). The technique of DNA-PAINT has recently been refined in several ways. Relevant for the topic at hand are qPAINT, which achieves target quantification by analyzing the binding kinetics of imagers even of targets unresolvable in DNA-PAINT. Excha nge-PAINT, which enables sequential multiplexing by performing multiple rounds of imaging using different imagers that bind different target oligonucleotides. The amount of plex in the initial Excha nge-PAINT implementation scales linearly in time. SPEED-PAINT describes optimized sequences and buffer conditions for up to lOOx speed increase. All published variations of DNA-PAINT rely on primary binders to be highly specific and affine to the target structure.

[0008] Furthermore, two almost identical approaches, SUM-PAINT and FLASH-PAINT have been published recently. These two approaches aim to improve the capabilities and performance of DNA-PAINT in terms of multiplexing by using transient intermediary adapter strands in combination with so called eraser strands. In particular, SUM-PAINT relates to a secondary probe-based DNA-PAINT implementation, wherein the direct labeling of a target with a DNA-PAINT docking strand is replaced by a longer primary strand on the binder, which facilitates achieving a speed-optimized Exchange-PAINT multiplexing.

[0009] RESI (Resolution enhancement by sequential imaging) is a variation of DNA-PAINT to improve the resolution of fluorescence microscopy down to the sub-nm (Angstrom) scale. RESI multiplexes a single target species by separating it into multiple, sparser subsets. By imaging the subsets sequentially, sufficiently spaced and isolated groups of localizations are measured. Determining the center of each group of localizations yields a resolution enhancement.

[0010] Moreover, there are other implementations of super-resolution microscopy not concerning the refinements of DNA-PAINT. For instance, IRIS uses protein fragment-based probes that directly associate with and dissociate from their targets over durations on the order of tens of milliseconds. IRIS employs exchangeable probes that repeatedly associate with and dissociate from their endogeneous targets. This exchangeable single-molecule labeling by IRIS probes enables high-density labeling of the targets. IRIS can also be extended to multiplexed imaging because of the exchangeable nature of the probes.

[0011] Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy (xHTLs) uses exchangeable ligands that reversibly bind to HaloTag. These Ligands can be coupled to fluorescent dyes, such as rhodamines of different colors. xHTLs allow STED imaging with reduced photobleaching as compared to covalent labeling. Transient binding of xHTLs to HaloTag fusion proteins enables PAINT and Minflux microscopy. The approach has been shown to allow dual-color PAINT and STED microscopy.

[0012] Peptide-PAINT is related to DNA-PAINT but applies short peptide tags at the protein of interest instead of short single-stranded DNA strands. More specifically, short coiled-coil peptide sequences are used as binding site and dye-labeled imager coils which bind transiently and repetitively. The main advantage of Peptide-PAINT compared to DNA-PAINT is the fact that the peptide-tag is directly expressed with the protein of interest and no additional target staining is required. Furthermore, the peptide tag is small compared to other affinity reagents such as antibodies, which leads potentially to higher spatial resolutions and reduced linkage error.

[0013] Stimulated emission depletion (STED) microscopy is a super-resolution microscopy technique based on confocal microscopy. Briefly, a Gaussian-shaped excitation beam excites fluorophores in a diffraction limited spot and in addition a Donut-shaped depletion beam which depletes the excited dye molecules where it is above a threshold intensity, thus leading to a non-diffraction-limited remaining excitation volume which can be detected. The sample is then scanned in analogy to confocal microscopy.

[0014] Minflux imaging is an advanced microscopy technique combining concepts of STED imaging with concepts from localization based super-resolution imaging. Minflux uses an illumination scheme which significantly reduces photobleaching and allows spatial resolution < 5 nm. Current limitations of Minflux are low throughput and demanding expertise during data acquisition and data analysis.

[0015] However, the approach of the state of the art is still facing a plurality of technical challenges, such as the availability of binders against protein targets which can fulfill the selection criteria, inter alia, of specificity, high labeling efficiency, compatibility with protocols for fixation and post-fixation, size, quantitative degree of labeling and sitespecific labeling. Summary

[0016] In light of the above, it is therefore an object of the present invention to overcome or at least to alleviate the shortcomings and disadvantages of the prior art. More particularly, it is an object of the present invention to provide an improved bispecific compound and method for detecting a target molecule.

[0017] These objects are met by the present invention.

[0018] In a first aspect, the invention relates to a bispecific compound capable of specifically binding to a target molecule, wherein the bispecific compound comprises at least one target molecule binding moiety capable of binding to the target molecule, and at least one docking module capable of binding to at least one imager.

[0019] The at least one target molecule binding moiety may correspond to the target molecule.

[0020] In one embodiment, the bispecific compound may comprise a binder. The binder may comprise the at least one target molecule binding moiety capable of binding to the target molecule, and the at least one docking module capable of binding to the at least one imager.

[0021] It should be understood that the term "correspond to" may be intended to imply that a specific and favorable interaction between two molecules due to their matching shapes, structures, or sequences. For instance, in the context of nucleic acids or proteins, corresponding to would often involve specific base pairing or matching of amino acid sequences, which can also be referred to as being complementary. Matching of epitopes via shape and molecular forces, as present in antigen-antibody interaction may be understood as "corresponding" and "complementary" here, as well.

[0022] In a further embodiment, the bispecific compound may comprise further at least one adapter. The at least one adapter may comprise the at least one docking module capable of binding to the at least one imager. The at least one adapter may comprise at least one adapter attachment module.

[0023] In another embodiment, the binder may comprise a binder attachment module.

[0024] Moreover, the bispecific compound may comprise: the binder comprising the at least one target molecule binding moiety capable of binding to the target molecule, and the binder attachment module, wherein the binder attachment module may be capable of binding the at least one adapter attachment module of the at least one adapter; and the at least one adapter comprising the at least one docking module capable of binding to the at least one imager, and the at least one adapter attachment module wherein the at least one adapter attachment module may be capable of binding to the binder attachment module of the binder. The at least one docking module may comprise at least at least one of: a nanobody; an antibody; an antigen; and a nucleotide. The nucleotide may comprise an oligonucleotide.

[0025] The at least one docking module may comprise at least two, preferably at least three, more preferably at least four of: a nanobody; an antibody; an antigen; and a nucleotide.

[0026] The at least one docking module may be capable of transiently binding to the at least one imager.

[0027] In one embodiment, the at least one imager may be capable of permanently binding to at least one oligonucleotide to form an imager-oligonucleotide construct, wherein the imager- oligonucleotide construct may be capable of transiently binding to the bispecific compound.

[0028] Moreover, the at least one imager may be capable of permanently binding to at least one oligonucleotide to form an imager-oligonucleotide construct, wherein the imager- oligonucleotide construct may be capable of binding transiently to the at least one docking module.

[0029] Furthermore, the at least one imager may comprise at least one imaging strand; and at least one dye comprising at least one of: a fluorescent dye, a radio isotope, a metal isotope, and a scattering probe.

[0030] In one embodiment, the at least one imager may comprise at least two dyes, preferably at least three dyes, more preferably at least four dyes.

[0031] Furthermore, the at least one imager may comprise at least one quencher.

[0032] Further, the at least one docking module may correspond to the at least one imager.

[0033] In one embodiment, the at least one imager has an on-rate to the at least one docking module of 105M-1s1to 109M-1s-1, more preferably 106M-1s1to 109M-1s-1. The at least one imager has an off-rate from the at least one docking module of 0.1 s1to 1000 s ~1, more preferably 1 s1to 100 s’1. These rates depend on various environmental conditions. For instance, typical conditions may comprise: temperature between 21-23°C, wherein off- rate increases with increased temperature and on-rate marginally increases with increased temperature. For example, an imager can have an off-rate of 0.15 s1at 21°C, an off-rate of 0.2 s1at 22 °C, an off-rate of 0.27 s1at 23°C, and an off-rate of 0.36 / s at 24°C, while the on-rate may be nearly constant with values between 6.2 x 106M-1s1to 6.9 x 106M_1s’1salt concentration, for example, 10-75 mM MgCI2 or 0.3 - I M NaCI, the off-rate decreases with increased salt concentration and the on-rate increases with increased salt concentration. For example, an imager can have an on-rate of 2.5 x 106M-1s1at 5 mM MgCI2, an on-rate of 6.5 x 106M-1s1at 10 mM MgCI2, and an on-rate of 7.5 x 106M-1s-1at 15 mM MgCI2, while it may have an off-rate of 0.31 s1at 5 mM MgCI2, an off-rate of 0.26 s1at 10 mM MgCI2, and an off-rate of 0.26 s1at 15 mM MgCI2; formamide concentration, typically OmM Formamide. The off-rate increases with increased formamide concentration, and the on-rate decreases with increased formamide concentration; ethylene carbonate concentration, typically OmM ethylene carbonate. The off-rate increases with increased ethylene carbonate concentration, and the on-rate decreases with increased ethylene carbonate concentration pH: typically, pH 6.5-8.5, preferably 7-8. It should be understood that this also the case when the adapters are oligonucleotides.

[0034] Furthermore, the at least one docking module may comprise at least one DNA sequence. The at least one DNA sequence may be at least partially complementary to the at least one imager.

[0035] An on-rate of the binder to the target molecule may be between 103and 108M-1s-1, more preferably between 104and 108M-1s-1. An off-rate of the binder may be between 0.0001 and 100 s ~1, more preferably between 0.001 and 10 s ~1, even more preferably between 0.002 and 1 s’1. This is particularly advantageous, as it allows for simpler removal of the bispecific compound compared to cases with negligible off-rate, as it is the case in the state of the art. The removal of the bispecific compound is beneficial in multiple methods as detailed below. Furthermore, this is advantageous, as it allows for stochastic binding and unbinding events of the bispecific compound, preferably at different timescales than the imager binding and unbinding, which in turn allows for more detailed analysis as described in the corresponding methods embodiments. This is also particularly advantageous, as it allows a large availability of binders that can be used, whereas the number of binders with negligible off-rate is very limited.

[0036] In one embodiment, the target molecule binding moiety may be a nanobody, antibody, or fragment, wherein a size of the target molecule binding moiety is between 1 and 150 kDa, more preferably between 15 and 150 kDa.

[0037] In another embodiment, the target molecule binding moiety may be a small molecule or peptide, wherein a size of the target molecule binding moiety is between 0.1 and 10 kDa.

[0038] In a further embodiment, the target molecule binding moiety may be an antibody comprising an antibody mimetic, more preferably an antibody mimetic selected from a list of Affibody molecules, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Gastrobodies, Kunitz domain peptides, Monobodies, nanoCLAMPs, Optimers, Repebodies, Pronectin, Centryins, Obodies, more preferably an affibody, an affimer or a DARpin, wherein when the target molecule binding moiety is an antibody, the target molecule binding moiety has a size between 2 and 20 kDa.

[0039] Moreover, the target molecule binding moiety may be a protein comprising at least one of: a protein A / G, a protein A, a protein G, or a protein L, wherein a part of the target molecule binding moiety binds to Fc regions, and wherein when the target molecule binding moiety is a protein, the target molecule binding moiety has a size between 60 and 120 kDa. Furthermore, the target molecule binding moiety may be an aptamer, wherein when the target molecule binding moiety is an aptamer, the target molecule binding moiety has a size between 30 and 150 nt.

[0040] The binder binding to the target molecule may comprise at least one of: establishment of a hydrogen bond, a Van der Waals interaction, and a Watson-Crick pairing.

[0041] Furthermore, the bispecific compound may comprise at least one marker molecule. The marker molecule may comprise at least one of: a dye such as a fluorescent dye, a radio isotope, and a metal isotope. This is particularly advantageous, as it allows to provide, in case of a fluorescent dye and in presence of an excitation source, continuous fluorescence, which is beneficial as it provides information regarding the expression level of a target molecule in the sample. For example, the sample can be cells, and the experiment should be done on cells that express a certain level of target molecules. Hence, the permanent presence of a dye on the bispecific compound and thus continuous fluorescence of cells with targets may facilitate finding suitable cells, and when such a cell is found and positioned under the microscope, for instance, DNA-PAINT in a different spectral region can be performed.

[0042] In presence of an excitation source, continuous fluorescence is beneficial as it provides information regarding the expression level of a target molecule in the sample. For example, the sample can be cells, and the experiment should be done on cells that express a certain level of target molecules. Hence, the permanent presence of a dye on the bispecific compound and thus continuous fluorescence of cells with targets may facilitate finding suitable cells, and when such a cell is found and positioned under the microscope, for instance, DNA-PAINT in a different spectral region can be performed.

[0043] In one embodiment, the at least one imaging strand may comprise at least one nucleotide. The at least one nucleotide may comprise at least one oligonucleotide. The at least one imaging strand may comprise at least one oligonucleotide. The at least one imaging strand may comprise at least one amino acid. The at least one imaging strand may comprise at least one peptide. The at least one imaging strand may comprise at least one protein.

