Methods and markers for analyzing sample

Abstract

There is provided a method for analyzing a sample, the sample comprising: a plurality of affinity reagents (5302a), each affinity reagent (5302a) configured to be attached to an analyte (900), at least one of the affinity reagents (5302a) being attached to the analyte (900); and a first plurality of dye combinations, each of the first plurality of dye combinations being unique and each of the dye combinations including at least two dyes having different characteristics with respect to at least one of excitation and emission. At least one of the unique dye combinations is attached to an associated affinity reagent (5302a) according to a first mapping. At least another one of the unique dye combinations is attached, preferably randomly, to the bound affinity reagent (5302a) according to a second mapping. The first mapping is different from the second mapping. With respect to the first mapping and the second mapping, the method includes, in a chronological manner, directing excitation light toward the sample, the excitation light having characteristics for at least exciting the at least two dyes, the at least two dyes having different characteristics with respect to at least one of excitation and emission; and generating at least one first readout from the emitted light emitted by the excited dye.

Description

Technical Field

[0001] The present invention relates to a method for analyzing a sample, particularly a biological sample. Furthermore, the present invention relates to an apparatus for analyzing a biological sample. The method or apparatus may also be used for chemical compounds or chemical elements. Furthermore, the present invention relates to a linker configured to bind to an affinity reagent, a reporter comprising a linker and at least one dye, and a marker comprising an affinity reagent and a reporter. Background Art

[0002] To address key questions in the life sciences, it is crucial to accurately detect the presence of analytes or molecular targets in a variety of samples: biological samples (e.g., tissue samples or cell cultures), environmental samples (soil samples), water samples, diagnostic procedures (e.g., solid or liquid biopsies or samples prepared therefrom), or lysates or extracts of such samples. This can be achieved by introducing markers into the sample that bind to specific structures (e.g., specific biomolecules). These markers typically include an affinity reagent that attaches to the structure in question and a fluorescent dye that is conjugated to the affinity reagent directly or through a secondary affinity reagent. Various techniques exist for analyzing biological samples prepared in this manner. The level of multiplexing in fluorescence microscopy, i.e., the number of different fluorescent dyes that can be read simultaneously, is typically low and, in the case of fluorescence microscopy, is typically in the range of 1-5 dyes for channel-based readout, and in the range of 5-12 dyes for spectral detectors using dispersive optical elements (such as prisms or gratings combined with multiple detectors or array detectors). Somewhat higher levels of multiplexing have been achieved in fluorescence-based cytometry and sorting techniques, but here too, multiplexing is limited to the small number of dyes and hence markers that can be read in one experiment.

[0003] "Fluorescent cell barcoding" is a multiplexing technology developed by Krutzig and Nolan in 2006 and is based on the use of different mixtures of three fluorescent dyes as described in the literature Nat Methods. 2006 May; 3(5): 361-8. doi: 10.1038 / nmeth872.

[0004] Tsai et al., 2020, noted that "Fluorescent cell barcoding (FCB) is a multiplexing technique for high-throughput flow cytometry (FCM). Although powerful in minimizing staining variability, it remains a subjective FCM technique due to inter-operator variability and differences in data analysis" (J Immunol Methods 2020 Feb;477:112667. doi:10.1016 / j.jim.2019.112667. Epub 2019 Nov 11). Both the technical subjectivity and inter-operator variability of this method are inherently related to the fact that the method is based on encoding partial information in dye hues (i.e., intensity variations such as light green, green, dark green), which severely limits the use of this technology.

[0005] However, there is no technology that allows for fluorescence-based readout of a large number of different markers, where the readout can refer to image-based readout or non-image-based readout.

[0006] Fluorescence microscopy allows the imaging of samples with high spatial resolution, but involves only a small number (usually between 1 and 5) different fluorescent dyes. The markers available in the same experiment must cover markers for identifying cell types, functional markers (such as proteins of interest) and general morphological markers. This means that in most imaging experiments, cell types are only rarely identified. This means that a fairly wide range of multi-cell type populations are being studied, severely limiting the predictive power and translational value of the results. Although modern methods exist that allow more reliable and robust identification of cell types, such as analysis based on gene regulatory networks (GRNs), they require a much larger number of different markers to be read out from the sample.

[0007] While neighboring techniques in the field of cytometry, mass cytometry and imaging mass cytometry, can distinguish between approximately 12 to 30 different markers, they do so with low spatial resolution.

[0008] Spatial profiling techniques are able to distinguish orders of magnitude higher numbers of different markers, although with even lower spatial resolution, as the technique is based on hybridization of oligonucleotides to the sample, followed by selective release of the bound oligonucleotides in a regioselective manner, and subsequent next-generation sequencing of the released oligonucleotides.

[0009] Spatial biology techniques based on combinatorial labeling have been developed, in which the identity of the marker is encoded using a combination of dyes, such as the nanoreporters or nanostrings used in Nanostring's CosMx imager, or the HuluFiSH probes of PixelBiotech (Heidelberg, Germany), which are compact combinatorial fluorescent labels based on oligonucleotides and can label a large number of markers using a limited number of fluorescent dyes. However, in both cases, the readout of the marker requires that only one labeled marker is present in the readout volume at the same time. If multiple markers are read out from the same readout volume at the same time, such as one marker protein K and another marker protein M, reliable decoding is no longer possible. This poses a significant limitation because it basically means that reliable readout relies on sparse labeling of the sample. In practice, this will result in the readout being extended to multiple rounds to avoid the simultaneous appearance of two different markers in the same readout volume at the same readout time (i.e., during the same round of iterative staining, imaging and inactivation). This is already a problem when trying to analyze a large number of target mRNAs, for example, but the problem becomes even more severe when trying to analyze a comparable number of protein analytes, as crowding is more common due to the higher protein content in cells.

[0010] The present invention allows for the readout of very large numbers of markers using a fluorescence-based optical readout, which can be based on a continuous data readout stream or a discrete readout (digital or analog), and can be based on a point detector, a line detector, or an area detector (such as a camera or a hyperspectral camera). Therefore, the method is widely applicable in life sciences, diagnostics, environmental sciences, healthcare, and quality control, and can be combined with a wide variety of optical readouts, including but not limited to cell counters, microplate readers, microscopes, and imaging systems.

[0011] The present invention achieves the marker discrimination capability and coverage currently achievable using next-generation sequencing-based readout based on optical fluorescence readout, and can be implemented on commercially available fluorescence imaging systems (such as the STELLARIS 8 confocal microscope platform (from Leica Microsystems)).

[0012] The present invention is based on viewing the "observation" of microscopy as an encoding / decoding problem rather than as a problem of recording spatial position intensities in an image, which is essentially a matrix of intensity values. Although the methods described in the present invention are compatible with image-based readout, the "images" generated by the methods and devices described in the present invention should be viewed as probabilistic mathematical models of the authenticity of the sample under investigation, where the presence of a target molecule is detected or called (presence call) based on the user's decision to accept its presence based on a measure of statistical confidence and a certain level of statistical confidence in the presence of the corresponding target molecule or analyte in the readout volume.

[0013] The step of accepting the presence of a specific marker, a specific affinity reagent, and a specific target molecule based on a statistical confidence value and / or a certain statistical confidence level (i.e., a presence call) yields mathematical truth. This is an important aspect of the present invention because it also means that according to the presence call, one operates in the axiomatic realm of mathematics, which is inherently unaffected by the measurement complications in non-mathematical fields (i.e., physics, chemistry, biology). Therefore, this fact is of great significance and also shows that this method can realize a completely new microscopy paradigm.

[0014] Statistical methods that provide a measure of statistical confidence on a per-marker or per-target basis are likely to be a combined measure and in many respects may be similar or identical to the methods used in transcriptomics and genomics, where enrichment scores and p-values ​​are commonly used.

[0015] The present invention uses a combination fluorescent marker, which includes a unique combination of dyes, which is essentially a code. Therefore, the term "code" and the term "combination fluorescent marker" and the term "combination marker" are used interchangeably in this document. As mentioned above, there are other methods in the prior art that use such combination markers, such as nano-reporters (nano strings) described in US 9,376,712 B2 and US 8,519 115 B2. Similarly, there are such methods in the prior art that encode the identity of the marker with a specific combination of dyes or a specific sequence of 0s and 1s in a cycle of multiple iterative staining, imaging and inactivation processes. The latter readout method is referred to as reading in a "time-sequential manner" because the code is not read out in a single cycle, but read out in multiple cycles, which is different from the so-called "spatial readout" (which means that the code is read out in a single cycle).

[0016] Examples of techniques employing a "temporal readout" include MERFISH, SeqFISH or the method disclosed in US 10,227,639 B2, while examples of techniques employing a "spatial readout" include nanoreporters (NanoStrings) described in US 9,376,712 B2 or HuluFiSH (PixelBiotech, Heidelberg).

[0017] Conceptual elements of the prior art as explained in application PCT / EP2021 / 073819 (the entire content of which is incorporated herein by reference) and / or in application PCT / EP2021 / 066645 (the entire content of which is incorporated herein by reference), the conceptual elements of which are incorporated herein relate to: Multiple unique combinatorial fluorescent labels, which are physical structures comprising a backbone onto which unique combinations of dyes can be assembled before, during, or after introduction into a sample; A linker or connecting element or connecting structure, which mediates the connection between the combined label and the affinity reagent; • Mapping, where each affinity reagent is assigned to a unique combinatorial label for each round.

[0018] The core idea set out in applications PCT / EP2021 / 073819 and PCT / EP2021 / 066645 is the recognition that the main limitation of the prior art (i.e. the inability to reliably read out the identity of multiple markers present in the same read volume at the same time) can be overcome by introducing "code swapping" (i.e. a change in the mapping between affinity reagents and combination labels from round n to round n+1). Using "code swapping" and an iterative or cyclic process of staining, imaging and inactivation, a read volume that simultaneously includes multiple markers (e.g. labeled antibodies against 5 different proteins) can be reliably decoded with a certain statistical confidence within a limited number of rounds. Therefore, the decoding strength (i.e. how fast the confidence in the decoding result rises) depends on the distance of the codes in the code space, or in other words, on how different the overlapping combination labels are with respect to their dye composition, or in other words, on the number of dyes that can be generated in combination and the combination selected from all possible combinations. While the methods disclosed in applications PCT / EP2021 / 073819 and PCT / EP2021 / 066645 are very powerful, they may still rely on an iterative or cyclic process of staining, imaging, and inactivation.

[0019] It is therefore an object of the present invention to improve this method and to eliminate the need for an iterative process.

[0020] The methods described in applications PCT / EP2021 / 073819 and PCT / EP2021 / 066645 may essentially rely on labeling the sample with markers comprising a combination of markers, which may be exchanged (code swapping) from one round to the next, and accepting the presence of the marker in the read volume ("presence call") based on a statistical analysis of a measure of statistical confidence.

[0021] A key aspect of the present invention is the recognition that "code swapping" does not require a cyclic process of staining, imaging, and inactivation. Using oligonucleotide-barcoded affinity reagents, where each affinity reagent comprises a unique oligonucleotide barcode and a combinatorial tag configured to bind to the oligonucleotide barcode, "code swapping" can be performed without the need for inactivation and re-staining. This is particularly feasible when the following conditions are met: (A) assigning a subset of unique combinatorial tags in the plurality of combinatorial tags to affinity agents in the plurality of affinity agents; (B) no subset of the unique combinatorial tags is assigned to more than one affinity reagent; and (C) The linkage between the oligonucleotide barcoded affinity reagent and the unique combinatorial tag is based on transient or reversible hybridization and / or dynamic association / dissociation or reversible association / dissociation.

[0022] In this case, when observing a particular read volume, which includes a marker, which includes an affinity reagent, such as an antibody against protein X, which is labeled with the first combination label k of a set of unique combination labels assigned to it, then the label k will dissociate after a period of time, and the oligonucleotide barcode of the antibody will be free to bind to other labels in the set, such as label f, etc.