[0044] In a further embodiment, the at least one adapter attachment module may comprise at least one nucleotide complementary to at least one of the least one nucleotide of the binder attachment module of the binder. Moreover, the at least one adapter attachment module may comprise at least one oligonucleotide complementary to at least one of the least one nucleotide of binder attachment module of the binder. Additionally or alternatively, the at least one adapter attachment module may comprise at least one peptide. Furthermore, the at least one adapter attachment module may comprise at least one amino acid. Additionally or alternatively, the at least one adapter attachment module may comprise at least one protein. In one embodiment, an on-rate of the at least one adapter may be between 103and 109M-1s-1, preferably between 5 x 103and 5 x 108M-1s-1, further preferably between 104and 108M-1s-1. Furthermore, an off-rate of the at least one adapter may be less than 10 s-1, preferably less than 5 s-1, further preferably less than 1 s’1. In one embodiment, the at least one adapter may be an oligonucleotide, and wherein a size of the at least one adapter may be between 10 and 250 nucleotides, preferably between 10 and 100 nucleotides, further preferably between 10 and 50 nucleotides.

[0045] Moreover, the at least one adapter may be at least one of: a nanobody, antibody, or fragment, wherein a size of the at least one adapter may be between 1 and 150 kDa, more preferably between 15 and 150 kDa.

[0046] In a further embodiment, the at least one adapter may be a small molecule or peptide, wherein when the at least one adapter may be a small molecule, the at least one adapter has a size between 0.1 and 10 kDa.

[0047] Furthermore, the at least one adapter may be an antibody mimetic, more preferably an antibody mimetic of the list of Affibody molecules, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Gastrobodies, Kunitz domain peptides, Monobodies, nanoCLAMPs, Optimers, Repebodies, Pronectin, Centryins, Obodies, more preferably an affibody, an affimer or a DARpin, wherein when the at least one adapter may be an antibody mimetic, the at least one adapter has a size between 2 and 20 kDa.

[0048] In one embodiment, the at least one adapter may be a protein comprising at least one of: A / G, a protein A, a protein G, or a protein L, wherein a size of the target molecule binding moiety binds to Fc regions, and wherein when the at least one adapter may be a protein, the at least one adapter has a size between 60 and 120 kDa.

[0049] Furthermore, the at least one adapter binding to the binder may comprise at least one of: establishment of a hydrogen bond, a Van der Waals interaction, and a Watson-Crick pairing. Further, binding may comprise establishment of a covalent bond and / or establishment of a hydrogen bond.

[0050] In one embodiment, the bispecific compound may comprise at least two binders. The at least two binders may comprise a first binder and a second binder. The first binder and second binder may comprise the same docking module, and wherein the first binder and second binder may comprise each one of the at least one target molecule binding to the same target molecule on different epitopes.

[0051] Moreover, the bispecific compound may be suitable for visualizing the target molecule bound to the bispecific compound by means of points accumulation for imaging in nanoscale topography (PAINT) microscopy via at least one imager.

[0052] In a second aspect, the invention relates to a method for detecting a target molecule, the method comprising: introducing into a sample chamber a sample containing the target molecule; introducing into the sample chamber a bispecific compound capable of binding the target molecule; allowing the bispecific compound to bind to the target molecule; and visualizing the target molecule bound to the bispecific compound. The sample chamber may be a sample carrier compatible with microscopy in aqueous environments. For example, a sample chamber may be a chamber containing the sample embedded in an aqueous environment, a cover-slip mounted on a carrier comprising an aqueous environment, a cover-slip carrying the aqueous buffer without providing any chambers or compartments, a cover-slip carrying one or several chambers or compartments, such as a multi-well slide, a multiwell or multititer or SBS plate with e.g. 96 or 384 or a different number of wells, a cover-slip providing a channel system for fluidic exchange, e.g. an ibidi slide. It may comprise a thin and flat surface accessible to a microscope objective, which forms a border between the aqueous environment and the surrounding of the microscope objective, for example made of glass or plastic, and maybe with a specified thickness as common for microscope such as for example #0, #1, #1.5, or #1.51-1.

[0053] The bispecific compound may be a bispecific compound as recited herein.

[0054] The method may comprise visualizing the target molecule bound to the bispecific compound by means of points accumulation for imaging in nanoscale topography (PAINT) microscopy via at least one imager.

[0055] Moreover, the method may comprise allowing the bispecific compound to transiently bind to the target molecule. Transiently binding to the target molecule is particularly advantageous, because the transient binding of the bispecific compound can be used to easily remove the bispecific compound from the target molecule. Furthermore, kinetic analysis of bispecific compound binding and imager binding can disentangle multiple target molecules.

[0056] Furthermore, the bispecific compound may be capable to semi-permanently bind to the target molecule. Semi-permanently binding to the target molecule is particularly advantageous, because the semi-transient binding of the bispecific compound can be used to easily remove the bispecific compound from the target molecule. Furthermore, kinetic analysis of bispecific compound binding and imager binding can disentangle multiple target molecules.

[0057] In another embodiment, the method may comprise allowing the bispecific compound to permanently bind to the target molecule.

[0058] The step of introducing into the sample chamber the bispecific compound capable of binding the target molecule may precede the step of visualizing the target molecule bound to the bispecific compound.

[0059] Moreover, the step of visualizing the target molecule bound to the bispecific compound comprising at least one of: capturing at least one image data, and processing the at least one image data. The method may comprise adding the bispecific compound to the sample at a concentration between 1 pM and 1 pM, preferably between 1 nM and 100 nM. This is particularly advantageous, as the bispecific compounds in solution bind during the experiment, which is particularly beneficial, as it allows for a higher labeling efficiency of target molecules, for instance, when an off-rate of the binder is between 0.0001 and 100 s ~1, more preferably between 0.001 and 10 s ~1, even more preferably between 0.002 and 1 s’1.

[0060] In one embodiment, the method may comprise adding the at least one imager to the sample at a concentration between 1 pM and 100 nM, preferably between 25 pM and 50 nM, further preferably between 50 pM and 10 nM. A concentration at which the at least one imager is added to the sample is based, at least in part, on an on-rate of the binder. A concentration at which the at least one imager is added to the sample based, at least in part, on an on-rate of the adapter. A concentration at which the at least one imager is added to the sample is based, at least in part, on an off-rate of the binder. A concentration at which the at least one imager is added to the sample is based, at least in part, on an off-rate of the adapter.

[0061] Moreover, the step of capturing the at least one image data may comprise imaging the sample. The method may comprise imaging the sample after adding the bispecific compound. Imaging the sample may comprise directing light at the sample. Imaging the sample may comprise directing light at the sample which excites the at least one imager.

[0062] Furthermore, when the bispecific compound comprises a binder comprising at least one marker molecule, the step of imaging the sample may comprise directing light at the sample which excites the at least one marker molecule. A wavelength of the light is between 300 and 1000 nm, preferably between 350 and 900 nm, further preferably between 400 and 800 nm, further preferably between 550 and 650 nm.

[0063] A result of directing light at the sample may be emission of light from the at least one imager.

[0064] A result of directing light at the sample may be emission of light from the binder or the at least one adapter of the bispecific compound.

[0065] The at least one image data may be based, at least in part, on the light emitted from the at least one imager.

[0066] In a further embodiment, the method may comprise determining a location of the target molecule in the sample based on the at least one image data.

[0067] Moreover, imaging the sample may comprise capturing at least two image data, preferably at least 100 image data, more preferably at least 1000 image data.

[0068] Furthermore, the method may comprise directing light at the sample before capturing each of the at least two, at least 100 or at least 1000 image data of the sample. In one embodiment, the method may comprise capturing the at least two image data at a capturing time interval, wherein the capturing time interval between two consecutive image data is based, at least in part, on at least one of: an on-rate of the binder, an off- rate of the binder, an on-rate of the at least one adapter, an off-rate of the at least one adapter, an on-rate of the least one imager, and an off-rate of the at least one imager.

[0069] The sample may comprise at least two target molecules, wherein at least two of the at least two target molecules are different target molecules.

[0070] In one embodiment, the method may comprise determining locations of each of the two target molecules based, at least in part, on the at least two image data.

[0071] Moreover, the method may comprise adding the bispecific compound comprising at least two binders to the sample. The at least two binders may comprise a first binder and a second binder. The first binder and second binder may comprise the same docking module, and wherein the first binder and second binder may comprise each one of the at least one target molecule binding to the same target molecule on different epitopes. This approach is particularly advantageous, as it significantly increases the labeling efficiency.

[0072] The at least two target molecules may comprise a first target molecule and a second target molecule, wherein the first target molecule is different from the second target molecule.

[0073] The at least two target molecules may comprise a first target molecule and a second target molecule, wherein the first target molecule is identical to the second target molecule.

[0074] The first binder is capable of specifically binding to the first target molecule and the second binder is capable of specifically binding to the second target molecule.

[0075] Moreover, the first docking module comprised in the first bispecific compound comprising the first binder may differ from the second docking module comprised in the second bispecific compound comprising the second binder.

[0076] Furthermore, the first docking module comprised in the first bispecific compound comprising the first binder may be identical to the second docking module comprised in the second bispecific compound comprising the second binder. This is particularly advantageous, as having the same docking module I imager combination used to localize one type of target molecule can be used for localizing different types of target molecules, when the corresponding bispecific compounds are added to and removed from the sample between multiple imaging rounds.

[0077] The method may comprise, between consecutively adding any two of the at least two binders, washing the sample with an unbinding reagent, wherein the unbinding reagent is configured to unbind a first of the two binders from the target molecule. This is particularly advantageous, as the binding moiety is removed from the target. For example, a nearby target that was sterically blocked by the bispecific compound is now accessible for another round of labeling and imaging. Moreover, this approach is beneficial, as the same docking modules can be used for different target molecules. The unbinding reagent is a buffer solution. The method may comprise the buffer solution increasing the off-rate of the target molecule binding moiety by at least one of: using low or high pH, protein denaturing conditions, or high or low salt conditions. This approach is particularly advantageous, as the target molecule binding moiety is hindered from binding to the target molecules, such that less of the bispecific compound is bound to the sample and more of it can be washed out. For example, the buffer solution may be comprised of one or more of the following : ionic strength such as 3.5 M KCI or MgCI2; low pH such as 50 - 100 mM glycine-HCI, pH 2-3; high pH such as 100 mM glycine, NaOH, pH 10 or 150 mM NH4OH, pH 10.5; denaturing such as 25 mM glycine-HCI, 10% SDS pH 2 or 62 mM Tris, 2% SDS pH 6.75 or beta-mercaptoethanol, pH 6.75 or 1-6 M guanidine-HCI or 2-8 M urea; organic such as 10% dioxane or 50% ethylene glycol, pH 8 - 11.5.

[0078] Moreover, the unbinding reagent may be a solution comprising free target molecules, preferably at a concentration of 10 pM to 100 mM, more preferably 1 nM to 1 mM. This is particularly advantageous, as the free target molecules compete with the target molecules in the sample for binding with the bispecific compound, such that less of the bispecific compound is bound to the sample and more of it can be washed out.

[0079] Further, the unbinding reagent may be a solution comprising unlabeled target molecule binding moiety molecules, preferably at a concentration of 10 pM to 100 mM, more preferably 1 nM to 1 mM. This is particularly advantageous, as the unlabeled target molecule binding moiety molecules compete with the bispecific compound for binding with the target molecules, such that less of the bispecific compound is bound to the sample and more of it can be washed out.

[0080] In one embodiment, the method may comprise washing the sample with the unbinding reagent for a defined time interval. This method is especially advantageous, as the time interval can be calibrated in test experiments, where the efficiency of removing bispecific compound from the sample is evaluated for different time intervals. The defined time interval may be based, at least in part, on an off-rate of the bispecific compound. This method is especially advantageous, as the remaining concentration of bispecific compound in the sample after a time interval can be approximated by rate calculations, for example c_remaining = c_0 * exp(-k_off*t) with c_0 the initial concentration of the bispecific compound and the time interval t, when assuming that all bispecific compound unbinding from the sample is immediately washed out.

[0081] The of rate of the target molecule binding moiety is in the range of 10’5s1to 101s ~1, preferably in the range of 10’4s1to 10’2s’1.