[0023] We refer to this as "code exchange" within a round, and the implementation process as a "barcode cycle." This barcode cycle can be performed in a variety of ways to achieve transient or reversible binding between the oligonucleotide barcode and the corresponding oligonucleotide portion of the combination tag. These various methods include, but are not limited to, the following (1) to (7): (1) DNA-Exchange imaging, which is closely related to DNA-PAINT and Exchange-PAINT, can be used to achieve dynamic intra-round code exchange or repeated intra-round code exchange. (2) DNA-PAINT, in which the labeled oligonucleotide moiety and the corresponding oligonucleotide barcode of the corresponding affinity reagent are configured to bind to each other transiently, allowing for “dynamic intra-round code exchange.” This can be achieved, for example, by using complementary sequence segments of approximately 9–13 nt in length. (3) Exchange-PAINT, which is a combination of DNA-PAINT and exchange imaging. (4) Strand displacement, in which the labeled oligonucleotide portion that hybridizes to the oligonucleotide barcode of the corresponding affinity reagent binds poorly due to mismatches and can be displaced by introducing a free oligonucleotide that binds perfectly (no mismatches) to the labeled oligonucleotide portion that exhibits incomplete complementarity to the oligonucleotide barcode of the corresponding affinity reagent. (5) NCDA for optical switching, which is a molecular glue for DNA composed of "two guanine-recognizing naphthyridine moieties connected to a photochromic azobenzene unit that undergo cis / trans isomerization upon light irradiation". NCDA can be used to achieve hybridization or reverse hybridization of oligonucleotides containing short GG mismatches. Switching is performed using 360nm and 430nm light. The reversible binding mediated by NCDA can also allow different combination tags to repeatedly bind / dissociate with the same barcoded affinity reagent without the need for an iterative staining process. The use of NCDA as a DNA molecular glue is described in Dohno et al., 2007 (Journal of the American Chemical Society 2007 129(39), 11898-11899). (6) Controllable DNA hybridization by host–guest complexation-mediated ligand invasion can also be used as a strategy to achieve reversible binding / dissociation or transient binding. Ligand-controlled DNA hybridization was achieved using cucurbit[7]uril (Xiao, L., Wang, LL., Wu, CQ. et al., Controllable DNA hybridization by host–guest complexation-mediated ligand invasion. Nat Commun 13, 5936 (2022)). (7) Temperature cycling, which also allows longer predetermined sequences or attachment sites to be used for intra-cycle "code swapping." Depending on the buffer conditions and the G / C content of the predetermined sequence or attachment site, the melting temperature can be in the range of 30-72°C, preferably 50-72°C.

[0024] the term

[0025] In the meaning of this document, the following terms are used in the following manner:

[0026] "Sample": For the purposes of this document, a "sample" refers to a biological sample, which may also be referred to as a biological specimen, and includes, for example, blood, serum, plasma, tissue, body fluids (e.g., lymph, saliva, semen, interstitial fluid, cerebrospinal fluid), feces, solid biopsies, liquid biopsies, explants, whole embryos (e.g., zebrafish, fruit flies), whole model organisms (e.g., zebrafish larvae, fruit fly embryos, Caenorhabditis elegans), cells (e.g., prokaryotes, eukaryotes, archaea), multicellular organisms (e.g., Volvox), suspension cell cultures, monolayer cell cultures, 3D cell cultures (e.g., spheroids, tumoroids, organoids derived from various organs (e.g., small intestine, brain, heart, liver), lysates of any of the above, and viruses. For the purposes of this document, a "sample" further refers to the volume surrounding a biological sample. For example, in analyses of secreted proteins (e.g., growth factors) and extracellular matrix components, the extracellular environment surrounding cells within a certain distance relevant to the analysis is also referred to as the "sample." In particular, an affinity reagent introduced into this surrounding space is referred to as being "introduced into the sample" in the sense of this document.

[0027] "Affinity reagent": In the sense of the present document, the term "affinity reagent" may in particular be an antibody, a single domain antibody (also called nanobody), a combination of at least two single domain antibodies, an aptamer, an oligonucleotide, a morpholino, a peptide nucleic acid (PNA) complementary to a predetermined RNA or DNA target sequence, a ligand (e.g. a drug or drug-like molecule) or a toxin (e.g. phalloidin, a toxin that binds to actin filaments). In the sense of the present document, an affinity reagent is configured to bind to a target molecule or analyte with a certain affinity and specificity, whereby the affinity reagent can be said to be substantially specific for the target molecule or predetermined target structure. In the sense of the present document, a "plurality of affinity reagents" (S2) includes the following affinity reagents (a1, a2, a3 ... a n), these affinity reagents are configured to specifically bind to a predetermined target structure within the biological sample, or to a predetermined chemical compound, or to a predetermined chemical element or analyte. At least some of the plurality of affinity reagents (A) are "introduced into the sample" so that the affinity reagents can attach to corresponding predetermined target structures within the sample. In this context, within the meaning of this document, and as described above, "introduced into the sample" can mean physically introduced into the sample volume or introduced into a volume surrounding and allocated to the sample. An example of the latter case can be for analysis of secreted molecules, e.g., secreted molecules that are best assessed in the extracellular space, which can be external to the sample but within or near a certain spatial range of the sample. Within the meaning of this document, in the case of nucleic acid targets or analytes (such as DNA targets or RNA targets, etc.), an affinity reagent (e.g., an oligonucleotide probe) can refer to a set of different affinity reagents, such as a set of oligonucleotide probes that bind to the same analyte (e.g., the same gene, the same locus, or the same mRNA target) at different overlapping positions or sequence segments or non-overlapping positions or sequence segments. In this context, code-switching refers to labeling the same analyte nucleic acid with different combinations of dyes attached to different affinity reagents, such as oligonucleotide probes that bind to different sites on the same nucleic acid target (e.g., mRNA).

[0028] “Predetermined target structure”: In the sense of this document, “predetermined target structure” refers to a target molecule, target structure or analyte, which can be, for example, a protein (e.g. a certain protein), an RNA sequence (e.g. mRNA of a certain gene), a peptide (e.g. somatostatin), a DNA sequence (e.g. a gene site or element), a metabolite (e.g. lactate), a hormone (e.g. estradiol), a neurotransmitter (e.g. dopamine), a vitamin (e.g. cobalamin), a micronutrient (e.g. biotin), a metal ion (e.g. metal and heavy metal ions such as Cd(II), Co(II), Pb(II), Hg(II), U(VI)).

[0029] "Dye": In the sense of this document, the terms "fluorescent dye", "fluorophore", "fluorochrome", "dye" interchangeably refer to a fluorescent compound or structure, and may specifically be one of the following substances: fluorescent organic dyes, fluorescent quantum dots, fluorescent dyads, fluorescent carbon dots, graphene quantum dots or other carbon-based fluorescent nanostructures, fluorescent proteins, fluorescent DNA origami-based nanostructures. The term "fluorescent dye" refers to an organic fluorescent dye, in particular a derivative of a xanthene (e.g., fluorescein, rhodamine, Oregon Green, Texas), a cyanine (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiocarbocyanine, merocyanine), a derivative, a squaryl rotaxane derivative, a naphthalene, a coumarin, an oxadiazole, anthracene (anthraquinone, DRAQ5, DRAQ7, CyTRAK Orange), a pyrene (Cascade Blue), an oxazine (Nile Red, Nile Blue, Cresyl Violet, Oxazine 170), an acridine (proflavine, acridine orange, acridine yellow), an arylmethine (auramine, crystal violet, malachite green), a tetrapyrrole (porphine, phthalocyanine, bilirubin), a dipyrromethene (BODIPY, azaBODIPY), a phosphorescent dye or a luminescent dye.Commercially available fluorescent dyes are designated by the following trademark groups, which may include dyes belonging to different chemical families: CF dyes (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), EverFluor (Setareh Biotech), Alexa Fluor (Invitrogen), Bella Fluore (Setareh Biotech), DyLight Fluor (Thermo Scientific), Atto and Tracy (Sigma-Aldrich), FluoProbes (Interchim), Abberior Dyes (Abberior Dyes), Dy and MegaStokes Dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau and Square Dyes (SETA BioMedicals), Quasar and Cal Fluor dyes (Biosearch Technologies), SureLight Dyes (Columbia Biosciences), Vio Dyes (Milteny Biotec) [List modified from: https: / / en.wikipedia.org / wiki / Fluorophore]. In the group of fluorescent proteins, in particular members of the green fluorescent protein (GFP) family, including GFP and GFP-like proteins (e.g. DsRed, TagRFP) and their (monomeric) derivatives (e.g. EBFP, ECFP, EYFP, Cerulaen, mTurquoise2, YFP, EYFP, mCitrine, Venus, YPet, Superfolder GFP, mCherry, mPlum) are referred to in the present document as "fluorescent dyes". Furthermore, in this group of fluorescent proteins, the term "fluorescent dye" in the present document may include fluorescent proteins whose absorbance or emission properties change upon binding a ligand (such as BFPms1) or in response to changes in the environment (e.g. redox-sensitive roGFP or pH-sensitive variants). Furthermore, within this group of fluorescent proteins, the term "fluorescent dye" in the sense of this document may include the small super-red fluorescent protein smURFP, a derivative of phycobiliprotein from cyanobacteria, as well as fluorescent protein nanoparticles that may be derived from smURFP.A review of fluorescent proteins can be found in Rodriguez et al., 2017, Trends Biochem Sci. 2017 Feb; 42(2): 111–129. In the context of this document, the term “fluorescent dye” may also refer to fluorescent quantum dots. In the context of this document, the term “fluorescent dye” may also refer to fluorescent carbon dots, fluorescent graphene quantum dots, fluorescent carbon-based nanostructures, as described in Yan et al., 2019, Microchimica Acta (2019) 186: 583 and Iravani and Varma 2020 in Environ Chem Lett. 2020 Mar 10: 1–25. In the context of this document, the term “fluorescent dye” may also refer to fluorescent polymer dots (Pdots) or nanodiamonds. In the sense of this document, the term "fluorescent dye" may also refer to fluorescent dyads, such as dyads of perylene antenna and triangelium emitters, as described in Kacenauskaite et al., 2021, J. Am. Chem. Soc. 2021, 143, 1377-1385.

[0030] In the sense of this document, the term "fluorescent dye" may also refer to organic dyes, dyads, quantum dots, polymer dots, graphene dots, carbon-based nanostructures, DNA origami-based nanostructures, nanorulers, dye-doped polymer beads, fluorescent proteins, inorganic fluorescent dyes, SMILEs, or microcapsules pre-loaded with any of the above.

[0031] In the sense of this document, the term "fluorescent dye" may also refer to a FRET pair having at least one fluorescent dye as a FRET donor and at least one fluorescent dye as a FRET acceptor or a FRET pair for generating a three-component FRET triplet for resonance energy transfer. Specifically, the FRET pair or FRET triplet is connected by a complementary linker or connecting element.

[0032] In the sense of this document, the term "fluorescent dye" may also refer to a FRET tandem of physically linked dyes.

[0033] "Plurality of combinations of dyes" (S1): In the sense of this document, the term "plurality of combinations of dyes" (S1) refers to a plurality of combinations of dyes wherein each of the dye combinations (s1, s2, s3, ... s1) in the plurality of combinations of dyes (S1) n ) are unique, each dye combination (s1, s2, s3...s n) includes at least two different dyes (|s|>=2); wherein the composition of the plurality of dye combinations (S1) is such that each dye (y1, y2, y3 ... y σ ) can be read out by a readout device; wherein the dye can be separated into multiple channels by a readout device; each channel corresponds to one of the dyes (y1, y2, y3...y σ ).

[0034] "Marker": In the sense of this document, "marker" is used to denote both a single molecule used as a marker and a collection of the same molecules used as markers. In the sense of this document, a "marker" is a combination of affinity reagents configured to attach to a predetermined structure (also called a target molecule or analyte and / or a "reporter"). Therefore, a "marker" is a virtual assignment or mapping of an affinity reagent and a specific dye combination (virtual marker) as well as a physical assembly of an affinity reagent and a dye combination (physical marker). In the sense of this document, the physical assembly of the affinity reagent and dye combination can be performed before, during or after the introduction of the corresponding affinity reagent into the sample. For example, when using an antibody barcoded with an oligonucleotide sequence as an affinity reagent, it can be brought into the sample and attached to its predetermined target structure, for example, by introducing a unique dye combination (s) before or after at least some of the multiple affinity reagents (A) into the sample, into a compound or into a chemical element, or before generating a readout of the emission light emitted by the excited dye. i ) physically attached to the distributed affinity reagent (a i During the iterative staining-imaging-dye inactivation process, affinity reagents that bind to predetermined target structures can be cyclically linked to a series of different dye combinations, such as using a first dye combination in the first iteration and a second dye combination in the second iteration. We call this strategy "first qualitative iterative multi-species readout volume decoding by reassigning codes between iterations ("code swapping"). In other words, one or more of the markers in the sample will change between iterations.