[0082] In one embodiment, the method may comprise determining a location of the at least one imager transiently hybridized to the bispecific compound corresponding to the target molecule in the sample based on the at least one image data. The location of the at least one imager bound to a target molecule is determined on at least two image data. The at least two locations of the at least one imager bound to a target molecule determined on at least two image data are combined to determine the location of the target molecule. The method may comprise combining the location of the at least one imager for determining the location of the target molecule, at least in part, by their relative temporal occurrence in the imaging session. The method may comprise combining the location of the at least one imager for determining the location of the target molecule, at least in part, by the off- rate of the bispecific compound. This approach is particularly advantageous, as it provides resolution enhancement by combining multiple imager binding events, which can be used simultaneously with the sequential probing of nearby target molecules by the bispecific compound. For example, a bispecific compound can bind one target molecule but not a nearby target molecule. Then, multiple imager binding events generate multiple binder locations that can be combined to determine the location of the first target molecule. Then the bispecific compound unbinds stochastically due to its off-rate, leading to no imager location data. Then, the same or a different bispecific compound stochastically binds to the second target molecule, and the location of the latter can be precisely determined by the accompanying multiple imager binding events. The temporal analysis of all imager binding events allows for disentangling imager binding events related to the first bispecific compound binding event from those related to the second bispecific compound binding event. Moreover, the approach is also beneficial, as especially with the high resolution, it allows, by (semi)transient labeling by the bispecific compounds, avoiding potential problems of steric hindrance of labels on target molecules in very close proximity.

[0083] In one embodiment, the method may comprise analyzing multiple cells.

[0084] In another embodiment, the method may comprise clustering and / or subtyping of cells based on the localization results of the one or more target molecules.

[0085] Moreover, the method may comprise adding to the sample at least two different bispecific compounds, wherein each of the at least two bispecific compounds may be as recited herein.

[0086] Furthermore, the method may comprise imaging the sample after adding the at least one imager. In a further embodiment, the method may comprise adding the bispecific compound and the at least one imager simultaneously. In a further embodiment, the method may comprise adding one of imager and bispecific compound one after the other, without completely displacing the other from solution. This is particularly advantageous, as the presence of the bispecific compound in the solution during the measurement allows for its repetitive binding and thus the benefits of the bispecific compound as described herein.

[0087] In third aspect, the invention relates to a system for detecting a target molecule in a sample, the system comprising: a sample chamber configured to receive the sample and a bispecific compound; and a super-resolution microscope configured to perform points accumulation for imaging in nanoscale topography (PAINT) microscopy measurements. In one embodiment, the system may be configured to perform the method according to any of the preceding method embodiments.

[0088] In another embodiment, the system may be configured to automatically perform fluid exchange steps of the method as recited herein. The system may be configured to automatically record image data after the fluid exchange steps.

[0089] In a further embodiment, the system is configured to automatically analyze recorded image data.

[0090] Furthermore, the method may comprise using the system as recited herein to carry out the method according as recited herein.

[0091] In a fourth aspect, the invention relates a kit for use in a method for detecting a target molecule, the kit comprising a bispecific compound as recited herein.

[0092] In one embodiment, the bispecific compound may be in a solution.

[0093] In another embodiment, the bispecific compound may be freeze-dried.

[0094] Moreover, the kit may comprise at least one buffer for hybridization comprising at least one of: fixation buffer, wash buffer, and hybridization buffer.

[0095] Furthermore, the kit may be suitable for performing the method as recited herein in the system as recited herein.

[0096] In a further embodiment, the kit may comprise at least one imager.

[0097] Moreover, the kit further may comprise at least one adapter.

[0098] The invention also relates to the use of the system as recited herein for carrying out the method as recited herein.

[0099] The approach of the present invention is particularly advantageous, as the state of the art describes bispecific compounds assuming that these permanently binding with a high labeling efficiency, however, the present invention allows that the bispecific compounds are optionally washed out before imaging. To the contrary, in the state of the art, washing out is always performed, as this is preferential when the bispecific compounds are attaching permanently with a high labeling efficiency. However, the present invention allows that imager and bispecific compound are added simultaneously, because then the bispecific compound cannot be washed out without washing out the imager as well, which is definitely needed for the experiment. Therefore, the present invention also allows to first load the sample with more bispecific compound than needed, and in a separate addition, dilute it with the at least one imager, or vice versa.

[0100] The present technology is also described by the following numbered embodiments.

[0101] Below, bispecific compound embodiments will be discussed. These embodiments are abbreviated by the letter "B" followed by a number. When reference is herein made to a bispecific compound embodiment, those embodiments are meant.

[0102] Bl. A bispecific compound capable of specifically binding to a target molecule, wherein the bispecific compound comprises at least one target molecule binding moiety capable of binding to the target molecule, and at least one docking module capable of binding to at least one imager.

[0103] B2. The bispecific compound according to the preceding embodiment, wherein the at least one target molecule binding moiety corresponds to the target molecule.

[0104] B3. The bispecific compound according to any of the two preceding embodiments, wherein the bispecific compound comprises a binder.

[0105] B4. The bispecific compound according to the preceding embodiment, wherein the binder comprises the at least one target molecule binding moiety capable of binding to the target molecule, and the at least one docking module capable of binding to the at least one imager.

[0106] B5. The bispecific compound according to any of embodiments Bl to B3, wherein the bispecific compound comprises further at least one adapter.

[0107] B6. The bispecific compound according to the preceding embodiment, wherein the at least one adapter comprises the at least one docking module capable of binding to the at least one imager.

[0108] B7. The bispecific compound according to the two preceding embodiments, wherein the at least one adapter comprises at least one adapter attachment module.

[0109] B8. The bispecific compound according to any of the preceding embodiment except to embodiment B4, wherein the binder comprises a binder attachment module.

[0110] B9. The bispecific compound according to the 5 preceding embodiments, wherein the bispecific compound comprises the binder comprising the at least one target molecule binding moiety capable of binding to the target molecule, and the binder attachment module, wherein the binder attachment module is capable of binding the at least one adapter attachment module of the at least one adapter; and the at least one adapter comprising the at least one docking module capable of binding to the at least one imager, and the at least one adapter attachment module wherein the at least one adapter attachment module is capable of binding to the binder attachment module of the binder.

[0111] BIO. The bispecific compound according to any of any of the preceding embodiments, wherein the at least one docking module comprises at least at least one of: a nanobody; an antibody; an antigen; and a nucleotide.

[0112] Bll. The bispecific compound according to the preceding embodiment, wherein the nucleotide comprises an oligonucleotide.

[0113] B12. The bispecific compound according to any of the preceding bispecific compound embodiments and with the features of embodiments BIO, wherein the at least one docking module comprises at least two, preferably at least three, more preferably at least four of: a nanobody; an antibody; an antigen; and a nucleotide.

[0114] B13. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the at least one docking module is capable of transiently binding to the at least one imager.

[0115] B14. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the at least one imager is capable of permanently binding to at least one oligonucleotide to form an imager-oligonucleotide construct, wherein the imager- oligonucleotide construct is capable of transiently binding to the bispecific compound.

[0116] B15. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the at least one imager is capable of permanently binding to at least one oligonucleotide to form an imager-oligonucleotide construct, wherein the imager- oligonucleotide construct is capable of transiently to the at least one docking module.

[0117] B16. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the at least one imager comprises at least one imaging strand; and at least one dye comprising at least one of: a fluorescent dye, a radio isotope, a metal isotope, and a scattering probe.

[0118] B17. The bispecific compound according to the preceding embodiment, wherein the at least one imager comprises at least two dyes, preferably at least three dyes, more preferably at least four dyes.

[0119] B18. The bispecific compound according to any of the two preceding embodiments, wherein the at least one imager comprises at least one quencher.

[0120] B19. The bispecific compound according to any of the preceding embodiments, wherein the at least one docking module corresponds to the at least one imager. B20. The bispecific compound according to any of the preceding embodiments, wherein the at least one imager has an on-rate to the at least one docking module of 105M-1s1to 109M-1s-1, more preferably 106M-1s1to 108M-1s-1.

[0121] B21. The bispecific compound according to any of the preceding embodiments, wherein the at least one imager has an off-rate from the at least one docking module of 0.1 s1to 1000, more preferably 1 s1to 100 s’1.

[0122] B22. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the at least one docking module comprises at least one DNA sequence.

[0123] B23. The bispecific compound according to the two preceding embodiments, wherein the at least one DNA sequence is at least partially complementary to the at least one imager.

[0124] B24. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein an on-rate of the binder to the target molecule is between 103and 108M-1s-1, more preferably between 104and 108M-1s-1.

[0125] B25. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein an off-rate of the binder is between 0.0001 and 100 s ~1, more preferably between 0.001 and 10 s ~1, even more preferably between 0.002 and 1 s’1.

[0126] B26. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the target molecule binding moiety is a nanobody, antibody, or fragment, wherein a size of the target molecule binding moiety is between 1 and 150 kDa, more preferably between 15 and 150 kDa.

[0127] B27. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the target molecule binding moiety is a small molecule or peptide, wherein a size of the target molecule binding moiety is between 0.1 and 10 kDa.

[0128] B28. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the target molecule binding moiety is an antibody comprising an antibody mimetic, more preferably an antibody mimetic selected from a list of Affibody molecules, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Gastrobodies, Kunitz domain peptides, Monobodies, nanoCLAMPs, Optimers, Repebodies, Pronectin, Centryins, Obodies, more preferably an affibody, an affimer or a DARpin, wherein when the target molecule binding moiety is an antibody, the target molecule binding moiety has a size between 2 and 20 kDa.

[0129] B29. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the target molecule binding moiety is a protein comprising at least one of: a protein A / G, a protein A, a protein G, or a protein L, wherein a part of the target molecule binding moiety binds to Fc regions, and wherein when the target molecule binding moiety is a protein, the target molecule binding moiety has a size between 60 and 120 kDa.

[0130] B30. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the target molecule binding moiety is an aptamer, wherein when the target molecule binding moiety is an aptamer, the target molecule binding moiety has a size between 30 and 150 nt.

[0131] B31. The bispecific compound according to any of the preceding binder embodiments, wherein the binder binding to the target molecule comprises at least one of: establishment of a hydrogen bond, a Van der Waals interaction, and a Watson-Crick pairing.

[0132] B32. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the bispecific compound comprises at least one marker molecule.

[0133] B33. The bispecific compound according to the preceding embodiment, wherein the at least one marker molecule comprises at least one of: a dye such as a fluorescent dye, a radio isotope, and a metal isotope.

[0134] B34. The bispecific compound according to any of the preceding bispecific compound embodiments and with the features of embodiment B16, wherein the at least one imaging strand comprises at least one nucleotide.

[0135] B35. The bispecific compound according to the preceding embodiment, wherein the at least one nucleotide comprises at least one oligonucleotide.

[0136] B36. The bispecific compound according to any of the preceding bispecific compound embodiments and with the features of embodiment B16, wherein the at least one imaging strand comprises at least one oligonucleotide.

[0137] B37. The bispecific compound according to any of the preceding bispecific compound embodiments and with the features of embodiment B16, wherein the at least one imaging strand comprises at least one amino acid.

[0138] B38. The bispecific compound according to any of the preceding bispecific compound embodiments and with the features of embodiment B16, wherein the at least one imaging strand comprises at least one peptide. B39. The bispecific compound according to any of the preceding bispecific compound embodiments and with the features of embodiment B16, wherein the at least one imaging strand comprises at least one protein.

[0139] B40. The bispecific compound according to any of the preceding bispecific compound embodiments and with the features of embodiment B9, wherein the at least one adapter attachment module comprises at least one nucleotide complementary to at least one of the least one nucleotide of the binder attachment module of the binder.

[0140] B41. The bispecific compound according to any of the preceding bispecific compound embodiments and with the features of embodiment B9, wherein the at least one adapter attachment module comprises at least one oligonucleotide complementary to at least one of the least one nucleotide of binder attachment module of the binder.

[0141] B42. The bispecific compound according to any of the preceding bispecific compound embodiments and with the features of embodiment B7, wherein the at least one adapter attachment module comprises at least one peptide.

[0142] B43. The bispecific compound according to any of the preceding bispecific compound embodiments and with the features of embodiment B7, wherein the at least one adapter attachment module comprises at least one amino acid.

[0143] B44. The bispecific compound according to any of the preceding bispecific compound embodiments and with the features of embodiment B7, wherein the at least one adapter attachment module comprises at least one protein.

[0144] B45. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein an on-rate of the at least one adapter is between 103and 109mol’xs ~1, preferably between 5 x 103and 5 x 108molds’1, further preferably between 104and 108molds’1.

[0145] B46. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein an off-rate of the at least one adapter is less than 10 s ~1, preferably less than 5 s ~1, further preferably less than 1 s’1.

[0146] B47. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the at least one adapter is an oligonucleotide, and wherein a size of the at least one adapter is between 10 and 250 nucleotides, preferably between 10 and 100 nucleotides, further preferably between 10 and 50 nucleotides.

[0147] B48. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the at least one adapter is at least one of: a nanobody, antibody, or fragment, wherein a size of the at least one adapter is between 1 and 150 kDa, more preferably between 15 and 150 kDa. B49. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the at least one adapter is a small molecule or peptide, wherein when the at least one adapter is a small molecule, the at least one adapter has a size between 0.1 and 10 kDa.

[0148] B50. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the at least one adapter is an antibody mimetic, more preferably an antibody mimetic of the list of Affibody molecules, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Gastrobodies, Kunitz domain peptides, Monobodies, nanoCLAMPs, Optimers, Repebodies, Pronectin, Centryins, Obodies, more preferably an affibody, an affimer or a DARpin, wherein when the at least one adapter is an antibody mimetic, the at least one adapter has a size between 2 and 20 kDa.

[0149] B51. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the at least one adapter is a protein comprising at least one of: A / G, a protein A, a protein G, or a protein L, wherein a size of the target molecule binding moiety binds to Fc regions, and wherein when the at least one adapter is a protein, the at least one adapter has a size between 60 and 120 kDa.

[0150] B52. The bispecific compound according to any of the preceding bispecific compound embodiments, wherein the at least one adapter binding to the binder comprises at least one of: establishment of a hydrogen bond, a Van der Waals interaction, and a Watson-Crick pairing.

[0151] B53. The bispecific compound according to the preceding embodiment, wherein binding comprises establishment of a covalent bond.

[0152] B54. The bispecific compound according to any of the 2 preceding embodiments, wherein binding comprises establishment of a hydrogen bond.

[0153] B55. The bispecific compound according to any of the preceding embodiments and with the features of embodiments Bl to B3, wherein the bispecific compound comprises at least two binders.

[0154] B56. The bispecific compound according to the preceding embodiment, wherein the at least two binders comprise a first binder and a second binder.

[0155] B57. The bispecific compound according to the preceding embodiment, wherein the first binder and second binder comprise the same docking module, and wherein the first binder and second binder comprise each one of the at least one target molecule binding to the same target molecule on different epitopes. B58. The bispecific compound according to any of the preceding embodiments, wherein the bispecific compound is suitable for visualizing the target molecule bound to the bispecific compound by means of points accumulation for imaging in nanoscale topography (PAINT) microscopy via at least one imager.

[0156] Below, method embodiments will be discussed. These embodiments are abbreviated by the letter "M" followed by a number. When reference is herein made to a method embodiment, those embodiments are meant.

[0157] Ml. A method for detecting a target molecule, the method comprising introducing into a sample chamber a sample containing the target molecule; introducing into the sample chamber a bispecific compound capable of binding the target molecule; allowing the bispecific compound to bind to the target molecule; and visualizing the target molecule bound to the bispecific compound.

[0158] M2. The method according to the preceding embodiment, wherein the bispecific compound is according to any of the preceding bispecific compound embodiments.

[0159] M3. The method according to any of the two preceding embodiments, wherein the method comprises visualizing the target molecule bound to the bispecific compound by means of points accumulation for imaging in nanoscale topography (PAINT) microscopy via at least one imager.

[0160] M4. The method according to any of the preceding method embodiments, wherein the method comprises allowing the bispecific compound to transiently bind to the target molecule.

[0161] M5. The method according to any of the preceding method embodiments, wherein the method comprises allowing the bispecific compound to semi-permanently bind to the target molecule.

[0162] M6. The method according to any of the preceding method embodiments, wherein the method comprises allowing the bispecific compound to permanently bind to the target molecule.

[0163] M7. The method according to any of the preceding method embodiments, wherein the step of introducing into the sample chamber the bispecific compound capable of binding the target molecule precedes the step of visualizing the target molecule bound to the bispecific compound.

[0164] M8. The method according to any of the preceding method embodiments, wherein the step of visualizing the target molecule bound to the bispecific compound comprising at least one of: capturing at least one image data, and processing the at least one image data. M9. The method according to any of the preceding method embodiments, wherein the method comprises adding the bispecific compound to the sample at a concentration between 1 pM and IpM, preferably between 1 nM and 100 nM.

[0165] MIO. The method according to the preceding embodiment, wherein the method comprises adding the at least one imager to the sample at a concentration between 1 pM and 100 nM, preferably between 25 pM and 50 nM, further preferably between 50 pM and 10 nM.

[0166] Mil. The method according to any of the 2 preceding embodiments, wherein a concentration at which the at least one imager is added to the sample is based, at least in part, on an on-rate of the binder.

[0167] M12. The method according to any of the 3 preceding embodiments and with the features of embodiment M4, wherein a concentration at which the at least one imager is added to the sample based, at least in part, on an on-rate of the adapter.

[0168] M13. The method according to any of the 2 preceding embodiments, wherein a concentration at which the at least one imager is added to the sample is based, at least in part, on an off-rate of the binder.

[0169] M14. The method according to any of the 3 preceding embodiments and with the features of embodiment M4, wherein a concentration at which the at least one imager is added to the sample is based, at least in part, on an off-rate of the adapter.

[0170] M15. The method according to any of the preceding method embodiments and with the features of embodiment M8, wherein the step of capturing the at least one image data comprises imaging the sample.

[0171] M16. The method according to the preceding embodiment, wherein the method comprises imaging the sample after adding the bispecific compound.

[0172] M17. The method according to any of the 2 preceding embodiments, wherein imaging the sample comprises directing light at the sample.

[0173] M18. The method according to any of the 3 preceding embodiments, wherein imaging the sample comprises directing light at the sample which excites the at least one imager.

[0174] M19. The method according to any of the 4 preceding embodiments, wherein when the bispecific compound comprises a binder comprising at least one marker molecule, the step of imaging the sample comprises directing light at the sample which excites the at least one marker molecule. M20. The method according to the preceding embodiment, wherein a wavelength of the light is between 300 and 1000 nm, preferably between 350 and 900 nm, further preferably between 400 and 800 nm, further preferably between 550 and 650 nm.

[0175] M21. The method according to any of the preceding method embodiments and with the features of embodiment M18, wherein a result of directing light at the sample is emission of light from the at least one imager.

[0176] M22. The method according to any of the preceding method embodiments and with the features of embodiment M19, wherein a result of directing light at the sample is emission of light from the binder or the at least one adapter of the bispecific compound.

[0177] M23. The method according to embodiment M21, wherein the at least one image data is based, at least in part, on the light emitted from the at least one imager.

[0178] M24. The method according to any of the preceding method embodiments and with the features of embodiment M8, wherein the method comprises determining a location of the target molecule in the sample based on the at least one image data.

[0179] M25. The method according to any of the preceding method embodiments and with the features of embodiment M8, wherein imaging the sample comprises capturing a at least two image data, preferably at least 100 image data, more preferably at least 1000 image data.

[0180] M26. The method according to the preceding embodiment and with the features of embodiment M17, wherein the method comprises directing light at the sample before capturing each of the at least one image data of the sample.

[0181] M27. The method according to any of the preceding method embodiments and with the features of embodiment M25, wherein the method comprises capturing the at least two image data at a capturing time interval, wherein the capturing time interval between two consecutive image data is based, at least in part, on at least one of: an on-rate of the binder, an off-rate of the binder, an on-rate of the at least one adapter, an off-rate of the at least one adapter, an on-rate of the least one imager, and an off-rate of the at least one imager.

[0182] M28. The method according to any of the preceding method embodiments, wherein the sample comprises at least two target molecules, wherein at two of the at least two target molecules are different target molecules.

[0183] M29. The method according to the preceding embodiment and with the features of embodiment M24, wherein the method comprises determining locations of each of the two target molecules based, at least in part, on the at least one image data. M30. The method according to any of the preceding method embodiments, wherein the method comprises adding the bispecific compound comprising at least two binders to the sample.

[0184] M31. The method according to the preceding embodiment, wherein the at least two binders comprise a first binder and a second binder.

[0185] M32. The method according to the preceding embodiment, wherein the first binder and second binder comprise the same docking module, and wherein the first binder and second binder comprise each one of the at least one target molecule binding to the same target molecule on different epitopes.

[0186] M33. The method according to any of the preceding embodiments and with the features of embodiments M28, wherein the at least two target molecules comprise a first target molecule and a second target molecule, wherein the first target molecule is different from the second target molecule.

[0187] M34. The method according to any of the preceding embodiments and with the features of embodiments M28, wherein the at least two target molecules comprise a first target molecule and a second target molecule, wherein the first target molecule is identical to the second target molecule.

[0188] M35. The method according to the two preceding embodiments, wherein the first binder is capable of specifically binding to the first target molecule and the second binder is capable of specifically binding to the second target molecule.

[0189] M36. The method according to any of the three preceding embodiments, wherein the first docking module comprised in the first bispecific compound comprising the first binder differs from the second docking module comprised in the second bispecific compound comprising the second binder.

[0190] M37. The method according to any of the three preceding embodiments, wherein the first docking module comprised in the first bispecific compound comprising the first binder is identical to the second docking module comprised in the second bispecific compound comprising the second binder.

[0191] M38. The method according to any of the preceding method embodiments and with the features of embodiment M30, wherein the method comprises, between consecutively adding any two of the at least two binders, washing the sample with an unbinding reagent, wherein the unbinding reagent is configured to unbind a first of the two binders from the target molecule.

[0192] M39. The method according to the preceding embodiment, wherein the unbinding reagent is a buffer solution. M40. The method according to the preceding embodiment, wherein the method comprises the buffer solution increasing the off-rate of the target molecule binding moiety by at least one of: using low or high pH, protein denaturing conditions, or high or low salt conditions.

[0193] M41. The method according to any of the three preceding embodiments, wherein the unbinding reagent is a solution comprising free target molecules, preferably at a concentration of 10 pM to 100 mM, more preferably 1 nM to 1 mM.

[0194] M42. The method according to any of the four preceding embodiments, wherein the unbinding reagent is a solution comprising unlabeled target molecule binding moiety molecules, preferably at a concentration of 10 pM to 100 mM, more preferably 1 nM to 1 mM.

[0195] M43. The method according to any of the five preceding embodiments, wherein the method comprises washing the sample with the unbinding reagent for a defined time interval.

[0196] M44. The method according to the preceding embodiment, wherein the defined time interval is based, at least in part, on an off-rate of the bispecific compound.

[0197] M45. The method according to any of the embodiments M37 to M41 and with the features of embodiment M37, wherein the of rate of the target molecule binding moiety is in the range of 10’5s1to 101s ~1, preferably in the range of 10’4s1to 10’2s’1.

[0198] M46. The method according to any of the preceding method and with the features of embodiment M8, wherein the method comprises determining a location of the at least one imager transiently hybridized to the bispecific compound corresponding to the target molecule in the sample based on the at least one image data.

[0199] M47. The method according to the preceding embodiment, wherein the location of the at least one imager bound to a target molecule is determined on at least two image data.

[0200] M48. The method according to the preceding embodiment, wherein the at least two locations of the at least one imager bound to a target molecule determined on at least two image data are combined to determine the location of the target molecule

[0201] M49. The method according to the preceding embodiment, wherein the method comprises combining the location of the at least one imager imagers for determining the location of the target molecule, at least in part, by their relative temporal occurrence in the imaging session

[0202] M50. The method according to the preceding embodiment and with the features of at least one of the embodiments M4 or M5, wherein the method comprises combining the location of the at least one imager for determining the location of the target molecule, at least in part, by the off-rate of the bispecific compound.

[0203] M51. The method according to any of the preceding method embodiments, wherein the method comprises analyzing multiple cells.

[0204] M52. The method according to any of the preceding method embodiments, wherein the method comprises clustering and / or subtyping of cells based on the localization results of the one or more target molecules.

[0205] M53. The method according to any of the preceding method embodiments, wherein the method comprises adding to the sample at least two different bispecific compounds, wherein each of the at least two bispecific compounds is according to any of the preceding bispecific compound embodiments.

[0206] M54. The method according to any of the preceding method embodiments and with the features of any of embodiments M15 to M16, wherein the method comprises imaging the sample after adding the at least one imager.

[0207] M55. The method according to the preceding embodiment and embodiment M16, wherein the method comprises adding the bispecific compound and the at least one imager simultaneously.

[0208] M56. The method according to embodiments M16 and M51, wherein the method comprises adding one of imager and bispecific compound one after the other, without completely displacing the other from solution.

[0209] Below system embodiments will be discussed. These embodiments are abbreviated by the letter "S" followed by a number. When reference is herein made to a system embodiment, those embodiments are meant.

[0210] 51. A system for detecting a target molecule in a sample, the system comprising a sample chamber configured to receive the sample and a bispecific compound; and a super-resolution microscope configured to perform points accumulation for imaging in nanoscale topography (PAINT) microscopy measurements.

[0211] 52. The system according to the preceding embodiment, wherein the system is configured to perform the method according to any of the preceding method embodiments.

[0212] 53. The system according to any of the preceding embodiments, wherein the system is configured to automatically perform fluid exchange steps of the method according to any of the preceding method embodiments.

[0213] 54. The system according to the preceding embodiment, wherein the system is configured to automatically record image data after the fluid exchange steps. S5. The system according to any of the preceding embodiments, wherein the system is configured to automatically analyze recorded image data.

[0214] M57. The method according to any of the preceding method embodiments, wherein the method comprises using the system according to any of the preceding system embodiments to carry out the method according to any of the preceding method embodiments.

[0215] Below, kit embodiments will be discussed. These embodiments are abbreviated by the letter "K" followed by a number. When reference is herein made to a kit embodiment, those embodiments are meant.