[0035] "Reporter," "Combination Label": In the context of this document, "reporter" is used to refer to both a single molecule / structure acting as a reporter and a collection of identical molecules / structures acting as reporters. In the context of this document, a "reporter" is a unique combination of a "dye combination" and a "linker," configured to connect the "dye" to the "affinity reagent" combination.

[0036] "Code," "Code Swapping": Within the meaning of this document, a "reporter" or "combinatorial marker" comprises a unique "dye combination" that, analogously, encodes the identity of a specific affinity reagent, thereby encoding the identity of a specific marker (marker = affinity reagent + combinatorial marker), and thus the identity of a certain analyte (such as the mRNA of gene X or protein X). Within the meaning of this document, a "subset of unique dye combinations" is assigned to a specific affinity reagent, along with a "subset of combinatorial markers," to form a "subset of markers," where one affinity reagent is assigned to multiple "dye combinations" or "codes." The corresponding affinity reagent is then brought into contact with its analyte in a biological sample, and at least one combinatorial marker from the "subset of combinatorial markers" is allowed to bind to the affinity reagent, forming the first marker of the "subset of markers." The fluorescent dye of the attached "combinatorial marker" is read out, the "combinatorial marker" is allowed to separate from the affinity reagent, and a second "combinatorial marker" from the "subset of combinatorial markers" assigned to the affinity reagent is allowed to bind to it. This process, which results in a change in the binding between the affinity reagent and its assigned combinatorial marker, is referred to as "code swapping." In previous patent applications, this " code exchange " is to carry out between multiple rounds of cyclic dyeing, imaging, inactivation process. In the present document, this " code exchange " is carried out in " round ", promptly does not need cyclic dyeing, imaging, inactivation process, and basically occurs in part first round (this round is only made up of dyeing and imaging). " code exchange in round " can use the instantaneous combination between " combinatorial label " and the affinity reagent of distribution and their random combination to realize, this is referred to as " dynamic round intra-code exchange " in the present document, perhaps can realize by the conversion process between the stable combination and the release between " combinatorial label " and the affinity reagent of distribution, this can realize in the various ways of describing in detail in the specification sheets. The latter pattern is referred to as " code exchange in the round of repetition " in this article.

[0037] "Linker": In the sense of this document, a "linker" refers to a single chemical structure (e.g., a monomeric molecule or polymer) or a multi-part chemical structure assembly that connects a fluorescent dye combination to an affinity reagent. For example, the linker can be directly or covalently coupled to the dye and the affinity reagent, or can be indirectly coupled through, for example, an affinity tag-affinity ligand combination (such as a streptavidin-biotin interaction), a hapten, or an oligonucleotide. In the case of covalent coupling, this can be site-selective coupling. Common coupling chemistries, such as NHS-, maleimide, azide-alkyne, and a range of other so-called click chemistry methods, can be used to couple the linker to the affinity reagent and / or to couple the linker to the dye. Linkers can specifically include oligonucleotides (e.g., DNA, RNA, LNA, PNA, morpholino, other artificial oligonucleotides), peptides, DNA origami-based structures (such as, for example, nanorulers), microbeads / nanobeads, polymers, microcapsules / nanocapsules, microcrystals / nanocrystals, carbon tubes, carbon-based nanostructures (e.g., graphene). Linkers may specifically include oligonucleotides and other elements of the group described above, such as including oligonucleotides and peptides.

[0038] "Readout Device": For the purposes of this document, a "readout device" refers to a device used for performing multicolor fluorescence readout or imaging. A readout device typically comprises at least one excitation light source, a detection system comprising at least one detection channel, and may also include filters and / or dispersive optical components (such as prisms and / or gratings) to direct the excitation light to the sample and / or to direct the sample's emission light to a detector or appropriate region of the detector. For the purposes of this document, the detection system may comprise multiple detection channels and may be a spectral detector that detects multiple spectral bands in parallel, or a hyperspectral detector that detects a continuous portion of the spectrum. The detection system includes at least one detector, which may be a point detector (e.g., a photomultiplier tube, avalanche diode, hybrid detector), an array detector, a camera, or a hyperspectral camera. The detection system may record the intensity of each channel (as is typically the case in a cytometer) or may be an imaging detection system that records an image (as is the case in a microplate reader or microscope). A readout device with a single detection channel (such as a camera or photomultiplier tube) can generate a readout with multiple detection channels using, for example, distinct excitation and emission bands. The readout device allows a certain number of dyes to be analyzed from a given biological sample in a given run. A "run" can refer to an "iteration" or "round", i.e. at least one readout is generated for a given set of dye combinations and a given mapping of affinity reagents to dye combinations, wherein the affinity reagents are attached to the analyte. This number typically depends on the number n of detection channels that the readout device is configured to provide (i.e., capable of spectrally resolving). In the case of microscopes, for camera-based widefield detection (e.g. widefield epi-fluorescence microscopes, spinning disk microscopes, light sheet fluorescence microscopes), the number of detection channels is typically 4-5; for microscopes using spectral detection concepts (typically relying on excitation or emission fingerprinting and (spectral / linear) unmixing), the number of detection channels is typically 5-12. In contrast, hyperspectral imaging, which can distinguish a large number of dyes by providing high spectral resolution over a wide and continuous spectral range, has not yet been widely used in the field of microscopy. In addition to spectral properties, the lifetime of fluorescent dyes can also be used to discriminate between multiple dye species and distinguish them from autofluorescence, effectively increasing the number of detection channels y, which also corresponds to the maximum number of dyes that can be reliably separated within the group (i.e., excited by the same excitation light).

[0039] "Oligonucleotide": in the sense of this document refers to DNA, RNA, peptide nucleic acid, morpholino or locked nucleic acid, glycol nucleic acid, threose nucleic acid, hexitol nucleic acid or another form of artificial nucleic acid.

[0040] "Spot": in the sense of this document, refers to the volume in or around a sample that is read out. The size and shape of the spot is determined by the effective point spread function of the imaging system used to acquire the data.

[0041] “Point spread function”: – In the sense of this document, the term “point spread function” is used to denote the principal maximum of the point spread function and, unless otherwise stated, refers to the effective point spread function (PSF) of the imaging system, which is generally elliptical, i.e., lateral resolution is better than axial resolution, but can approach an almost spherical shape as more views are acquired from preferably equidistant angles.

[0042] "Readout": In the sense of this document, the term "readout" refers to an image-based readout, which can be acquired on a microscope (such as a point scanning confocal microscope) or a camera-based / wide-field imaging system (such as a spinning disk microscope, a light sheet fluorescence microscope, a light field microscope and a stereo microscope). Furthermore, the term "readout" also refers to a non-image-based readout, such as a cytometer or a flow-based readout device with at least one point detector or line detector. The readout can consist of discrete readouts, such as a single acquisition of an emission spectrum or an image stack; the readout can be a substantially continuous stream of readout data, such as in a point scanning confocal microscope or a cytometer). In addition, the readout can be a series of images, such as a spectral or hyperspectral image stack, where a different band of fluorescence emission is recorded in each image.

[0043] "Readout Volume": For the purposes of this document, the term "readout volume" refers to the volume that an optical system (such as a microscope or cytometer) can effectively detect at a given moment. For systems with a continuous data stream, the "readout volume" is determined by a clock, such as a "pixel clock," that divides the continuous data stream into blocks of data, which are then assigned to specific points in time or spatial locations. The readout volume can depend on the effective point spread function of the imaging system; for example, the effective point spread function can define or limit the maximum range of the readout volume.

[0044] "Readout sequence": In the sense of this document, the term "readout sequence" is used to denote the readout of a "readout volume" that reads out the plurality of dyes Y constituting the dye combination of the plurality of dye combinations (S1). D All dyes (y1, y2, y3...y σ ) at least once, i.e., multiple dyes Y D All dyes (y1, y2, y3...y σ ) is excited at least once, and the emitted fluorescence is detected and separated into multiple channels by a readout device, each channel corresponding to a dye (y1, y2, y3...y σ). Thus, after obtaining the read sequence, the presence of the dye in the read volume can be assessed qualitatively and / or quantitatively, wherein qualitatively refers to calling the dye present in the read volume when the intensity in the corresponding channel is above a certain user-defined threshold; wherein quantitatively refers to calling the dye present in the read volume and assigning it a relative intensity value or an absolute number of molecules. The threshold can be a fixed threshold, a fixed channel-specific threshold, or a dynamically adjusted threshold. The threshold can be a combination of multiple thresholds, such as an intensity threshold and a statistical confidence level for the dye separation result. A decision to call the presence of the dye (presence call) can be made depending on whether the combination of multiple thresholds passes.

[0045] "Readout trace": In the sense of this document, the term "readout trace" refers to the time sequence of acquiring a "readout sequence". In other words, it is used to represent the readout of a "readout volume" that reads out all the dyes (y1, y2, y3, ..., y2) of the plurality of dyes constituting the plurality of dye combinations (S1). σ ) at least twice. The difference between acquiring multiple "readout sequences" and acquiring a "readout trajectory" during a cycle is that the "readout trajectory" is not interrupted by pauses for inactivation, washing and re-staining of the markers. In a preferred embodiment, the biological sample is analyzed by performing block scans and recording a short time series for each block. Such "readout trajectories" can be acquired at intervals ranging from milliseconds to minutes, or can be acquired as a continuous acquisition (streaming acquisition) of a specific position (block). A "readout trajectory" of a given volume can also be performed by acquiring a time series at a specific XY position while moving the sample in the Z direction relative to the readout. In a particularly preferred embodiment of the present invention, a readout is used that includes an orientation of the object plane that is at an angle to the (optical) surface of the sample carrier (e.g., a microscope slide, an imaging well, the bottom of a well plate). In particular, this can be a SCAPE, an OPM, a microscope or a readout device, as described in PCT application number PCT / EP2022 / 077378, the entire contents of which are incorporated herein by reference.

[0046] Thus, in the present invention, a "readout sequence" is acquired as a time series that is continuous at every position in the sample imaging area. In applications PCT / EP2021 / 073819 and PCT / EP2021 / 066645, a "readout sequence" is acquired cyclically or iteratively, because "code swapping" relies on the inactivation of bound combinatorial labels from round n, washing out unbound or non-inactivated combinatorial labels from round n, and then re-labeling with at least some of the combinatorial labels that have been redistributed to at least some of the affinity reagents in round n+1. This can be referred to as inter-round "code swapping" because the codes are reassigned from one round to the next, whereas the present invention eliminates the need for a cyclic process and "code swapping" is performed "intra-round" in the following manner: · dynamically ("dynamic intra-round code swapping"), where multiple different combinatorial tags assigned to a particular affinity reagent are allowed to dynamically bind and dissociate from the oligonucleotide-barcode of the affinity reagent; or · repeatedly ("repeated intra-round code swapping"), where multiple different combinatorial tags assigned to a particular affinity reagent are allowed to repeatedly bind and dissociate from the oligonucleotide-barcode of the affinity reagent.

[0047] Can be by using DNA-Exchange imaging technology, controllable DNA hybridization strategy and simply by raising the temperature to the melting temperature of the oligonucleotide-barcode attachment part close to the linker interaction, "dynamic round code exchange", wherein the binding constant depends largely on the length of the complementary segment, the number of mismatches (if any mismatches are introduced) and the ionic strength of the buffer. It is important to note that if the oligonucleotide-based linker or backbone structure of the combinatorial labeling has been stabilized before using a suitable strategy (such as, for example, connection (ligation) or ultraviolet-mediated cross-linking or chemical cross-linking), the temperature can be raised to close to the melting temperature in their presence. Further, by maintaining the ligand at a suitable concentration, the "dynamic round code exchange" controllable DNA hybridization described by Xiao et al. in 2022 is achieved (Xiao, L., Wang, LL., Wu, CQ. et al., Controllable DNA hybridization by host–guest complexation-mediated ligand invasion, Nat Commun 13, 5936 (2022)). In “dynamic internal code exchange,” different combinatorial tags assigned to specific affinity reagents randomly bind and dissociate from the corresponding oligonucleotide-barcode.