[0216] KI. A kit for use in a method for detecting a target molecule, the kit comprising a bispecific compound according to any of the preceding bispecific compound.

[0217] K2. The kit according to the preceding embodiment, wherein the bispecific compound is in a solution.

[0218] K3. The kit according to any of the two preceding embodiments, wherein the bispecific compound is freeze-dried.

[0219] K4. The kit according to any of the preceding kit embodiments, wherein the kit comprises at least one buffer for hybridization comprising at least one of: fixation buffer, wash buffer, and hybridization buffer.

[0220] K5. The kit according to any of the preceding kit embodiments, wherein the method is according to any of the preceding method embodiments.

[0221] K6. The kit according to any of the preceding kit embodiments, wherein the kit is suitable for performing the method according to any of the preceding method embodiments in the system accordingly to any of the preceding system embodiments.

[0222] K7. The kit according to any of the preceding kit embodiments, wherein the kit further comprises at least one imager.

[0223] K8. The kit according to any of the preceding kit embodiments, wherein the kit further comprises at least one adapter.

[0224] Ul. Use of the system according to any of the preceding system embodiments for carrying out the method according to any of the preceding method embodiments. The present invention will now be described with reference to the accompanying drawings which illustrate embodiments of the invention. These embodiments should only exemplify, but not limit, the present invention.

[0225] Fig. 1 schematically depicts a bispecific compound comprising a binder according to embodiments of the present invention;

[0226] Fig. 2 schematically depicts a bispecific compound comprising a binder and at least one adapter according to embodiments of the present invention;

[0227] Figs. 3A-D schematically depicts an implementation example according to embodiments of the present invention;

[0228] Figs. 4A-F schematically depicts a multiplexed implementation example according to embodiments of the present invention;

[0229] Fig. 5 schematically depicts a method for detecting a target according to embodiments of the present invention;

[0230] Figs.6a-b depict protein visualized using transient-VHH-PAINT;

[0231] Fig. 7 depicts labeling efficiencies of mEos2 and mCherry VHH using an old and new method.

[0232] It is noted that not all the drawings carry all the reference signs. Instead, in some of the drawings, some of the reference signs have been omitted for sake of brevity and simplicity of illustration. Embodiments of the present invention will now be described with reference to the accompanying drawings.

[0233] Fig. 1 depicts a bispecific compound 100 according to embodiments of the present invention. In simple terms, the bispecific compound may comprise at least one target molecule binding moiety 114 capable of binding to a target molecule, and at least one docking module 112 capable of binding to at least one imager 120.

[0234] In embodiment, the bispecific compound may comprise, for instance, a binder 110. When the bispecific 100 compound only comprises a binder 100, the binder 100 comprises the at least one target molecule binding moiety 114 capable of binding to the target molecule 300, and the at least one docking module 112 capable of binding to the at least one imager 120

[0235] It should be understood that the at least one imager 120 may comprise at least one imaging strand 122 and at least one dye 124. The at least one imager 120 may also comprises at least two dyes, preferably at least three dyes, more preferably at least four dyes. The at least one dye may comprise, inter alia but not limited to, at least one of: fluorescent dye, a radio isotope, a metal isotope, and a scattering probe. Furthermore, the at least one imager 120 may also comprise a quencher (not depicted) which may be capable on controlling the fluorescence the imager-bispecific compound complex.

[0236] The at least one docking module 112 may be capable of binding to at least one imaging strand 122 of the at least one imager 120. In more simple terms, the binder 110 may be capable of specifically interacting with the target molecule 300, for instance, through a non-covalent interaction. The at least one target molecule binding moiety 114 may comprise, inter alia, at least one of: a protein, a nanobody, an antibody, a nucleotide and / or an oligonucleotide, which is capable of binding the target molecule 300.

[0237] For example, the binder 110 may comprise a target molecule binding moiety 114 capable of specifically binding to the target molecule 300. The target molecule binding moiety 114 may, in particular, correspond to the target molecule 300. For instance, when the target molecule binding moiety 114 comprises, for example, (oligo) nucleotides, these may be are complementary to, for instance, the (oligo) nucleotides of the target molecule 300, so that a specific binding can be established between the two. This may be of advantage in selectively ensuring interaction between the binder 110, particularly the target molecule binding moiety 114, and the target molecule 300. Further, this may also be of advantage in making the binder 110 target-specific, as the binder 110 may bind only to targets 300 which have, for example, the nucleotide bases conjugate to those in the target molecule binding moiety 114.

[0238] The binder 110 may further comprise the at least one docking module 112 configured to allow the at least one imager 120 to bind to the binder 110. The docking module 112 may, for example, comprise (oligo) nucleotides that are complementary to the (oligo) nucleotides comprised in the at least one imager 120.

[0239] In one embodiment, the at least one imager 120 may comprise at least one image strand 122 and at least one dye 124. The at least one image strand 122 may at least partially correspond to the docking module 122 of the binder 110.

[0240] In further embodiments, the at least one imager 120 may comprise at least two dyes, preferably at least three days, more preferably at least four dyes. It should be understood that the dyes may be different types, and even different type of dyes other than chromophores comprising fluorophores, such as the example described above.

[0241] For instance, the binder 110 may comprise, for example, a heavy chain-variable (VHH) antibody.

[0242] Embodiments of the present invention may be of particular relevance for preparation and / or analysis, particularly imaging, of samples, particularly biological samples such as cells, or tissues. The at least one imager 120 may comprise a molecule that may be labelled with a dye, i.e., a fluorescent molecule may be attached to the at least one imager 120. Thus, by binding the binder 110 to the target molecule 300, and the at least one imager 120 to the binder 110, the target molecule 300 may be imaged.

[0243] A time between binding and subsequent unbinding between the binder 110 and the target molecule 300 may be called an "on-time" of the binder 110. An "on-rate" of the binder 110 may be based, at least in part, on the "on-time" of the binder 110. In particular, the "on- rate" may be inversely proportional to the "on-time" of the binder 110. The "on-rate" of the binder 110 may be between 104and 108M-1s-1, preferably between 104and 107M_1s’1, further preferably between 104and 106M-1s-1.

[0244] A time between unbinding and subsequent binding between the binder 110 and the target molecule 300 may be called an "off-time" of the binder 110. An "off-rate" of the binder 110 may be based, at least in part, on the "off-time" of the binder 110. In particular, the "off-rate" may be inversely proportional to the "off-time" of the binder 110. The "off-rate" of the binder 110 may be between 10’4and 100 s ~1, preferably between 10’3and 10 s ~1, further preferably between 0.002 and 1 s’1.

[0245] A first kinetics kl may relate to the attachment of the binder 110 to the target molecule 300. A second kinetics k2 may relate to the attachment of the at least one imager 120 to the binder 110, particularly the docking module 112. The time scale over which the at least one imager 120 attaches to the binder 110 may be lower than the time scale over which the binder 110 attaches to the target molecule 300. Thus, the overall kinetics may be dominated by the k2, i.e., of the at least one imager 120 attaching to the binder 110. By accounting for this kinetics explicitly, embodiments of the present invention may allow using high "off-rate" binders for imaging targets.

[0246] Fig. 2 schematically depicts a bispecific compound 200 according to embodiments of the present invention. In simple terms, the bispecific compound 200 may comprise a binder 210 and at least one adapter 230 comprising at least one docking module 232 of binding to the at least one imager 220. The at least one imager 220 may comprise at least one imaging strand 222 and at least one dye 224. The at least one imager 220 may also comprises at least two dyes 224, preferably at least three dyes 224, more preferably at least four dyes 224. The at least one dye 224 may comprise, inter alia but not limited to, at least one of: fluorescent dye, a radio isotope, a metal isotope, and a scattering probe. Furthermore, the at least one imager 220 may also comprise a quencher (not depicted) which may be capable on controlling the fluorescence the imager-bispecific compound complex. Moreover, the at least one adapter 230 comprises at least one adapter attachment module 234, and the binder 210 may comprise a binder attachment module 212.

[0247] Thus, the bispecific compound 200 may comprise the binder 210 comprising the at least one target molecule binding moiety 214 capable of binding to the target molecule 300, and the binder attachment module 212, wherein the binder attachment module 212 is capable of binding the at least one adapter attachment module 234 of the at least one adapter 230; and the at least one adapter 230 comprising the at least one docking module 232 capable of binding to the at least one imager 220, and the at least one adapter attachment module 234 wherein the at least one adapter attachment module 234 is capable of binding to the binder attachment module 212 of the binder 210. Similar as described in Fig. 1, the bispecific compound 200 may also have an "on-rate and / or "off-rate".

[0248] A first kinetics k3 may relate to the attachment of the binder 210 to the target molecule 300. A second kinetics k4 may relate to the attachment of the at least one imager 220 to the at least one adapter 230, particularly the docking module 212. The time scale over which the at least one imager 220 attaches to the at least one adapter 230 may be lower than the time scale over which the binder 210 attaches to the target molecule 300. Thus, the overall kinetics may be dominated by the k4, i.e., of the at least one imager 220 attaching to the at least one adapter 230. By accounting for this kinetics explicitly, embodiments of the present invention may allow using high "off-rate" binders for imaging targets.

[0249] Figs. 3A-D schematically depicts an example application according to embodiments of the preset invention comprising multiplexed VHH-DNA-PAINT. In simple words, Fig. 3A depicts a first target molecule 300a and a second target molecule 300b to be visualized in a sample, indicated by a sketched cell, which is represented by three thick lines. Fig. 3B depicts a first binder 110a, and a first at least one imager 120a are added to the sample. The first binder 110a (semi)-transiently binds to the first target molecule 300a, and the first at least one imager 120a transiently binds to the first binder 110a, allowing for detecting and potentially localizing the first target molecule 300a. Fig. 3C depicts that upon removal of the first binder 110a, and optionally the first at least one imager 120a, the sample only shows background signal and no target is detected. Fig. 3B discloses a second binder 110b, comprising a target binding moiety, and optionally a second at least one imager 120b, comprising an oligonucleotide and dye, any of which may or may not be identical to the first at least one imager 120a and its parts, are added to the sample. The second binder 110b (semi)transiently binds to the second target molecule 300b, and the second at least one imager 120b transiently binds to the second binder 110b, thus allowing for detection and potentially localizing the second target molecule 300b.

[0250] Figs. 4A-F depicts an implementation example according to embodiments of the present invention. In simple words, Figs. 4A-F depicts an example of transient R.ESI VHH-DNA- PAINT. Fig. 4 depicts a plurality of targets 300 (300a, 300b, 300c) to be visualized in a sample, indicated by a sketched cell, which is represented by three thick lines. Fig. 4A depicts the targets 300 in potentially close proximity. Fig. 4A further depicts panels Al, A2, A3, A4, which represent a coarse time series, in which the slow binding events of the binder 110 are depicted. During each time interval, the at least one imager 120 can repetitively and transiently bind to the binder 110 and therefore be detected and localized. By spatio-temporal analysis, imager binding events of diffraction limited areas (represented by the circle in panels A) can be analyzed as signal over time (as depicted in panels Bl, B2, B3 and B4, respectively) and binder binding events can be determined. By assigning imager localizations to the binder binding events (panels Cl, C2, C3 and C4, assignment depicted by different symbols), an overall binder localization can be calculated from the assigned imager localizations (panels DI, D2, D3 and D4). Taken together in an image, the binder localizations (F) show a better localization precision than the imager localizations (E).

[0251] Embodiments of the method, as described herein, may, thus, be of particular advantage for improving a labeling efficiency of the binder 110 or the bispecific compound 100, 200. The labeling efficiency of the binder 110 or the bispecific compound 200 may be defined as the percentage of targets of interest to which at least one binder is attached.

[0252] Further, embodiments of embodiments of the present invention may be of particular advantage for binders that are not compatible with post-fixation, as no post-fixation may be needed for preparation and / or analysis of samples according to embodiments of embodiments of the present invention.

[0253] Fig. 5 schematically depicts a method for detecting a target molecule 300 according to embodiments of the present invention. The method may also comprise preparing and / or analyzing a sample. In particular, the method may be directed towards detecting a target molecule 300 in a sample. The target molecule 300 may be target molecule 300, as described above. In other words, the target molecule 300 may be detected by means of the visualizing the target molecule 300 bound to bispecific compound 100, 200 as described above.

[0254] In a first step, Pl, the bispecific compound 100, 200 may be introduced into the sample. The sample may comprise the target molecule 300 of interest. The bispecific compound 100, 200 may be introduced into the sample at a concentration as described above. In the following, reference will be made to the at least one imager 120, the binders 110, 210, the at least one adapter 230, or the target molecule 300. As may be appreciated by the skilled person, the sample may comprise a plurality of targets 300, and, correspondingly, a plurality of any of the binders 110, 210, the imagers 120, or the adapters 230, may be used in the method.

[0255] In a second step, Pla, that may only be performed in embodiments wherein the bispecific compound 200 is used, the at least one adapter 230 may be introduced into the sample. The at least one adapter 230 may be introduced into the sample at a concentration as described above.

[0256] Then, in a third step, P2, the at least one imager 120 may be introduced into the sample. The at least one imager 120 may be introduced into the sample at a concentration as described above.

[0257] In a fourth step, P3, the sample may be imaged by directing light at the sample. A wavelength of the light may be as described above. A result of directing light at the sample may be fluorescence of the dye comprised in the at least one imager 120 as described above. In a fifth step, P4, the light emitted by the fluorescing dye may be captured for further analysis.

[0258] In some embodiments, the method may comprise repeating the steps P3, and P4, regularly, to obtain time-resolved images of the sample. The non-zero-time interval between capturing the images of the sample may allow some of the imagers 120 to unbind from their targets 300 and subsequently bind to some other targets 300. This may be of advantage in improving the resolution of the images. For example, the analysis may be similar to that carried out in single-molecule localization microscopy (SMLM).

[0259] In yet other embodiments, the method may comprise a step, P5, comprising adding an unbinding reagent to the sample, after imaging the sample. The unbinding reagent may comprise any reagent that may effect the unbinding of the binder 110 or the bispecific compound 100, 200 from the target molecule 300. Then, in a further step, P6, a next binder 110 or bispecific compound 100, 200 may be added to the sample. This may be of advantage in targeting different compounds / proteins in the sample. For example, a first binder 110 or first bispecific compound 100, 200 may target a first compound / protein in the sample. Steps Pla to P4 may be carried out to image the first compound / protein. Then, step P5 may be carried out to unbind the first binder 110 or first bispecific compound 100, 200. Further, a second binder 110 or second bispecific compound 100, 200 may be added to the sample targeting a second compound / protein in the sample, comprising the step P6, followed by repeating steps Pla to P4 as described above, to obtain data relating to the second compound / protein. Note, in particular, that the at least one imager 120 used may be different for the first binder 110 or first bispecific compound 100, 200 and the second binder 110 or second bispecific compound 100, 200.

[0260] Below some examples related to the invention are briefly explained. ine

[0261] VHHs containing an ectopic cysteine at their C-terminus were conjugated using a Dibenzocyclooctyne (DBCO)-PEG4-Maleimid crosslinker (Cat No 760676-5MG, Merck) and azide-functionalized DNA docking strands (SeqID No 7 - 12, purchased from metabion international AG). 1 mg of VHHs (MW = 15 kDa) were thawed at 1-4 °C, and reacted with 15 molar equivalents of DBCO-PEG4-Maleimid crosslinker (40mg / ml in DMF, MW=674.75g / mol), diluted such that the final volume is between 300 and 400 pl. Incubation was performed for 90 min at 1 - 4 °C. To remove precipitates, the reaction mixture was centrifuged at 20000 g for 10 min at 10 °C. A 10 MWCO Amicon centrifugal filter (2 ml, Cat No UFC201024, Merck, Germany) was used to remove unreacted linker by exchanging the buffer to PBS according to the manufacturer's instruction. The concentration of the VHH-DBCO conjugate was determined by measuring the absorption at 280 nm (Amax, protein) and 309 nm (Amax,DBco) using a Nanodrop One instrument. DBCO- functionalized VHHs were then frozen in lOOpg aliquots and stored at -80 °C.

[0262] 100 pg of DBCO-modified VHH were then reacted with 3 molar equivalents of azide- modified DNA. The mixture was reacted overnight at 4 - 8 °C. Unreacted VHH and DNA were removed by anion exchange chromatography using a Knauer liquid chromatography system equipped with a Resource Q 1 ml (Cytiva) column. The collected fractions corresponding to the VHH-DNA conjugate were concentrated with a 10 MWCO Amicon centrifugal filter (2 ml).

[0263] Used VHHs comprising a single ectopic cysteine at the C-terminus (all purchased from Nanotag Biotechnologies) comprise: ALFA: clone 1G5, cat: N1505; mEOS: clone 1x108, cat: N3105; GFP: clone 1H1, cat: N0305; GFP: clone 1B2, custom; mRFP / mCherry: clone 2B12, cat: N0401

[0264] Sortase-mediated conjugation of VHHs

[0265] VHHs comprising Sortase tags were conjugated with GlyGlyGly-modified docking strands (SeqID No 15: 7xR3 docking strands were used for target molecule labeling and purchased from biomers.net) : 100 pg VHH was incubated with a 0.5x molar equivalents of Sortase A (Tebubio, Cat No 100666-2), and 2.5x molar equivalents of docking strands. The mixture was incubated for 45 min at 37 °C during shaking at 500 rpm. Subsequently, the mix was centrifuged with 20,000 g for 4 minutes. The supernatant was subjected to anion exchange chromatography using a Knauer liquid chromatography system equipped with a Resource Q 1 ml column. The collected fractions corresponding to the VHH-DNA conjugate were concentrated with a 10 MWCO Amicon centrifugal filter (2 ml).

[0266] Fluorescence imaging was performed on a TIRF-optimized inverted microscope (Eclipse Ti2 with Perfect Focus System, Nikon Instruments), using objective-type TIRF microscopy with an oil-immersion objective (Nikon Instruments, Apo SR TIRFx lOO, NA 1.49, Oil). A 488- nm (IBEAM-SMART-488-S-HP, Toptica) and 560-nm laser (MPB Communications, 1 W) was used for excitation. The laser beams were passed through cleanup filters (ZT488 / 10, ZET561 / 10, Chroma Technology) and coupled into the microscope objective using beam splitters (ZT488rdc, ZT561rdc, Chroma Technology,). Fluorescence was spectrally filtered with emission filters (ET525 / 50m, ET600 / 50m and ET575lp, Chroma Technology,) and imaged on an sCMOS camera (ORCA-Fusion BT, Hamamatsu) without further magnification. Imaging

[0267] The samples were mounted on the microscope stage and the focus was set using the Perfect Focus System (PFS) of the Nikon Eclipse Ti2 microscope. Further fine adjustments of the focus were performed by changing the position of the PSF offset lens. The TIRF arm was set to TIRF illumination mode. Imaging was performed at 22 - 23 °C with 400 pl of Imaging solution in the sample comprising Imaging buffer (IxPBS, pH 7.4, 500 mM NaCI, 0.02% (v / v) Tween-20), Cy3B modified DNA imager strands and VHHs as applicable.

[0268] For all measurements, the camera readout speed was set to readout mode 2. Images were acguired by choosing a region of interest with a size of 576x576 pixels and 2x2 binning, resulting in an effective pixel size of 130 nm.

[0269] Data analysis

[0270] Raw fluorescence data were subjected to super-resolution reconstruction using the Picasso software package (latest version available at https: / / github.com / junqmannlab / picasso) (Schnitzbauer et al, "Super-resolution microscopy with DNA-PAINT”, Nature Protocols 2017). For localizing the point spread functions (PSFs) of single molecules in the raw data the 'Picasso Localize' module was used. Here, the box side length was set to 7 and the 'Minimum Net Gradient' was adjusted such that all PSFs are detected (usually 3000 - 5000). Experimental settings and photon conversion parameters were set according to the specifications and chosen settings of the camera. Fitting of the PSFs was performed using the Least Square Gaussian method. Further image processing was performed using the 'Picasso Render' tool. Drift correction was conducted in two steps: First, a redundant cross correlation (RCC) algorithm was applied using a segmentation size of 1000 frames. Second, gold particles were picked and their trajectories used for drift correction. For multiplexed Excha nge-PAINT data, channels were aligned using the RCC method followed by a gold particle fiducial alignment. Filtering of localizations was performed with the 'Picasso Filter’ module.

[0271] Example 1: Imaging of Nup96-mEGFP using an anti-GFP VHH

[0272] In this example the applicability of VHH-PAINT in cellular imaging was demonstrated by targeting the nuclear pore complex, a well-characterized structure that is commonly used as a standard in super-resolution imaging. The nuclear pore subunit Nup96-mEGFP was targeted by an anti-GFP VHH. The VHH binding to GFP and its common variants (Cat: N0305, clone 1H1) was obtained from Nanotag with a single ectopic cysteine at the C- terminus. The VHH was conjugated to a 5xRl docking site (SeqID 7). Cell culture and fixation

[0273] U2OS-CRISPR-Nup96-mE GFP cells (Cat. No: 300174, CLS) were cultured in McCoy's 5A medium supplemented with 10% (v / v) FBS, at 37 °C, 5% CO2, in a standard humidified cell culture incubator. Approximately 30,000 U2OS-CRISPR-Nup96-mEGFP cells were seeded in each well of an 8-well chambered coverslip with glass bottom (Cat. No 80827, ibidi) and grown overnight. On the next day, cell fixation was performed with a solution of 4% (w / v) paraformaldehyde in phosphate buffered saline (PBS) that was pre-warmed to 37 °C before addition. The cell medium was aspirated and replaced by 200 pl of the fixative solution in each well, followed by incubation for 15 minutes at 23 °C. The wells were then washed with PBS three times. Subsequently, cells were permeabilized with 0.25% (v / v) Triton X-100 in PBS for 5 minutes, followed by washing with PBS. Next, the cell sample was passivated for 60 min with 2% (w / v) BSA in PBS, followed by incubating 200pl of a 1 : 1 dilution of gold nanoparticles (Cat No G-90-100, 90 nm Standard Gold Nanoparticles, Cytodiagnostics, Inc) in each well for 5 min, serving as fiducial markers for drift correction and channel alignment.

[0274] Imaging

[0275] 5 nM anti-GFP VHH with 5xRl docking site and 0.5 nM R1 Cy3B imager strands (SeqID 33) were diluted in Imaging buffer and added to the cells. Cells were selected and focus was set by locating the mEGFP fluorescent signal at the nuclear envelope using the 488 nm laser at approximately ImW laser power. Then the laser and filters were changed to the 560 nm channel to detect Cy3B labeled imager strands. The camera exposure time was set to 100 ms, the laser power to 30 mW, and 40,000 frames were recorded.

[0276] Fig. 6 depicts proteins visualized using transient-VHH-PAINT. In particular, a transient- VHH-PAINT was applied to visualize Nup96-mEGFP proteins using anti-GFP VHH conjugated to a docking strand. Fig. 6a depicts an overview image of the cell with specific signal at the nuclear envelope. Fig. 6b depicts a zoom-in of the overview image. Single nuclear pore rings are resolved according to their well-characterized structure. Scale bars are 5 pm and 500 nm for Fig. 6a and Fig. 6b, respectively.

[0277] Example 2: Improvement in labeling efficiency

[0278] In this example, the improvement in labeling efficiency of VHHs specific to mEOS, mCherry, and multiple membrane proteins was tested. To that end, labeling is performed using the state of the art, and the invented method as described herein. The labeling efficiency is then tested in both cases by comparing signals of two targets of a fusion protein consisting of the target protein and reference tag.

[0279] VHHs are modified with a tag at the C-terminus for site-specific and quantitative DNA attachment such as a sortase tag or an ectopic cysteine. Labeling efficiency testing is performed on cells that ideally do not express the target of interest. These cells are transiently transfected with the target protein fused to a reference tag (e.g. ALFA-mEGFP tag) at the intracellular part of the protein. The reference tag is sub-stoichiometrically stained using a low concentration of a specific VHH (e.g. anti-ALFA) with minimum nonspecific labeling. By performing DNA-PAINT imaging of the target and ALFA and / or GFP as a reference with known labeling efficiency, the labeling efficiency of VHHs against the target of interest can be evaluated. Results of separate experiments using the published labeling method or using the novel labeling method are compared.

[0280] Most steps in the experiment were done following general cell culture, microbiology, and biochemistry practice as known in the art. For reference, standard textbooks, such as "Bioconjugate Techniques" (Hermanson 2013, ISBN: 978-0-12-382239-0, DOI: 10.1016 / C2009-0-64240-9) can be used.

[0281] Plasmid cloning

[0282] For evaluating the labeling efficiency of mEos2 and mCherry, the following fusion proteins were cloned into a mammalian expression vector: ALFA-CD86-mCherry (SeqID 39); ALFA- CD86-mEos2 (SeqID 40).

[0283] For each target, a plasmid containing the cDNA sequence was cloned in-house with a combined ALFA-mEGFP tag at the C-terminus. The plasmid was designed using SnapGene software. Gene Fragments of the respective proteins were purchased from IDT (gBIocks) and cloned into a pcDNA™3.1 (+) Mammalian Expression Vector (cat: V79020, Thermo Fisher Scientific). Cloning was performed using a Gibson Assembly Cloning Kit (E5510S, New England BioLabs) according to the manufacturer's instructions.

[0284] All further plasmids were purchased from Eurofins GmbH, who cloned given membrane proteins into the vector sequence containing ALFA and mEGFP (SeqID 41).