[0048] “Repeated intra-round code exchange” can be performed by controllable DNA hybridization using temperature cycling (melting-reannealing) as described by Xiao et al. in 2022 (Xiao, L., Wang, LL., Wu, CQ. et al., Controllable DNA hybridization by host–guest complexation-mediated ligand invasion by changing the ligand concentration repeatedly, Nat Commun 13, 5936 (2022)), or by using photoconvertible NCDA described by Dohno et al. in 2007 (Journal of the American Chemical Society 2007 129 (39), 11898-11899).

[0049] Both ("dynamic intra-round code exchange") and ("repeated intra-round code exchange") can be performed by introducing all necessary reagents into the sample before acquiring the "readout trajectory", or adding at least some reagents or changing at least some physical parameters (such as UV light, 360nm, 430nm light) or changing the temperature during acquisition of the "readout trajectory".

[0050] Preferably, the readout sequence is obtained from the following steps: exciting the sample with a first excitation light A, detecting the emitted fluorescence, and assigning it to the corresponding dye A1 、Dye A2 、Dye A3 ...Dye Ayn of y A channel, excites the sample with the second excitation light B, detects the emitted fluorescence, and assigns it to the corresponding Dye B1 、Dye B2 、Dye B3 ...Dye Byn of y B channel, and repeat the process until Dye n1 、Dye n2 、Dye n3 ...Dye nyn (i.e., all σ A variety of dyes (Y D )) has been read at least once.

[0051] For the purposes of this document, a "code" is defined as follows: S and T are two finite sets, where S is called the "source alphabet" and T is called the "target alphabet". C: S→T* is a total function or algorithm that uniquely represents the elements of S as a sequence of symbols over T. An extension C' of C is S * to T * A homomorphism that maps every sequence of source symbols to a sequence of target symbols naturally. In the language used in computer science, code usually refers to an algorithm, and a sequence of symbols refers to a coded string (adapted from: Code. (nd) In Wikipedia. Retrieved June 17, 2021 from https: / / en.wikipedia.org / wiki / Code). In the sense of this document, a finite set S1 is also called a "multiple dye combinations", and T1 * is a finite set of strings on T1, corresponding to "multiple affinity reagents", which can also be called A or S2.

[0052] Alternatively, in addition to encoding / decoding the dye combination, the user can also encrypt / decrypt the dye combination using the password X. X: S → T *

[0053] Two different cases a and b are distinguished and can be viewed as different directions of encoding / encryption.

[0054] The method disclosed in this document is compatible with both the alpha and beta cases. n and / or password X1, X2, X3...X n are used, as long as they are total functions and as long as the resulting mapping is injective or bijective. In both cases, the codes C1, C2, C3, ... n and / or password X1, X2, X3...X n In a particularly preferred embodiment of the present invention, a bijective mapping (encoding or encryption) is used, which means that in the elements (a) of the plurality of affinity reagents (S2), i )(Also called (A) or (T1 * )) with multiple dye combinations (S1) elements (s i ) there is a one-to-one correspondence between the dye combinations contained in the readout volume. This allows for particularly easy decoding of the dye combinations (s l to s k ), thereby identifying its bound affinity reagent (a based on the readout sequence l to a k), which evaluates qualitatively (presence call, e.g., "yes" = 1, "no" = 0) and / or quantitatively (presence call with relative or absolute quantification) all dyes (y1, y2, y3, ... y σ ) presence. As described herein, microscopy of the readout volume can be viewed as an encoding / decoding problem that is solved by labeling target molecules with affinity reagents that are (dynamically) linked to and bind to dye combinations that encode those target molecules in the readout volume that are labeled in this way. Retrieving the identity of target molecules that have a one-to-one mapping (bijective binding) to the affinity reagents from a plurality of affinity reagents is therefore a decoding problem. It is important to note that if the presence of a particular dye from a plurality of dyes is accepted in the readout sequence based on a certain statistical confidence level, then this presence becomes a mathematical truth. This is achieved when it is observed that the readout sequence does not allow all possible dye combinations (s1, s2, s3, ... s1) from a plurality of dye combinations (S1) to be included in the readout volume. n ) are summarized under it, then the dye combination (s l to s k In other words, if it is observed that the readout sequence can decode all possible combinations (s1, s2, s3, ... s1) of the multiple dye combinations (S1), n ) are summarized under it, one cannot gain knowledge about the content of the read volume. If the read sequence indicates multiple dyes (P D ) of all dyes (y1, y2, y3...y σ ) is called "present" in the readout volume. However, this is unlikely even for the case where a large number of affinity reagents are used, because the base number of multiple dye combinations (S1) increases exponentially, while the number of available affinity reagents is limited by the number of target molecules of interest. For example, the entire human genome contains about 20,000 coding genes, so even if one were to use 20,000 affinity reagents from the multiple affinity reagents in order to label these target molecules with unique dye combinations from the multiple dye combinations (S1), it would be easy to define a sufficiently large multiple dye combination (P D ) to ensure that the number of elements in S1 is >> 20000, i.e., several orders of magnitude higher, such as 10 6 to 10 10. Thus, the following condition can be easily defined: in which the fraction of the dye combinations actually assigned among the plurality of dye combinations (S1) over all available dye combinations becomes very small. In this case, the probability of observing false positives (i.e., dye combinations that are not assigned to markers (type I false positives) and / or dye combinations that are assigned to affinity reagents that are not physically present in the read volume (type II false positives)) becomes lower. If the condition is that a single iteration cannot produce a satisfactory statistical confidence level for the presence of any of the following: the dye combination; the affinity reagent; and the target molecule contained in the read volume, then this analysis can be significantly improved by different means to be described herein.

[0055] In a particularly preferred embodiment of the present invention, in a first step, a first readout sequence is obtained and a "set of first dye combinations" that can be summarized under the first readout sequence is stored in a storage device. In a next step, the encoding / encryption can be changed for at least some of the affinity reagents in the plurality of affinity reagents (A). This can be achieved by inactivating the dye introduced in the first step by eluting the affinity reagent, bleaching the dye, or cutting off the connection between the dye combination and the affinity reagent. Depending on the choice of method, in a second step, the target molecule is then re-labeled using at least some of the affinity reagents in the plurality of affinity reagents (A), wherein at least some of the affinity reagents are assigned to different second dye combinations. In a next step, the "set of second dye combinations" is derived from a second readout sequence, i.e., a second readout sequence is generated in the same manner as the first readout sequence, and the dyes in the set of second dye combinations are identified in the second readout sequence. The retrieval results of all second dye combinations that can be summarized under the second readout sequence are stored in a storage device. In a next step, the "set of first dye combinations" and the "set of second dye combinations" can be compared to define an overlap region, and for each dye combination and / or affinity reagent and / or target molecule and / or analyte detected in the overlap region, at least one statistical confidence level is calculated. A specific dye combination and / or specific affinity reagent and / or specific target molecule and / or analyte is said or called to be present in the read volume when at least one statistical confidence level calculated for that particular specific dye combination and / or specific affinity reagent and / or specific target molecule and / or analyte is acceptable based on a criterion, which criterion can be fixed and defined a priori or dynamically derived and adjusted during the experiment.

[0056] In principle, this process can be repeated until an acceptable level of statistical confidence is reached to accept or reject the presence of a particular dye combination and / or affinity reagent and / or target molecule and / or analyte of interest. Importantly, while from a strictly mathematical point of view each iteration of this iterative process analyzes exactly the same read volume, particularly preferred embodiments of the present invention can tolerate small deviations in the spatial and / or temporal position of the read volume between the first and second read sequences (e.g., fractions of 1 / 10000, 1 / 1000, 1 / 100, 1 / 10, 1 / 4, 1 / 2 of the time it takes a sample to traverse the lateral extent of the effective PSF). In this case, according to the Bayesian theorem, the first readout sequence generates prior knowledge about the second readout sequence in the sense of Bayesian probability, which is similar to the pre-test probability in diagnostic tests, where symptomatic patients generally have a much lower false positive rate than asymptomatic patients. In a similar manner, if the affinity reagent a is detected in the first readout volume t , this will affect the probability of being detected in the overlapping second readout volume, where the overlapping area can be understood as spatial or temporal.

[0057] Preferably, a "code" in the sense of this document can be, for example, a linear code (e.g., a binary code), a fixed-length code, a variable-length code, or an error-correcting code. In a particularly preferred embodiment, the codes are "independent and identically distributed." In a particularly preferred embodiment of the present invention, a binary code is used.

[0058] "Set of sets of dye combinations that can be grouped under a readout sequence": In the context of this document, a "set of sets of dye combinations that can be grouped under a readout sequence" refers to a set that includes all dye combinations among the plurality of dye combinations that can be grouped under a particular readout sequence. A "set of dye combinations that can be grouped under a readout sequence" that can be grouped under a readout sequence includes multiple elements.

[0059] "Assignment rate": This is the proportion of unique codes (also called dye combinations) in the set of unique codes (also called multiple dye combinations (S1)) that are actually assigned to markers and is denoted as α. Summary of the Invention

[0060] It is therefore an object of the present invention to provide a method and a device for analyzing a sample, preferably a biological sample, which allows the analysis of a very large number of markers in a very short time.

[0061] The above objects are achieved by the subject-matter of the independent claims. Advantageous embodiments are defined in the independent claims and in the following description.

[0062] According to the present invention, there is provided a method for analyzing a sample, the sample comprising: a plurality of affinity reagents, each affinity reagent configured to attach to an analyte, at least one of the affinity reagents being attached to the analyte; and a first plurality of dye combinations, each dye combination within the first plurality of dye combinations being unique and each dye combination comprising at least two dyes having different properties with respect to at least one of excitation and emission, wherein at least one of the unique dye combinations is attached to a bound affinity reagent according to a first mapping, wherein at least another of said unique dye combinations is attached, preferably randomly attached, to said bound affinity reagent according to a second mapping, wherein the first mapping is different from the second mapping; For the first mapping and the second mapping, the method includes, in a chronological manner: i) directing excitation light toward the sample, the excitation light having characteristics for exciting at least the at least two dyes, the at least two dyes having different characteristics with respect to at least one of excitation and emission; and ii) generating at least one first readout of emission light emitted by the excited dye.

[0063] According to the present invention, there is provided a method for analyzing a sample, the sample comprising: a plurality of affinity reagents, each affinity reagent configured to attach to an analyte, at least one of the affinity reagents being attached to the analyte; and a first plurality of dye combinations, each dye combination within the first plurality of dye combinations being unique and each dye combination comprising at least two dyes having different properties with respect to at least one of excitation and emission, a second plurality of dye combinations, each dye combination within the second plurality of dye combinations being unique and each dye combination comprising at least two dyes having different properties with respect to at least one of excitation and emission; wherein the second plurality of dye combinations is different from the first plurality of dye combinations; wherein at least one of said unique dye combinations of said first plurality of dye combinations, in particular a first subset of dye combinations, is attached to a bound affinity reagent according to a first mapping, wherein at least one of said unique dye combinations of said second plurality of dye combinations, in particular a second subset of dye combinations, is attached, preferably randomly attached, to said bound affinity reagent according to a second mapping, wherein the first mapping is different from the second mapping; For the first mapping and the second mapping, the method includes, in a chronological manner: i) directing excitation light toward the sample, the excitation light having characteristics for exciting at least two dyes, the at least two dyes having different characteristics for at least one of excitation and emission; ii) generating at least one first readout of emission light emitted by the excited dye.

[0064] Preferably, each unique dye combination in the first plurality of dye combinations is attached to only one affinity reagent, such that no unique dye combination is bound to more than one affinity reagent in the first mapping.

[0065] Preferably, generating at least one first readout comprises: Emission light emitted by the excited dye is separated into detection channels corresponding to the emission characteristics of the dye.

[0066] Each dye combination can be selected so that each detection channel includes one dye.

[0067] Preferably, the at least two dyes have different excitation characteristics, and wherein excitation light having each excitation characteristic is directed to the sample at different times or simultaneously.

[0068] The method may further comprise: providing the plurality of affinity reagents; and The first plurality of dye combinations is provided.

[0069] The method may further comprise: providing said sample; and 1) attaching the plurality of affinity reagents to the first plurality of dye combinations to form a plurality of markers; and introducing the plurality of markers into the sample to allow attachment to analytes in the sample; or II) introducing the plurality of affinity reagents into the sample to allow attachment to analytes in the sample; and The plurality of affinity reagents are attached to the first plurality of dye combinations to form a plurality of markers attached to the analyte.

[0070] Preferably, attaching the plurality of affinity reagents to the first plurality of dye combinations comprises: providing a plurality of linkers, each linker comprising a plurality of binding sites, each configured to bind a dye; and For each affinity reagent, a linker is attached to the affinity reagent, and one of the dye combinations is bound to the linker, with each dye in the combination being bound to a binding site.