[0285] VHH-DNA conjugates used in Example 2 comprise: ALFA: clone 1G5 -SeqID 7 and 9; mEOS:clone IxlO8- SeqID 9; GFP: clone 1H1 - SeqID 9; mRFP / mCherry: clone 2B12 - SeqID 9, and anti-membrane protein VHH - SeqID 13-18. Cell culture and fixation

[0286] CHO-K1 cells were cultured in Ham's F12 medium supplemented with 10% (v / v) FBS at 37 °C, 5% CO2, in a standard humidified cell culture incubator. Approximately 10,000 CHO- K1 cells were seeded in each well of an 8-well chambered coverslip with glass bottom (Cat. No 80827, ibidi GmbH) and grown overnight. On the next day, transient transfection of the fusion proteins was performed using the Lipofectamine 3000 transfection kit (L3000- 001, Thermo Fisher Scientific) according to the manufacturer's recommendations. Cell fixation was performed with a solution of 4% (w / v) paraformaldehyde in phosphate buffered saline (PBS) that was pre-warmed to 37°C before addition. The cell medium was aspirated and replaced by 200pl of the fixative solution in each well, followed by incubation for 15 minutes at 23 °C. The wells were then washed with PBS three times. Subsequently, cells were permeabilized with 0.25% (v / v) Triton X-100 in PBS for 15 minutes, followed by washing with PBS. Next, the cell sample was passivated for 60 min with 2% (w / v) BSA in PBS.

[0287] Immunostaininq

[0288] For labeling using the state-of-the-art method, cells were incubated with 25 nM DNA- conjugated VHH and 1-5 nM of anti-ALFA VHH (reference) for Ih at 23 °C. The sample was then washed 3 times with 200 pl PBS each, and post-fixation was performed by incubating with 4% (w / v) paraformaldehyde and 0.2% (v / v) glutaraldehyde in PBS for 10 min at 23 °C. The samples were then washed twice in PBS.

[0289] For labeling using the novel method, 5 nM anti-ALFA VHH (reference) was incubated for 30 min at 23 °C. After immunostaining, a 1 : 1 dilution of gold nanoparticles in PBS were incubated in the wells for 5 min.

[0290] Imaging

[0291] Measurements were performed using 400pl Imaging solution in the sample. In an experiment using the state-of-the-art labeling method, the Imaging Solution consisted of 1 nM Cy3B-modified imager in Imaging Buffer. In an experiment using the novel labeling method, the Imaging Solution consisted of 1.5 nM Cy3B-labeled imager and 25 nM VHH- docking strand conjugate in Imaging Buffer.

[0292] In the first imaging round, imager R3 (SeqID No 35) was used to image the target VHH binder. Afterwards, the sample was washed twice with PBS and an imaging solution containing imager R1 (SeqID No 33) was introduced, binding to the anti-ALFA-tag VHH (reference). Transfected cells were identified by detecting the signal of the fluorescent proteins (mEGFP, mEos2, mCherry). The TIRF arm was set to TIRF illumination and the 488 nm (mEos2, mEGFP) or 560 nm (mCherry) laser was set to 1-2 mW. The transfection level was aimed to be low to moderate to see well separated PSFs from single molecules in the Cy3B channel.

[0293] For DNA-PAINT imaging, a 560 nm excitation laser power in the sample of 35 mW and 30 mW for target and reference imaging, respectively, were used. 15 000 frames were acquired at an exposure time of 75 ms resulting in approximately 13 frames per second.

[0294] Analysis

[0295] Localizations were filtered using the "Picasso Filter" module by discarding localizations with localization precisions (Ipx and Ipy) larger than 7 nm. The filtered data was then rendered in "Picasso Render" and homogeneous areas inside each cell were picked with pick diameters of 20 - 50 pixels. Localizations of the reference data was linked using the input parameters 0.5 pixels and 5 gap frames.

[0296] The picked data was clustered, using the Picasso Postprocessing clustering algorithm (SMLM clusterer) (latest version available at https: / / github.com / jungmannlab / picasso) for each target separately. In the process, circular clusters of localizations centered around local maxima are identified and grouped. Then, the centers of the localization groups are calculated as weighted mean by applying the squared inverse localization precisions as weights. A radius of 10 nm and 10 minimum number of localization were set as input for clustering. Additionally, clusters are filtered using the basic frame analysis function. This function discards clusters that do not contain repetitive binding events but rather events of non-specific sticking of imager strands to the sample. Specifically, the algorithm excludes all clusters with a mean frame in the first or last 20% of frames. Secondly, clusters without repetitive binding are identified by dividing the acquisition time into 20 time windows each containing 5% of frames. The cluster is excluded if any of these time windows contains more than 80% of localizations.

[0297] After clustering, the nearest neighbor distance was determined from each reference cluster center to its next target cluster center. Plotting these distances in a histogram result in two distributions: One at shorter distances (5 - 30 nm), representing the transfected fusion proteins labeled by both the target and reference VHHs, while the distribution at longer distances corresponds to fusion proteins labeled by the reference VHH only. The mean distance of the second peak depends on the density of target and reference proteins while the first peak is specific to the distance between the target and ALFA-tag VHH. To quantify the labeling efficiency of a given binder, the nearest neighbor distribution (NND) extracted from the data is compared to a simulation. The simulation consists of simulating monomers of the reference protein, monomers of the target protein and dimers of reference-target protein at different proportions. Subsequently, the NND distribution of a given simulation is calculated and compared to the experimental NND distribution. The most likely proportions of populations of monomers (pre / ) and dimers were obtained through a least-squares optimization procedure. The labeling efficiency is then Calculated as follows:

[0298] Fig. 7 depicts labeling efficiencies of mEos2 and mCherry VHH using an old and a new method according to embodiments of the present invention. Line indicates the median, boxes the quartiles, and whiskers the 10-90% confidence interval.

[0299] Example 3: VHH-Exchanqe-PAINT

[0300] Example 3 relates to the use of the bispecific compound for sequence-unlimited multiplexing. By washing out the bispecific compounds between imaging rounds, the same docking sequences and imagers can be used in every imaging round.

[0301] CHO cells may be transiently transfected with either membrane protein 1-ALFA-mEGFP, membrane protein 2-ALFA-mEGFP, and membrane protein 3-ALFA-mEGFP constructs as also used in Example 2.

[0302] All steps except "imaging" may be performed in analogy to Example 1.

[0303] All VHH binders (anti-membrane protein 1, 2, 3) used in this example may be conjugated to a 7xR3 docking strand (SeqID 15).

[0304] Bispecific Compound off-rate calibration

[0305] Measurements may be performed using a 400 pl Imaging solution in the sample. The Imaging Solution may consist of 1.5 nM Cy3B-labeled imager and 25 nM VHH-docking strand conjugate in Imaging Buffer.

[0306] In a first bispecific compound off-rate calibration experiment, the anti-membrane protein 1 VHH conjugated to a docking strand may be used in the Imaging solution with a freshly prepared sample. Imager R3 (SeqID No 35) may be used to image the target with antimembrane protein 1 VHH binder. Afterwards, the sample may be washed with 500pl Imaging solution consisting of 1.5 nM Cy3B-labeled imager R3 in Imaging Buffer, without VHH. The measurement may be run until a number of binding events is observed at background level, meaning that the VHHs have dissociated. The Imaging solution with Imager strands may be exchanged every 10 min. For example, 12 minutes may be required for the anti-membrane protein 1 VHH to completely dissociate.

[0307] In a second bispecific compound off-rate calibration experiment, the anti- membrane protein 2 VHH binder conjugated to GlyGlyGly of 5xRl docking strand (SeqID No 13) may be used in the imaging solution with a freshly prepared sample. Imager R1 (SeqID No 33) may be used to image the target with anti- membrane protein 2 VHH binder. Afterwards, the sample may be washed with 500pl Imaging solution consisting of 1.5 nM Cy3B-labeled imager R1 in Imaging Buffer, without VHH. The measurement may be run until a number of binding events is observed at background level, meaning that the VHHs have dissociated. The Imaging solution with Imager strands may be exchanged every 10 min. For example, 24 min may be required for the anti-membrane protein 1 VHH to completely dissociate.

[0308] In a third bispecific compound off-rate calibration experiment, the anti- membrane protein 3 VHH binder conjugated to the azide group of 5xRl docking strand (SeqID No 1) may be used in the imaging solution with a freshly prepared sample. Imager R1 (SeqID No 33) may be used to image the target anti- membrane protein 3 VHH binder. Afterwards, the sample may be washed with 500pl Imaging solution consisting of 1.5 nM Cy3B-labeled imager R1 in Imaging Buffer, without VHH. The measurement may be run until a number of binding events is observed at background level, meaning that the VHHs have dissociated. The Imaging solution with Imager strands may be exchanged every 10 min. For example, 5 min may be required for the anti-membrane protein 1 VHH to completely dissociate.

[0309] Measurements may be performed using 400pl Imaging solution in the sample. The Imaging Solution may consist of 1.5 nM Cy3B-labeled imager and 25 nM VHH-docking strand conjugate in Imaging Buffer. For the measurement protocol, the timing and wash volumes elucidated in the previous section may be used.

[0310] For imaging, a 560 nm excitation laser power in the sample of 35 mW and 30 mW for target and reference imaging, respectively, may be used. E.g., 15 000 frames may be acquired at an exposure time of 75 ms resulting in approximately 13 frames per second. In the first imaging round, Imaging solution containing anti-membrane protein 1 VHH and 1.5 nM imager R1 (SeqID No 33) may be used. Afterwards, the sample may be washed for 12 minutes with 2 ml Imaging buffer.

[0311] For the second imaging round, Imaging Solution with 400pl of anti-membrane protein 2 VHH and 1.5 nM imager R1 (SeqID No 33) may be flushed into the sample. After imaging, the sample may be washed for 24 minutes with 4 ml Imaging buffer.

[0312] For the third imaging round, Imaging solution with 500p I of anti-membrane protein 3 VHH and 1.5 nM imager R1 (SeqID No 33) may be flushed into the sample. After imaging, the sample may be discarded.

[0313] Example 4: VHH-Exchanqe-PAINT with adapters

[0314] Example 4 relates to the use of the bispecific compound for sequence-unlimited multiplexing. By washing out the bispecific compounds between imaging rounds, the same docking sequences and imagers can be used in every imaging round.

[0315] The sample with the fusion protein of membrane protein 1 / 2 / 3-ALFA-mEGFP, of Example 2 and 3 may be used in this example.

[0316] The VHH binders used in this example may be conjugated to binder attachment modules (SI - SeqID 19, S2 - SellD 20 and S3 - SeqID 21 respectively).

[0317] Imaging

[0318] Measurements may be performed using 400 pl Imaging solution in the sample. The Imaging Solution may consist of 1.5 nM Cy3B-labeled imager, 25 nM adapter and 25 nM VHH-docking strand conjugate in Imaging Buffer. For the measurement protocol, the timing and wash volumes elucidated in the previous section may be used.

[0319] For imaging, a 560 nm excitation laser power in the sample of 35 mW may be used. 15 000 frames may be acquired at an exposure time of 75 ms resulting in approximately 13 frames per second.

[0320] In a first imaging round, anti-membrane protein 1 VHH, SI to 5xRl adapter (SeqID No 25) and imager R1 (SeqID No 33) may be used. Afterwards, the sample may be washed for 12 minutes with 2 ml PBS, as determined in Example 3. For a second imaging round, 500 pl of anti-membrane protein 2, S2 to R1 adapter (SeqID No 26) and imager R1 (SeqID No 33) in Imaging Solution may be flushed into the sample. After imaging, the sample may be washed for 24 minutes with 4 ml PBS.

[0321] For a third imaging round, 500 pl of anti-membrane protein 3 VHH, S4 to R2 adapter (SeqID No 27) and imager R2 (SeqID No 34) in Imaging Solution may be flushed into the sample. After imaging, the sample may be discarded.

[0322] Example 5: Multi-Epitope VHH-PAINT

[0323] For an additional improvement in labeling efficiency, multiple epitopes of a protein of interest can be targeted. For example, using two different clones of anti-GFP VHHs that bind two distinct epitopes of GFP may improve the labeling efficiency in comparison to when the clones are used individually.

[0324] As described in Example 2, a transfected cell sample with a fusion protein of ALFA-CD86- mEGFP may be created.

[0325] All steps may be performed in analogy to Example 2 while adding the following components to the Imaging buffer:

[0326] • Sample 1 : 5 nM anti-GFP VHH clone 1H1, 5 nM anti-GFP VHH clone 1B2

[0327] • Sample 2: 5 nM anti-GFP VHH clone 1H1

[0328] • Sample 3: 5 nM anti-GFP VHH clone 1B2

[0329] All VHH binders (anti-GFP clone 1H1, anti-GFP clone 1B2) used in this example may be conjugated to a 7xR3 docking strand (SeqID 9).

[0330] The labeling efficiency may be determined using the procedure described in example 2 for all samples. An improved efficiency labeling of Sample 1 may be expected compared to Sample 2 and Sample 3, respectively.