[0071] The method may further comprise at least one of the following: deactivating at least one of the dyes in the first plurality of dyes; removing the attachment between at least one affinity reagent and at least one of the dye combinations; removing the attachment between at least one affinity reagent and at least one of the analytes; and waiting for a time longer than a fluorescence lifetime of at least one of the dyes in the first plurality of dye combinations; and Repeat steps i) to iii) of claim 1 for different second plurality of dye combinations or for the first plurality of dye combinations according to different second mappings.

[0072] The method may further comprise: Based on the at least one first readout, at least one dye of the second plurality of dye combinations and / or a combination of dyes and / or a rule of the second mapping is suggested by a computer processor.

[0073] The method may further comprise iteratively repeating the above steps for at least one of a series of multiple dye combinations and a series of mappings until all affinity agents attached to analytes in the sample are determined.

[0074] Preferably, stochastic labeling is achieved by at least one of DNA-PAINT, DNA-Exchange, Exchange-PAINT, and by using an oligonucleotide barcode attached to an affinity reagent and a complementary attachment oligonucleotide moiety attached to a dye combination configured to allow dynamic binding of the dye combination to its assigned affinity reagent.

[0075] Stochastic labeling can be achieved by at least one of DNA-PAINT, DNA-Exchange, Exchange-PAINT, and by using an oligonucleotide barcode attached to an affinity reagent and a complementary attachment oligonucleotide moiety attached to a dye combination configured to allow repeated association and dissociation of the dye combination from its assigned affinity reagent by at least one of: changes in the concentration of host molecules; changes in temperature; changes in lighting conditions; a change in the concentration of at least one capture strand; and Changes in enzyme concentration and / or activity.

[0076] Preferably, determining, by at least one computer processor, the presence of at least one affinity reagent in the sample comprises: comparing at least two reads selected from the group consisting of: the at least one first read, the at least one second read, and any other reads generated in step ii); and The presence of at least one affinity agent is determined based at least in part on the comparison.

[0077] Preferably, determining the presence of the at least one affinity agent is based on at least one measure of statistical confidence.

[0078] Preferably, the characteristic of the dye with respect to at least one of excitation and emission comprises at least one of: excitation wavelength; emission wavelength; fluorescence intensity; and fluorescence lifetime.

[0079] Preferably, determining, by at least one computer processor, the presence of at least one affinity reagent in the sample based on the readout comprises: converting the readout into a well-determined or overdetermined system of linear equations; and Solve the system of linear equations.

[0080] A device for analyzing a sample may be configured to perform the above method.

[0081] Linkers configured to bind to affinity reagents may include: at least one binding site configured to bind to at least one dye; and An oligonucleotide attachment moiety configured to bind to the oligonucleotide barcode of the affinity reagent.

[0082] The linker is configured to couple to the affinity reagent described above.

[0083] The connector may also include: A plurality of binding sites, wherein at least two of the binding sites are configured to bind to dyes having different properties with respect to at least one of excitation and emission.

[0084] Reports may include: a linker as described above; and At least one dye bound to the at least one binding site of the linker.

[0085] Preferably, the linker may comprise a combination of dyes, each dye being bound to one of the plurality of binding sites, wherein at least two of the dyes have different properties with respect to at least one of excitation and emission.

[0086] Markers may include: an affinity reagent configured to attach to the analyte; and The reporter described above is attached to the affinity reagent.

[0087] Preferably, the oligonucleotide attachment portion of the adaptor may be fully complementary or partially complementary to the oligonucleotide barcode of the affinity reagent.

[0088] Preferably, the oligonucleotide attachment portion of the linker is configured to have affinity for the oligonucleotide barcode of the affinity reagent, e.g., to transiently bind to the oligonucleotide barcode before dissociating from the oligonucleotide barcode again under at least one condition, wherein the at least one condition provides for separation of the oligonucleotide attachment portion depending on at least one of host-guest mediated ligand invasion, strand displacement, and photochemical control of hybridization.

[0089] According to specific embodiments, one of the oligonucleotide attachment portion of the linker and the oligonucleotide barcode of the affinity reagent includes at least one guest molecule, which is attached to its nucleobase and is configured to complex with a host molecule; and the other of the oligonucleotide attachment portion of the linker and the oligonucleotide barcode of the affinity reagent includes at least one nucleobase that is complementary to the nucleobase to which the guest molecule is attached.

[0090] Preferably, the host molecule may be a cucurbituril, such as CB[7].

[0091] According to another embodiment, the marker may comprise at least one base mismatch between the oligonucleotide attachment portion of the linker and the oligonucleotide barcode of the affinity reagent, thereby preventing stable binding therebetween, wherein the base mismatch is configured to be bridged by photoconversion.

[0092] For example, the base mismatch is a GG mismatch, which is configured to be bridged by photoconverting trans-NCDA to cis-NCDA.

[0093] According to a preferred embodiment, the affinity reagent is an oligonucleotide probe configured to bind to a nucleic acid analyte.

[0094] A plurality of markers as described above may be provided, wherein each reporter comprises a unique dye combination, and wherein each reporter is attached to an affinity reagent configured for attachment to an analyte such that no unique dye combination is bound to more than one affinity reagent.

[0095] A solution may include a linker as described above, a reporter as described above, a marker as described above, or a plurality of markers as described above.

[0096] A lyophilized solid can include a linker as described above, a reporter as described above, a marker as described above, or a plurality of markers as described above.

[0097] A computer program is provided, which has a program code and, when the computer program is run on a processor, executes the above method.

[0098] A computer-readable storage medium storing the above-mentioned computer program is provided.

[0099] A database is provided, the database including information corresponding to: Various affinity reagents; a first plurality of dye combinations; and First mapping, and optionally further comprising information corresponding to at least one of: a second plurality of dye combinations and / or any other plurality of dye combinations; each dye combination having different characteristics for at least one of excitation and emission of the dye; a second map or any other map; at least one first readout; at least one second readout and / or any other readout; The database is used to perform at least one of steps i) to ii) of the above method.

[0100] Additionally, a method for analyzing a sample may be provided, the sample comprising: a plurality of affinity reagents, each affinity reagent configured to bind to an analyte, at least one of the affinity reagents being bound to the analyte; and a first plurality of dye combinations, each dye combination in the first plurality of dye combinations being unique and each dye combination comprising at least two dyes having different properties with respect to at least one of excitation and emission, wherein each of the unique dye combinations is bound to a bound affinity reagent according to a first mapping, the method comprising: i) directing excitation light toward the sample, the excitation light having characteristics for exciting at least two dyes, the at least two dyes having different characteristics for at least one of excitation and emission; ii) generating at least a first readout of emission light emitted by the excited dye; and iii) determining, by at least one computer processor, the presence of at least one affinity reagent in the sample based on the at least one first readout.

[0101] The method may be a computer-implemented method.

[0102] In this manner, the method provides an improved method for detecting the presence of an analyte in a sample. Specifically, by directing excitation light having properties for exciting dyes having different excitation and / or emission properties, the resulting readouts can contain information that allows each readout to determine a greater number of analytes than can be determined using known methods, as will be described in greater detail herein.

[0103] The method can also be defined as follows: each unique dye combination in the first plurality of dye combinations is attached to only one affinity reagent, such that no unique dye combination is bound to more than one affinity reagent in the first mapping. In this manner, detection of a unique dye combination in the readout can be reliably used to determine the presence of an analyte in the sample. It can also be said that mapping the plurality of dye combinations to affinity reagents is at least single-shot, and preferably double-shot.

[0104] Generating at least one first readout may further comprise separating the emission light emitted by the excited dye into detection channels, wherein each detection channel substantially corresponds to one of the plurality of dyes, or wherein the detection channel corresponds to one of the plurality of dyes. Each dye combination may be selected such that each detection channel includes one dye.

[0105] In this way, a greater number of dyes can be read on a readout device, as will be described in more detail herein.

[0106] The at least two dyes can have different excitation characteristics, and the excitation light with each excitation characteristic is directed to the sample at different times. In this way, the information available to the readout device can be increased, and more dye combinations can be unique. For example, if light with two excitation characteristics is directed to the sample at the same time, dyes with different excitation characteristics and the same emission characteristics may be more difficult to distinguish. By separating the excitation light and paying attention to when light with a common emission characteristic is emitted, the dyes can be more easily distinguished. This can result in a greater number of viable dye combinations.

[0107] The at least two dyes may have different excitation properties, and excitation light having each excitation property is directed to the sample simultaneously. In this way, the method is more efficient in terms of both computational and run time. For smaller amounts of analytes in the sample, i.e., when fewer unique dye combinations are required, all dyes may be excited simultaneously.

[0108] The method may further include providing the plurality of affinity reagents; and providing the first plurality of dye combinations.

[0109] The method may further comprise: providing the sample; and 1) attaching the plurality of affinity reagents to the first plurality of dye combinations to form a plurality of markers; and introducing the plurality of markers into the sample to allow attachment to analytes in the sample; or II) introducing the plurality of affinity reagents into the sample to allow attachment to analytes in the sample; and The plurality of affinity reagents are attached to the first plurality of dye combinations to form a plurality of markers attached to the analyte.

[0110] In this way, it is possible to control how the attachment between the affinity reagent and the analyte and the attachment between the dye combination or reporter and the affinity reagent is achieved. Depending on the affinity reagent, analyte and dye and other variables, it may be advantageous to form the marker prior to attachment to the analyte. In other cases, it may be advantageous to attach the affinity reagent to the analyte and then add the dye to form the marker "in situ" in the sample.

[0111] Attaching the plurality of affinity reagents to the first plurality of dye combinations may comprise: providing a plurality of linkers, each linker comprising a plurality of binding sites, each configured to bind a dye; and For each affinity reagent, a linker is attached to the affinity reagent, and one of the dye combinations is bound to the linker, with each dye in the combination being bound to a binding site.

[0112] The method may further comprise at least one of the following: deactivating at least one of the dyes in the first plurality of dyes; removing the attachment between at least one affinity reagent and at least one of the dye combinations; removing the attachment between at least one affinity reagent and at least one of the analytes; and waiting for a time longer than a fluorescence lifetime of at least one of the dyes in the first plurality of dye combinations; and For different second plurality of dye combinations or for the first plurality of dye combinations, the above steps i) to iii) are repeated according to different second mappings.

[0113] In this way, more information related to the same sample can be generated. A single dye combination and / or a single mapping may not be able to produce a readout that can be used to determine any or all analytes. Therefore, it is desirable to obtain more information related to the original sample. Therefore, the method can inactivate at least one of the dyes or allow at least one of the dyes to be inactivated or attached so that when steps i) to iii) are run again, the (second) readout generated will be different from the original (first) readout. Then, the second readout with new information relative to the first readout can be used to determine other analytes.

[0114] The method may further comprise: suggesting, by a computer processor, at least one dye of the second plurality of dye combinations and / or a dye combination and / or a rule for the second mapping based on the at least one first readout. In this way, the number of iterations may be advantageously reduced, thereby optimizing the process.

[0115] The method may further comprise iteratively repeating the steps of deactivating, de-attaching, waiting, and making recommendations for a series of multiple dye combinations and a series of mappings until all affinity reagents attached to analytes in the sample are determined. In this manner, the method can determine all analytes present in the sample.

[0116] Determining, by at least one computer processor, the presence of at least one affinity reagent in the sample may comprise: comparing at least two reads selected from the group consisting of: the at least one first read, the at least one second read, and any other reads generated in step ii); and The presence of at least one affinity agent is determined based at least in part on the comparison.

[0117] In this way, the method can analyze differences between the reads and known differences between the mapping and / or dye combinations to assist in determining the analyte in the sample.

[0118] Determine that the existence of at least one affinity reagent can be based on the measured value of at least one statistical confidence.The measured value of statistical confidence can be used in any number of (including only one) stages of the method.For example, the measured value of statistical confidence can be used to determine whether dye has been excited, then the same or different measured values ​​can be used to determine whether a particular dye or dye combination exists, and the same or different measured values ​​can be used based on the existence of determined dye combination to determine the existence of analyte.This paper describes this "existence call" process and some exemplary probability thresholds in more detail.

[0119] The characteristics of the dye for at least one of excitation and emission may include at least one of: excitation wavelength; emission wavelength; fluorescence intensity; and fluorescence lifetime. In this way, the readout can be efficiently retrieved and analyzed, and the dyes can be distinguished from each other.

[0120] Determining, by at least one computer processor, the presence of at least one affinity reagent in the sample based on the readout may comprise: converting the readout into a well-determined or overdetermined system of linear equations; and Solve the system of linear equations.

[0121] In this way, the method provides a computationally efficient way to "decode" the information in each readout so that the presence of analyte in the sample can be determined.

[0122] According to the present invention, there is provided a device for analyzing a sample, said device being configured to perform a method according to the present invention.

[0123] According to the present invention, there is provided a linker configured to be coupled to an affinity reagent, the linker comprising: A plurality of binding sites, wherein at least two of the binding sites are configured to bind to dyes having different properties with respect to at least one of excitation and emission.

[0124] Preferably, the adaptor is configured to perform a method according to the invention or a method described herein.

[0125] According to the present invention, a reporter is provided, comprising: A joint according to the present invention; and A combination of dyes, each dye being bound to one of the plurality of binding sites, wherein at least two of the dyes have different properties with respect to at least one of excitation and emission.

[0126] According to the present invention, a marker is provided, comprising: an affinity reagent configured to attach to the analyte; and According to the present invention, the reporter is attached to the affinity reagent.

[0127] According to the present invention, a plurality of markers according to the present invention are provided, wherein each reporter comprises a unique dye combination, and wherein each reporter is attached to an affinity reagent configured for attachment to an analyte such that no unique dye combination is bound to more than one affinity reagent.

[0128] In these ways, the present invention provides building blocks for implementing the methods of the present invention.The linkers, reporters, markers and multiple markers described above allow for advantageous effects including the determination of a greater number of analytes per readout, as described in the methods.

[0129] According to the present invention, a solution is provided, comprising at least one of a dye combination according to the present invention, a linker according to the present invention, a reporter according to the present invention, a marker according to the present invention, and a plurality of markers according to the present invention. The solution comprising the linker, the reporter, the marker, or the plurality of markers can be prepared by any suitable method known to those skilled in the art. By way of example only, the solution can comprise water and / or saline (e.g., phosphate buffered saline), and in alternative formulations can comprise other minerals.

[0130] According to the present invention, a lyophilized solid is provided, comprising at least one of a dye combination according to the present invention, a linker according to the present invention, a reporter according to the present invention, a marker according to the present invention, and a plurality of markers according to the present invention. The lyophilized solid comprising the linker, reporter, marker, or plurality of markers can be prepared by any suitable method known to those skilled in the art. By way of example only, the lyophilized solid can be prepared by a freeze and dry process, optionally under vacuum.

[0131] According to the invention, a computer program is provided with a program code for performing the method according to the invention when the computer program is run on a processor.

[0132] According to the present invention, there is provided a computer-readable storage medium for storing the computer program according to the present invention.

[0133] According to the present invention, there is provided a database comprising information corresponding to: Various affinity reagents; a first plurality of dye combinations; and First mapping, and optionally further comprising information corresponding to at least one of: a second plurality of dye combinations and / or any other plurality of dye combinations; each dye combination having a characteristic with respect to at least one of excitation and emission of said dyes; a second map or any other map; at least one first readout; at least one second readout and / or any other readout; The database is used to perform at least one of the above steps i) to iii).

[0134] In this manner, the present invention can efficiently record trajectory-related information, which allows for rapid retrieval and storage, requiring only a minimal amount of memory on the storage device.

[0135] In other exemplary embodiments, a method for analyzing a biological sample comprises the following steps: a) providing a plurality of affinity reagents (S2), wherein each affinity reagent (a1, a2, a3...a n ) is configured to specifically bind to a predetermined target structure within a biological sample or to a predetermined chemical compound or to a predetermined chemical element; b) providing a plurality of dye combinations (S1) [from dyes having y σ A variety of dyes (Y D )], each dye combination (s1, s2, s3 ... s n ) are unique, each dye combination (s1, s2, s3...s n ) comprises at least two different dyes (|s|>=2); c) wherein the composition of the plurality of dye combinations (S1) is such that each dye (y1, y2, y3 ... y σ ) can be read out by a readout device; wherein the dye can be separated into multiple channels by a readout device; each channel corresponds to the dye (y1, y2, y3 ... y σ ); this means that each dye can be distinguished from other dyes in the same dye combination by the readout used for data acquisition based on any or a combination of the following properties: excitation fingerprint / spectrum, emission spectrum, intensity, fluorescence lifetime, fluorescence anisotropy, and an affinity reagent unique to the marker, the affinity reagent being configured to attach to a predetermined structure within the sample. In a particularly preferred embodiment of the present invention, the method disclosed in PCT / EP2021 / 063310, which we refer to as "IHP" (Iterative Hyperplexing), is used to substantially increase the total number of dyes y σ , these dyes can be reliably distinguished from each other and represented in different channels by appropriately configured readout devices. This is particularly advantageous because even if y σ A small increase in σSTD = 5 (standard widefield fluorescence microscope) increased to y using "IHP" σIHP =25) also results in a significantly higher cardinality of multiple dye combinations (S1) that can be generated using different functions or algorithms to construct dye combinations. The significant increase in the cardinality of multiple dye combinations (S1) leads to an exponential increase in the statistical power of the method and lays the foundation for decoding a very large number of dye combinations present in the read volume with high statistical confidence in the decoding result, i.e., presence call. d) Preferably, at least some of the plurality of affinity reagents (A) are introduced into the sample; the plurality of affinity reagents can be introduced en bloc or in several iterative steps. In the latter case, a priori knowledge can be used to define a subset of the plurality of affinity reagents to optimize the conditions for presence calling. e) before or after step d), each of the plurality of affinity reagents (S2) is preferably assigned to at least one of the plurality of dye combinations (S1); both the virtual assignment of markers (virtual markers) and the physical constitution (physical markers) (i.e., the physical assembly of the connection between the affinity reagent and the dye combination to which it is assigned) can be performed before the affinity reagent is introduced into the sample. In a particularly preferred embodiment of the present invention, the affinity reagent is introduced into the sample and attached to its predetermined target structure before the connection between the affinity reagent and the assigned dye combination is established. In another particularly preferred embodiment of the present invention, in an iterative process (repeating steps d)-f) at least twice), the connection between the affinity reagent and the dye combination assigned in the first round is severed so that a new connection is established between the affinity reagent and the second dye combination assigned in the second round. This can be called "code swapping" or introducing other mappings or changing the encoding of affinity reagents and / or markers, which is an effective strategy for decoding readout volume even if the sample contains a very large number of different dye combinations and bound affinity reagents. f) Preferably, excitation light having corresponding specific characteristics for exciting each dye in the plurality of dye combinations is directed to the sample to excite the corresponding dye. g) generating at least one readout from emission light emitted by an excited dye located in a readout volume of said sample, preferably by means of a detection channel for at least one readout volume; and h) Preferably, determining which affinity reagents are present in said readout volume based on said at least one readout.

[0136] In a particularly preferred embodiment of the invention, the presence or absence of an affinity reagent in the read volume is determined based on a measure of statistical confidence and a certain statistical confidence level.

[0137] The measure of statistical confidence is calculated for every kind of mark and / or affinity reagent and / or dye combination and / or predetermined target molecule.This can be the measure of the combination being made up of the measure of multiple statistical confidences, for assessing related aspects.The measure of statistical confidence can be in conjunction with prior knowledge, and use Bayes theorem, so that for example, based on the prior knowledge about given mark and / or affinity reagent and / or dye combination and / or predetermined target molecule, adjust the probability of observing this mark and / or affinity reagent and / or dye combination and / or predetermined target molecule.This prior knowledge can be generated before experiment or during experiment.This means, for example, for every kind of mark and / or affinity reagent and / or dye combination and / or predetermined target molecule, calculate p value, described p value assessment observes the probability of detected existence (qualitative decoding) and / or quantity (relative or absolute quantitative decoding) when null hypothesis is true (that is, corresponding mark and / or affinity reagent and / or dye combination and / or predetermined target molecule is not actually present in the read volume). The presence call, i.e. the user's acceptance of the decision that a given marker and / or affinity reagent and / or dye combination and / or predetermined target molecule is present in the read volume, is based on reaching a sufficient level of statistical confidence. The decision can be made automatically using thresholds, which can be fixed and the same for all markers, affinity reagents, dye combinations and target molecules, or they can be different thresholds, which can be based on prior knowledge. Other thresholds can be dynamically adjusted throughout the experiment. For example, they can be made more stringent or less stringent. This is advantageous because it allows a higher level of statistical confidence to be required for a target molecule of particular interest.

[0138] The present invention is based on viewing the "observation" of microscopy as an encoding / decoding problem rather than as a problem of recording spatial position intensities in an image (which is essentially a matrix of intensity values). Although the methods described in the present invention are compatible with image-based readout, the "images" generated by the methods and devices described in the present invention should be viewed as probabilistic mathematical models of the authenticity of the sample under investigation, where the presence of a target molecule is detected or called (presence call) based on the user's decision to accept its presence based on a measure of statistical confidence and a certain level of statistical confidence in the presence of the corresponding target molecule in the readout volume.

[0139] The step of accepting the presence of a specific marker, a specific affinity reagent, and a specific target molecule (i.e., an existence call) yields a mathematical truth based on a measure of statistical confidence and a certain level of statistical confidence. This is an important aspect of the present invention because it also means that according to the existence call, one operates in the axiomatic realm of mathematics, which is inherently unaffected by the measurement complications in non-mathematical fields (i.e., physics, chemistry, biology). Therefore, this fact is of great significance and also shows that the method can realize a completely new microscopy paradigm.

[0140] The statistical method that provides a measure of statistical confidence on a per-marker or per-target basis is likely to be a combined measure and in many ways may be similar or identical to the methods used in transcriptomics and genomics, where enrichment scores and p-values ​​are commonly used.

[0141] The present invention relates to a patent application entitled “Method and device for analyzing a biological sample” with application number PCT / EP2021 / 063310, which utilizes the ability of the “IHP method” to image or read out multiple dyes, imaging or reading out a large number of dyes in a single round. Unlike the method disclosed in the patent application entitled “Method and device for analyzing a biological sample” with application number PCT / EP2021 / 063310 (wherein there is a 1: 1 relationship between a given dye and a given affinity reagent, so that each marker is unique in one round), here we disclose a method in which this principle is combined with a combinatorial code so that there is a 1-to-many relationship between markers and dyes. The contents of PCT / EP2021 / 063310 are fully included in this article by reference.

[0142] In a particularly preferred embodiment of the method, a given affinity reagent (such as an antibody, single domain antibody, oligonucleotide probe, aptamer or toxin) can be assigned to a reporter comprising a unique combination of dyes and linkers, forming a virtual marker, i.e., wherein the affinity reagent (a i ) and the dye combination, either a doublet or a singlet binding, corresponds to one marker (μ) among the multiple markers (M). i ).

[0143] Furthermore, in this embodiment, the dyes among the plurality of dyes can be assigned to a plurality of groups of dyes A to n (n is 0 or a natural number element), wherein each group of dyes A to n includes y A to y n dyes, making multiple dyes (P D ) contains y A +y B +y C +…y n = y σ wherein the dyes assigned to one of the plurality of groups of dyes A to n can be excited with the same excitation light, for example with different corresponding wavelengths λ1 to λ nThe method is characterized in that at least all dyes in each group of dyes A to n can be separated into channels by a readout device, wherein each dye is read out in an individual channel, and each channel corresponds to one of the dyes; in this way, the number of dyes that can be reliably read out and separated is maximized using dyes and a readout device based on the "IHP method", i.e., increasing y σ , thereby increasing the base number of multiple dye combinations (S1). In this case, a unique dye combination (s i ) is assigned to the affinity reagent (a i In the next step, at least some of the plurality of affinity reagents (S2) are introduced into the sample. Excitation light is then directed to the sample to excite the fluorescent dyes of the markers (μ1, μ2, μ3, ..., μ n ), which means that all dyes in the plurality of dye combinations (S1) are excited at least once. After dye excitation, at least one readout (preferably a complete readout) is generated from the fluorescence emitted by the excited dye located in the sample readout volume, said readout comprising at least two channels, one channel corresponding to one of the dyes. In other words, in the method described in this document, each dye is read out in an individual channel. In a next step, based on the at least one readout sequence obtained in step (d), the presence of markers in the readout volume is determined, which can be made by relying on a measure of statistical confidence and achieving a certain statistical confidence level ("presence call").