[0331] Example 6: VHH-Transient RESI

[0332] This example may exploit the transient binding of VHH to link imager localizations to the same VHH while discriminating between different VHHs, for improved spatial resolution. Cell culture and fixation

[0333] U2OS-CRISPR-Nup96-mEGFP cells (Cat. No: 300174, CLS) may be cultured in McCoy's 5A medium supplemented with 10% (v / v) FBS, at 37 °C, 5% CO2, in a standard humidified cell culture incubator. Approximately 30,000 U2OS-CRISPR-Nup96-mEGFP cells may be seeded in each well of an 8-well chambered coverslip with glass bottom (Cat. No 80827, ibidi) and grown overnight. On the next day, cell fixation may be performed with a solution of 4% (w / v) paraformaldehyde in phosphate buffered saline (PBS) that may be prewarmed to 37°C before addition. The cell medium may be aspirated and replaced by 200 pl of the fixative solution in each well, followed by incubation for 15 minutes at 23°C. The wells may then be washed with PBS three times. Subsequently, cells may be permeabilized with 0.25% (v / v) Triton X-100 in PBS for 5 minutes, followed by washing with PBS. Next, the cell sample may be passivated for 60 min with 2% (w / v) BSA in PBS.

[0334] Imaging

[0335] Measurements may be performed using 400 pl Imaging solution in the sample. The Imaging Solution may consist of 1.5 nM Cy3B-labeled imager, 25nM adapter and 25 nM VHH-docking strand conjugate in Imaging Buffer. For the measurement protocol, the timing and wash volumes elucidated in the previous section may be used.

[0336] For locating the sample area, the sample may be mounted on a TIRF arm and the fluorescence signal of the fluorescent proteins (mEGFP) may be exploited to find transfected cells. The TIRF arm may be set to TIRF illumination and the 488 nm laser may be set to 1-2 mW to detect transfected cells.

[0337] The TIRF arm may be set to TIRF / HILO illumination and the 488 nm laser may be set to 1 mW in the sample, and cells with GFP signal at the nucleus searched and their positions saved.

[0338] For imaging, a 560 nm excitation laser power in the sample of 35 mW may be used. 100 000 frames may be acquired at an exposure time of 75 ms resulting in approximately 13 frames per second.

[0339] One long imaging round may be performed for this experiment, using anti-GFP VHH clone 1H1 with 5xRl docking strand (SeqID No 1) and imager R1 (SeqID No 33).

[0340] Analysis:

[0341] Localizations may be filtered using the "Picasso Filter" module by discarding localizations with localization precisions (Ipx and Ipy) larger than 7 nm. The filtered data may be clustered, using the Picasso Postprocessing clustering algorithm (SMLM clusterer) (latest version available at https: / / github.com / jungmannlab / picasso) for each target separately. In the process, circular clusters of localizations centered around local maxima may be identified and grouped. Then, the centers of the localization groups may be calculated as weighted mean by applying the squared inverse localization precisions as weights. A radius of 10 nm and 10 minimum number of localization may be set as input for clustering. Additionally, clusters may be filtered using the basic frame analysis function. This function discards clusters that do not contain repetitive binding events but rather events of non-specific sticking of imager strands to the sample. Specifically, the algorithm may exclude all clusters with a mean frame in the first or last 20% of frames. Secondly, clusters without repetitive binding may be identified by dividing the acquisition time into 20 time windows each containing 5% of frames. The cluster may be excluded if any of these time windows contains more than 80% of localizations.

[0342] Spatio-temporal analysis using Hidden Markov Modeling may be performed on the clustered groups of localizations, determining localizations belonging to the same imager binding event, and those belonging to the same VHH binding event. The average positions of localizations belonging to the same VHH binding event may be calculated. A visualization of these average localizations may confirm improved resolution.

[0343] Example 7: Steric Hindrance Reduction

[0344] To compare the effect of the new method on imaging of targets with epitopes in close proximity, samples may be imaged with the new and old methods, and results compared.

[0345] High off-rate binders are preferential for this use case because they provide more cycles of association and dissociation compared to high affinity binders. Thus, epitopes in close proximity are less likely sterically blocked by a binder bound to the nearby epitope. For a given high density target, the results of binders with high and low off-rate may be compared.

[0346] The experiment may be performed in analogy to Experiment 2, with EGFR as a target in A431 cells, and separately with Her2 as a target in SK-BR3 cells. As a readout, the nearest neighbor distances may be used.

[0347] Analysis

[0348] Nearest Neighbor analysis of the cluster center positions may, for example, reveal a higher fraction of detected targets in close proximity with the new method than with the old method. Example 8: Titration of VHH to find ideal concentration for imaging

[0349] The concentration of VHH in the imaging buffer may be titrated for at least one high affinity and low affinity binder. Binding kinetics of the imager strand to the targets may be determined (kon and koff). At low VHH concentrations, kon is limited by the VHH binding. At high VHH concentrations, kon is limited by the unbound fraction of imager strands because high excess of VHH can bind the imager in solution, effectively reducing the imager concentration. A sweet spot to maximize kon, thereby reducing background or increase imaging speed, may be found. Further, low VHH concentrations might reduce non-specific binding. konmay be defined using the following eguation: kon= with TDas the mean dark time and c, the applied imager concentration. The mean dark time may be defined as the average time between two consecutive binding events. koffmay be defined using the following equation: koff= with TBas the mean bright time. The mean bright time may be defined as the average time of a detected binding event.

[0350] Experiment 8a: In analogy to example 2, labeling efficiency, kon, and koff may be measured for a target binding moiety with KDbetween 1 and 50 nM at multiple concentrations (namely, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM). The imager concentration may always be kept to 1 nM.

[0351] Experiment 7b: the same experiment as Experiment 8a, but with a target binding moiety with a KDsmaller than 1 nM.

[0352] e table below comprises a list of sequences used in the examples of the present invention:

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365] 59

[0366] While in the above, a preferred embodiment has been described with reference to the accompanying drawings, the skilled person will understand that this embodiment was provided for illustrative purpose only and should by no means be construed to limit the scope of the present invention, which is defined by the claims.

[0367] Whenever a relative term, such as "about", "substantially" or "approximately" is used in this specification, such a term should also be construed to also include the exact term. That is, e.g., "substantially straight" should be construed to also include "(exactly) straight".

[0368] Whenever steps were recited in the above or also in the appended claims, it should be noted that the order in which the steps are recited in this text may be accidental. That is, unless otherwise specified or unless clear to the skilled person, the order in which steps are recited may be accidental. That is, when the present document states, e.g., that a method comprises steps (A) and (B), this does not necessarily mean that step (A) precedes step (B), but it is also possible that step (A) is performed (at least partly) simultaneously with step (B) or that step (B) precedes step (A). Furthermore, when a step (X) is said to precede another step (Z), this does not imply that there is no step between steps (X) and (Z). That is, step (X) preceding step (Z) encompasses the situation that step (X) is performed directly before step (Z), but also the situation that (X) is performed before one or more steps (Yl), ..., followed by step (Z). Corresponding considerations apply when terms like "after" or "before" are used.

Claims

Claims1. A bispecific compound capable of specifically binding to a target molecule, wherein the bispecific compound comprises at least one target molecule binding moiety capable of binding to the target molecule, and at least one docking module capable of binding to at least one imager, wherein the at least one target molecule binding moiety corresponds to the target molecule.

2. The bispecific compound according to the preceding claim, wherein the bispecific compound comprises a binder, wherein the binder comprises the at least one target molecule binding moiety capable of binding to the target molecule, and the at least one docking module capable of binding to the at least one imager.

3. The bispecific compound according to claim 1, wherein the bispecific compound comprises a binder and at least one adapter, wherein the at least one adapter comprises the at least one docking module capable of binding to the at least one imager, wherein the at least one adapter comprises at least one adapter attachment module, wherein the binder comprises a binder attachment module, wherein the bispecific compound comprises the binder comprising the at least one target molecule binding moiety capable of binding to the target molecule, and the binder attachment module, wherein the binder attachment module is capable of binding the at least one adapter module of the at least one adapter; and the at least one adapter comprising the at least one docking module capable of binding to the at least one imager, and the at least one adapter attachment module wherein the at least one adapter attachment module is capable of binding to the binder attachment module of the binder.

4. The bispecific compound according to any of the preceding claims, wherein the at least one docking module comprises at least one of: a nanobody; an antibody; an antigen; and a nucleotide, wherein the at least one docking module is capable of transiently binding to the at least one imager; and the at least one imager is capable of permanently binding to at least one oligonucleotide to form an imager-oligonucleotide construct, wherein the imager- oligonucleotide construct is capable of transiently binding to the bispecific compound, wherein the at least one imager comprises at least one imaging strand; and at least one dye comprising at least one of: a fluorescent dye, a radio isotope, a metal isotope, and a scattering probe.

5. The bispecific compound according to any of the preceding claims, wherein the at least one imager has an on-rate to the at least one docking module of 105M-1s1to 109M-1s-1, more preferably 106M-1s1to 109M-1s-1, andan off-rate from the at least one docking module of 0.1 s1to 1000 s-1, more preferably 1 s1to 100 s-1; an on-rate of the binder to the target molecule is between 103and 108M-1s-1, more preferably between 104and 108M-1s-1; an on-rate of the at least one adapter is between 103and 109molds’1, preferably between 5 x 103and 5 x 108M-1s-1, further preferably between 104and 108M-1s-1; and an off-rate of the at least one adapter is less than 10 s-1, preferably less than 5 s-1, further preferably less than 1 s’1.

6. A method for detecting a target molecule, the method comprising introducing into a sample chamber a sample containing the target molecule; introducing into the sample chamber a bispecific compound capable of binding the target molecule; allowing the bispecific compound to bind to the target molecule; and visualizing the target molecule bound to the bispecific compound, wherein the bispecific compound is according to any of the preceding bispecific compound claims, and wherein the method comprises visualizing the target molecule bound to the bispecific compound by means of points accumulation for imaging in nanoscale topography (PAINT) microscopy via at least one imager, wherein the step of visualizing the target molecule bound to the bispecific compound comprising at least one of: capturing at least one image data, and processing the at least one image data.

7. The method according to the preceding method claim, wherein the step of capturing the at least one image data comprises imaging the sample, preferably at least two image data, more preferably at least 100 image data, most preferably at least 1000 image data, wherein the method comprises directing light at the sample before capturing each of the at least one image data of the sample.

8. The method according to any of the preceding method claims, wherein the method comprises capturing the at least two image data at a capturing time interval, wherein the capturing time interval between two consecutive image data is based, at least in part, on at least one of: an on-rate of the binder, an off-rate of the binder, an on-rate of the at least one adapter, an off-rate of the at least one adapter, an on-rate of the least one imager, and an off-rate of the at least one imager.

9. The method according to any of the preceding method claims, wherein the sample comprises at least two target molecules, wherein at least two of the at least two target molecules are different target molecules, wherein the method comprises determining locations of each of the two target molecules based, at least in part, on the at least one image data.

10. The method according to any of the preceding method claims, wherein the method comprises, between consecutively adding any two of the at least two binders, washing the sample with an unbinding reagent, wherein the unbinding reagent is configured to unbind a first of the two binders from the target molecule, wherein the unbinding reagent is abuffer solution, wherein the method comprises the buffer solution increasing the off-rate of the target molecule binding moiety by at least one of: using low or high pH, protein denaturing conditions, or high or low salt conditions, wherein the method comprises washing the sample with the unbinding reagent for a defined time interval, wherein the defined time interval is based, at least in part, on an off-rate of the bispecific compound, wherein the off-rate of the target molecule binding moiety is in the range of 10’5s1to 10’1s ~1, preferably in the range of 10’4s1to 10’2s’1.

11. The method according to any of the preceding method claims, wherein the step of capturing the at least one image data comprises imaging the sample, wherein the method comprises imaging the sample after adding the bispecific compound, wherein the method comprises adding the bispecific compound and the at least one imager simultaneously.

12. The method according to any of the method claims 6 to 10, wherein the step of capturing the at least one image data comprises imaging the sample, wherein the method comprises imaging the sample after adding the bispecific compound and after adding the imager, wherein the method comprises adding one of imager and bispecific compound one after the other, without completely displacing the other from solution.

13. A system for detecting a target molecule in a sample, the system comprising a sample chamber configured to receive the sample and a bispecific compound; and a super-resolution microscope configured to perform points accumulation for imaging in nanoscale topography (PAINT) microscopy measurements, wherein the system is configured to perform the method according to any of the preceding method claims.

14. A kit for use in a method for detecting a target molecule, the kit comprising a bispecific compound according to any of the preceding bispecific compound, wherein the bispecific compound is at least one of: in a solution, and is freeze-dried.

15. The kit according to any the preceding kit claim, wherein the kit comprises at least one of: a buffer for hybridization comprising at least one of: fixation buffer, wash buffer, and hybridization buffer; an imager; and an adapter.

Images