[0144] In a particularly preferred embodiment of the method, at least one code C is used. α1 to C αn and / or at least one password X α1 To X αn , uniquely mapping multiple dye combinations (S1) to multiple affinity reagents (A = S2 = T1 * ), where C:S->T* or X:S->T* is a total function, which is preferably bijective or at least injective, where S is the "source alphabet" and T is the "target alphabet", and where S and T are finite sets (case α).

[0145] In a particularly preferred embodiment of the method, at least one code C is used. β1 to C βn and / or at least one password X β1 To X βn , multiple affinity reagents (A=S2=T1 *) uniquely maps to multiple dye combinations S1, where C:S->T* or X:S->T* is a total function, which is preferably bijective or at least injective, where S2 is the "source alphabet" and T1 is the "target alphabet", and where S2 and T1 are finite sets [case β].

[0146] In both cases α and β, as long as the codes C1, C2, C3, ..., C for encoding / decoding and / or encryption / decryption are n and / or password X1, X2, X3...X n The method disclosed in this article can be performed if it is a single-shot or bijective total function. The following preferred embodiment is proposed as an example, which uses a specific code C i, , but this does not mean that the method is limited to any specific code, because all codes C1, C2, C3...C n and / or password X1, X2, X3...X n (which is a total function and is injective or bijective) can be used for encoding / decoding and / or encryption / decryption to perform the methods disclosed herein.

[0147] Since the dye combination contains at least two dyes, there may be a 1:n relationship between a given affinity reagent and the dye to which it is assigned or attached. Importantly, in this case, n defines the number of different dye species used, not the number of dye molecules.

[0148] In a particularly preferred embodiment of the present invention, a dye combination is created by randomly selecting a dye from the group A to n dyes, where y A +y B +y C +…y n = y σ For example, if the method disclosed in the "IHP method" is used and each group A to n includes y A to y n markers, and n different excitation lights are used to generate n different readouts, then the number of unique markers that can be read out and distinguished from each other is y A +y B +y C +…y n For example, if the method disclosed in this document is used, and each marker is labeled with n dyes, and n different excitation lights are used to generate n different images, the number of unique markers that can be read out and distinguished from each other is y A xy B xy C …xy n In other words, this yields y A xyB xy C ...xy n We refer to this particularly preferred embodiment as "group-based encoding."

[0149] In a particularly preferred embodiment of the present invention, the plurality of dyes (Y) formed by all fluorescent dyes in the plurality of dye combinations (S1) D ) includes at least 10, 20, 50, 100, 1000 or 10,000 different fluorescent dyes.

[0150] In another particularly preferred embodiment of the method, a given affinity reagent (such as an antibody, single domain antibody, oligonucleotide probe, aptamer or toxin) is linked to a set of at most nxy tags, such that there is a variable and random relationship between the given affinity reagent and the dye to which it is attached. This results in a binary code, where the absence of a particular dye in the code is recorded as "0" and its presence as "1", with a maximum of nxy numbers, generating a set of 2 (y A +y B +y C ...+y n ) or 2 y σ We refer to this particularly preferred embodiment as "binary encoding".

[0151] Thus, the method described in this document greatly increases the number of markers and / or dye combinations and / or affinity reagents and / or predetermined target structures that can be read out without the need to remove or inactivate previous markers and without the need for additional staining.

[0152] Importantly, there are many other codes or ciphers, i.e., ways of encoding / encrypting and decoding / decrypting, that can be used in conjunction with the methods disclosed herein.

[0153] Each affinity reagent targets its dye combination to a predetermined structure (also referred to as a target molecule or analyte), such as a specific biomolecule, in a biological sample or lysate.

[0154] Because both methods employ combinatorial encoding, the number of dye combinations that can be considered unique codes increases exponentially and quickly exceeds the number of protein-coding genes (which is in the range of 20,000). This is an important reference value because proteins are responsible for most biological functions and are therefore of great interest. For this reason, the majority of fluorescence microscopy methods used today analyze protein targets.

[0155] In both embodiments, the methods are compatible with various amplification methods, including multiple binding site amplification or enzymatic reaction-based amplification strategies, such as rolling circle DNA amplification.

[0156] In both embodiments, the method is compatible with various analyte classes, since the affinity reagent can be, for example, an antibody (protein target) or an oligonucleotide (RNA / DNA target).

[0157] In a particularly preferred embodiment of the present invention, each marker (μ i ) comprises a linker having at least two different attachment sites, the combination of attachment sites being unique to the marker; and wherein each dye is linked to a complementary linker to form a reporter, the complementary linker being unique to the dye and being configured to attach to a predetermined attachment site.

[0158] In a particularly preferred embodiment of the present invention, the linker and / or complementary linker is an oligonucleotide, including DNA, RNA, peptide nucleic acid, morpholino or locked nucleic acid, glycol nucleic acid, threose nucleic acid, hexitol nucleic acid or other forms of artificial nucleic acid.

[0159] In a particularly preferred embodiment of the invention, the linker and / or the complementary linker comprises a site for enzymatic cleavage or photolysis. This allows efficient and particularly easy release of the first dye combination so that it is inactivated and can subsequently be relabeled with the second dye combination.

[0160] In a preferred embodiment of the present invention, the reporter is attached to its corresponding attachment site prior to introducing the marker into the sample.

[0161] In a particularly preferred embodiment of the present invention, at least two readouts are generated; and between the generation of the first and second readout signals, the reporter is dynamically bound to and / or dissociated from its corresponding attachment site to achieve stochastic labeling. This is a strategy for improving spatial resolution and simplifying decoding of multi-species readout volumes. Such stochastic labeling can be based on ultra-high resolution microscopy (such as STORM, PALM, GSDIM) or related methods utilizing scintillation.

[0162] In a particularly preferred embodiment of the present invention, random labeling is achieved by combining the method with DNA-PAINT.

[0163] In a particularly preferred embodiment of the present invention, the plurality of dyes formed by all fluorescent dyes of the marker is divided into groups having y A to y n A dye group A to n of members, where y A +yB +y C +……y n =y σ , where y is a natural number, and y σ For a variety of dyes (P D ) in a total number of dyes in the same group; wherein each dye in the same group can be excited by light of substantially one wavelength spectrum or by light of the same wavelength spectrum; wherein at least one excitation light for each group of dyes is directed to the sample so as to excite the fluorescent dyes of the corresponding group; wherein at least one readout of each group of dyes is generated by fluorescence emitted by the excited dyes located in the sample readout volume, the readout comprising at least two channels, each channel corresponding to one of the dyes in the corresponding group. This embodiment employs an "IHP approach" which substantially allows a greater number of dyes to be read out on a readout device. This is particularly advantageous because a higher y σ This results in a higher cardinality of multiple dye combinations (S1), leading to a lower partitioning rate α and higher statistical power.

[0164] In a particularly preferred embodiment of the present invention, the excitation light for exciting the dye groups A to n is directed onto the sample one by one in temporal sequence.

[0165] In a particularly preferred embodiment of the invention, the readout is an image or a microscopic image or a readout image data stream of the readout volume.

[0166] In a particularly preferred embodiment of the invention, the readout is or comprises a hyperspectral image of the sample. This is particularly advantageous because it allows the use of a high dye population y σ And a higher cardinality of multiple dye combinations (S1) is obtained, and thus a lower partitioning rate α and a higher statistical power of the method are obtained.

[0167] In a particularly preferred embodiment of the present invention, the method further comprises the step of stabilizing the fluorescence lifetime of at least one fluorescent dye. This can be achieved by placing the fluorescent dye in a shielded environment by at least one of encapsulation, embedding in a polymer matrix, and co-crystallization. In this regard, SMILE is a particularly advantageous class of dyes. In a particularly preferred embodiment of the present invention, the plurality of dyes (P D ) wherein at least one dye is SMILE.

[0168] In a particularly preferred embodiment of the present invention, the step of generating the channels is based on at least one of channel unmixing, spectral unmixing, excitation spectral imaging, spectral phasor analysis, spectral fluorescence lifetime imaging (FLIM) phasor, fluorescence lifetime of fluorescent dyes, and excitation fingerprinting of fluorescent dyes. This is particularly advantageous because it allows the use of a high number of dyes y σAnd a higher cardinality of multiple dye combinations (S1) is obtained, and thus a lower partitioning rate α and a higher statistical power of the method are obtained.

[0169] In a particularly preferred embodiment of the present invention, the step of generating the channel is based on at least two orthogonal contrasts. When orthogonal contrasts are obtained from these methods and used in combination, they can be used to significantly increase the total number of dyes y σ , that is, separating a larger number of dyes. For example, the excitation fingerprint information can be combined with the fluorescence emission spectrum information and / or the fluorescence lifetime information with any one or both of the above.

[0170] In a particularly preferred embodiment of the present invention, the step of generating said pathway is based on at least one of machine learning, deep learning or artificial intelligence.

[0171] In a particularly preferred embodiment of the present invention, the following steps are repeated at least twice to create a series of images or readouts of the sample: providing a second plurality of markers, introducing the second plurality of markers into the sample, directing at least one excitation light onto the sample, generating at least one readout, and determining the markers present in the readout volume; or wherein steps a) to e) of the above method are repeated at least twice.

[0172] In particularly preferred embodiments of the invention, said reporter labelling said second plurality of markers comprises a combination of dyes determined based on a first series of images or readouts of said sample.

[0173] In particularly preferred embodiments of the invention, the reporter is assembled by adding a dye mixture, wherein each dye is linked to a complementary linker to form a reporter having a linker molecule comprising dye-specific attachment sites for all dyes in the plurality of dyes, such that addition of a dye mixture corresponding to a unique combination of dyes to the linker molecule in the coupling reaction volume results in stoichiometric coupling.

[0174] In a particularly preferred embodiment of the invention, the reporter is assembled by adding a dye mixture, wherein each dye is linked to a complementary linker to form a reporter having a linker molecule comprising dye-specific attachment sites for all of the plurality of dyes, such that addition of dye mixtures corresponding to unique dye combinations to the linker molecule in the coupling reaction volume results in random coupling.

[0175] In a particularly preferred embodiment of the present invention, the excitation light is coherent light.

[0176] In a particularly preferred embodiment of the present invention, the excitation light comprises a wavelength range of less than 50 nm, less than 30 nm, less than 10 nm, or a single wavelength.

[0177] In a particularly preferred embodiment of the present invention, the device for analyzing a biological sample is adapted to perform a method according to one of the methods described above.

[0178] In a particularly preferred embodiment of the present invention, the apparatus comprises a microscope configured to generate the at least one readout, preferably a lens-free microscope, a light-field microscope, a wide-field microscope, a fluorescence wide-field microscope, a light-sheet microscope, a scanning microscope or a confocal scanning microscope, a microplate reader, a cell counter, an imaging cytometer or a fluorescence-activated cell sorter.

[0179] In a particularly preferred embodiment of the present invention, the apparatus is configured to determine fluorescence emission intensity, fluorescence lifetime, emission spectrum, excitation fingerprint, fluorescence anisotropy based on the fluorescent dye in the sample.

[0180] In a particularly preferred embodiment of the present invention, the device is configured to split the readout into at least two channels by means of at least one of a spectrometer comprising a prism or a grating and at least one detector.

[0181] In a particularly preferred embodiment of the present invention, the device is configured to split the readout into at least two channels by at least one of a spectrometer comprising a prism or a grating and at least one detector, and the device comprises a time-sensitive detector.

[0182] In a particularly preferred embodiment of the invention, the device may comprise a storage device for storing a unique identifier identifying the affinity reagent, the predetermined structure and the unique dye combination for each marker.

[0183] In a particularly preferred embodiment of the present invention, the device may comprise a calibration unit configured to receive fluorescence emitted by the excited dye and to generate calibration data based on the received fluorescence; wherein the at least one readout is generated based on the calibration data.

[0184] In a particularly preferred embodiment of the invention, no dye combination is assigned to more than one affinity reagent.

[0185] The following example illustrates the effectiveness of this method and also emphasizes its feasibility. In the following example, n=5 groups of fluorescent dyes are excited using n=5 different excitation light wavelengths (e.g., 405nm, 488nm, 560nm, 630nm, and 700nm), where y A =y B =yC =y D =y E = 5 members, so that the various dyes y used in this example σ is 25. In this case, due to the exponential nature of the combined code, the total number of unique codes is 5 5 =3125. In other words, the method described here can be used to read out the entire human secretome based on a simple 5-channel fluorescence-based readout (such as a microscope or cell counter) using commercially available dyes. Compared to being able to use an iterative process to multiplex within the range of 30-60 biomarkers (i.e., corresponding markers per round), this is a significant improvement of 50-100 times. Using the same dye set and an iterative process in which the sample is stained, imaged, and then blanked (i.e., the dye is removed or inactivated), 30,000 targets can be detected in 10 rounds, which is roughly equivalent to the number of coding genes in the human genome. Currently, approximately 20,380 human protein-coding genes have been registered in UNIPROT.

[0186] In the following example, n=6 different excitation lights (e.g., 350 nm, 405 nm, 488 nm, 560 nm, 630 nm, and 700 nm) are used to excite n=10 sets of fluorescent dyes, where y A =y B =y C =y D =y E =y F =y G =y H =y I =y J = 10 members, so that the various dyes y used in this example σ is 60. In this case, due to the exponential nature of the combined code, the total number of unique codes is 10 6 = 1,000,000. In other words, the method described here can be used to read out the entire human proteome in a single round, which is estimated to have on the order of 80,000-400,000 different proteins).

[0187] Furthermore, the method is easily adaptable to cytometers, microplate readers, and fluorescence microscopes, enabling the readout of a very large number of markers. In other words, the method is compatible with both image-based and non-image-based readouts.

[0188] Furthermore, the method is readily adaptable to fluorescence microscopy, enabling the readout of very large numbers of markers with extremely high spatial resolution.

[0189] In a preferred embodiment, the method is based on detecting each different spot, i.e., a spot that can be distinguished from each other by a readout device. Using a regional detector (such as, for example, a camera) that images the visual field, a large amount of spots can be read out simultaneously. The difference of the spot may be due to the different positions of a single visual field in the X and Y directions in the microscope image or the different time points T when passing through the flow cell (such as in a cell counter, an imaging cytometer, or a laser scanning microscope). In a system using image-based readout (such as a microscope and a microplate reader), such as when attempting to image a very large amount of markers in a cell, the difference in the structure of dense labeling can be achieved by flashing random dyes (i.e., time separation). This is a strategy commonly used in stochastic optical reconstruction microscopy (STORM) and related modalities thereof, which rely on, for example, Gaussian fitting to find the positions of different emitters in a sample of dense labeling.

[0190] The difference of spots may be a direct result of the detection method, such as in a flow-through detection based on microbeads, in which multiple microbeads pass through a flow cell in sequence (the readout device is applicable to the flow cell). Spots can be produced by structures that are larger or smaller than the readout volume. In biological samples such as cells, intracellular targets can be located at different X, Y, and Z axis positions. In some cases, when the labeling density is too high, an iterative method can be used to reduce the labeling density to an acceptable level. Other strategies for achieving spot differences may include random labeling, for example, by reducing the concentration of the labeling reagent. Other differences can be achieved by expanding the sample using a scheme called expansion microscopy, as described in Wassie et al., 2019 Nature Methods, Volume 16, pages 33–41 (2019). In addition, the spacing between suitable spots can be achieved by increasing the number of dye sets n. To this end, an adjustable light source or a continuous light source can be used in combination with this method. Other strategies for densely labeled samples will be discussed below.

[0191] Combination of the disclosed method with an iterative staining process

[0192] In another preferred embodiment, the following steps are repeated at least twice to create a series of readouts / images of the sample: staining the sample; directing a first excitation to the sample; generating a first readout / image; directing a second excitation to the sample; generating a second readout / image. The steps defined in claim 1 describe a single round of readout / image acquisition. Additional rounds can be performed to acquire a series of readouts / images of the sample. In particular, a series of subsequent readouts / images can be used to observe changes in the sample over time. In particular, a series of subsequent readouts / images can be used to further increase the number of markers that can be read out, or to ensure that the number of markers read out in a single round in a densely labeled sample is not too high. For example, in this case, the number of markers can be reduced to, for example, 1000 markers per round, but this is still significantly higher than the fluorescence-based imaging-compatible readout methods described in the prior art.

[0193] Single-species readout volume versus multi-species readout volume

[0194] When using this method, a spot in a sample (defined by the size of the principal maximum of the effective point spread function, also known as the confocal volume in confocal microscopy) can contain only one marker or multiple markers of a single specificity (single-species read volume), or it can contain markers with multiple specificities (multi-species read volume). The method described in this document enables robust decoding of single-species read volumes and provides a very large number of unique codes. However, for multi-species read volumes, decoding of markers with multiple specificities located in the same spot is not guaranteed, so only a single possible set of unique codes is found. That is, decoding a multi-species read volume yields multiple possible marker combinations. However, in the event of a multi-species read volume, the method reliably identifies this event and informs the user that a multi-species read volume has been encountered, but no clear solution has been found. The method can also identify a limited number of possible alternatives and, based on these solutions, recommend a labeling strategy for at least one additional round of staining and imaging that uses a subset of markers labeled with a new set of fluorescent combinatorial codes. Alternatively or additionally, the user can resort to the methods described in the "Strategies for Densely Labeled Samples" section.

[0195] The likelihood of encountering a multi-species readout volume depends on both the number of markers to be read in a single round and their subcellular location. Cells have multiple metacompartments, such as the nucleus, cytoplasm, and secretory pathway, and a series of compartments, including, for example, the nuclear membrane, nucleolus (about 7%, about 1300 proteins), nucleoplasm, actin filaments, intermediate filaments, centrosomes, microtubules, cytosol, mitochondria, endoplasmic reticulum, Golgi apparatus, plasma membrane, secretory proteins, vesicles, etc., which are further divided into subcompartments. For example, endosomes, lipid droplets, lysosomes, peroxisomes, and vesicles are grouped in the vesicle queue. While some proteins have a given location in the cell, other proteins that make up about 55% (n=7106) of the localized proteins in the cell atlas are multilocalizing proteins (MLPs) (source: https: / / www.proteinatlas.org / humanproteome / cell / multilocalizing). Therefore, the number of localized proteins is in the range of about 14,000, with about 55% localized in multiple (sub) compartments. The nucleolus is a particularly dense structure. To date, about 7% or 1361 proteins have been detected in one or more of the nucleolar subcompartments: the nucleolus (1008 proteins), the nucleolar fibrous center (300 proteins), and the nucleolar periphery (100 proteins) (Source: https: / / www.proteinatlas.org / humanproteome / cell / nucleoli). The diameter of a typical nucleolus can range from 0.2 to 3.5 μm, which means that a small nucleolus with a diameter of 0.2 μm has a volume of about 0.0335 μm. 3 , which is about 1.3 times larger than the effective PSF of a NA1.4 oil immersion objective. Therefore, the nucleolus may be considered a challenging structure in terms of multi-species readout volume and the present invention. It is estimated that if one wants to achieve this in a single round of imaging, in the worst case, about 1500 different localized proteins must be resolved simultaneously in the confocal volume (excluding non-localized proteins). Similarly, about 24% or 4770 proteins are detected in the human cytoplasm (composed of about 70% water and 20-30% proteins) (Source: https: / / www.proteinatlas.org / humanproteome / cell / cytosol Cytoplasmic proteins can be distributed uniformly throughout the cytoplasm or in punctate patterns, such as aggregates, cytoplasmic bodies, rods, and rings. Therefore, the chance of encountering multispecies readout volumes during iterative staining and imaging can be minimized by taking this a priori knowledge of protein localization into account when defining the marker set for each round, such that they are distributed across the maximum possible number of subcompartments.

[0196] Multi-species readout volume decoding

[0197] By obtaining a first read sequence and retrieving or calculating (from a storage device) all dye combinations from a plurality of dye combinations (S1) that can be summarized under the first read sequence, a multi-species read volume can be reliably detected by this method. When more than one dye combination in the plurality of dye combinations (S1) can be summarized under the first read sequence, a multi-species read volume is detected. In this case, the system notifies the user (e.g., through a software program) that the corresponding read volume includes multiple target molecular species. Based on the species that may be present in the spot, and preferably the prior knowledge obtained in the above-mentioned protein expression database, an optimized second set of assembled dye combinations in the plurality of dye combinations (S1) can be suggested so as to decode the multi-species read volume in the iterative decoding process and decode with a minimum number of iterations based on a certain acceptable statistical confidence level. Alternatively or additionally, a second set of independent and identically distributed dye combinations in the plurality of dye combinations (S1) can be assigned to a plurality of affinity reagents in a second round (this can be regarded as random repeated extraction and replacement).

[0198] First qualitative iterative multi-species read volume decoding by redistributing codes between iterations (“code swapping”)

[0199] However, it is worth noting that for multi-species read volumes where no clear solution can be found in a single round of readout, it is still possible to decode the spot and find a clear solution by multiple rounds of readout (using sets of different dye combinations attached to the same set of affinity reagents). It is worth noting that due to the exponential nature of combinatorial encoding, even when a limited number of dyes are used to generate codes, the cardinality of the set of unique codes can be very high relative to the cardinality of the gene set in the human genome or the cardinality of the analytes to be identified / decoded. Therefore, it is easy to perform experiments in which only a small fraction of the available codes are actually assigned to markers and target molecules. For example, the number of protein-coding genes in the human genome is estimated to be about 20,000. In a case where n=5 and y A =y B =y C =y D =y E = 10, 100,000 unique codes will be generated, which means that about 20% of the available codes will actually be assigned. Furthermore, this means that the proportion of codes actually assigned can be easily adjusted over a wide range by adding another set of dyes or increasing the number of dyes in the set. For example, when n = 6 and y A =y B =y C =y D =y E =y F=15, 10,000,000 unique codes will be generated, and the proportion of codes actually assigned required to encode 20,000 markers will be reduced to about 0.2%. Importantly, for example, on a commercial confocal microscope, 6 excitation lines (such as 360nm, 405nm, 488nm, 560nm, 630nm, 700nm) and a dye set including 15 dyes can be easily provided, with each set having about 5 dyes, which are divided into three major categories according to their fluorescence lifetime (e.g., <1ns, 1-5ns, >10ns), which can be obtained by modifying the basic structure of existing fluorescent dyes. Similarly, a variety of methods are available that combine the use of fluorescence emission spectral information and lifetime information, and include spectral and fluorescence lifetime, gating, unmixing, phasor-based methods, and machine or deep learning-based classification strategies.

[0200] Thus, particularly preferred embodiments of the methods disclosed herein can be used to reliably decode multi-species read volumes using an iterative process based on a certain statistical confidence level (e.g., a p-value). The p-value measures the probability of obtaining a test result equal to the actual measured value, assuming the null hypothesis is true. In the methods described herein, a p-value can be calculated for each marker, which measures the probability of observing the marker in the read volume (i.e., the confocal volume or effective point spread function of the readout device) even though the marker is not actually present in the read volume and the null hypothesis (i.e., the marker is not present in the read volume) is true. Importantly, the confidence in the decoding result grows rapidly with each iteration. Therefore, a generally acceptable statistical confidence, i.e., p-value, can be achieved with a limited number of iterations (e.g., 1-10 times).

[0201] This can be achieved by the following steps: (1) providing a first set of markers with a first set of affinity reagents labeled with a first set of dye combinations, the first set of dye combinations being selected (randomly or deterministically) from a set of dye combinations; reading the spots or read volumes and assembling a first set of all possible dye combinations that can be summarized under the first read sequence; (2) providing a second set of markers with a first set of affinity reagents labeled with a second set of dye combinations, the second set of dye combinations being selected (randomly or deterministically) from a set of dye combinations; again reading the spots or read volumes and assembling a second set of all possible dye combinations that can be summarized under the second read sequence; (3) comparing the first set of dye combinations that can be summarized under the first read sequence with the second set of dye combinations that can be summarized under the second read sequence; and removing all dye combinations that are not shared by the first and second sets of dye combinations from all possible dye combinations, i.e., defining an overlap region. (4) calculating a p-value and / or other suitable statistical confidence measure for each dye and / or affinity reagent and / or marker and / or target molecule combination detected in the overlap region. (5) comparing all calculated p-values ​​and / or other suitable statistical confidence measures to a user-defined threshold, and making a "presence call" for at least some of the dye combinations and / or affinity reagents and / or markers and / or target molecules detected in the overlap region based on a certain statistical confidence level. (6) repeating this process until all multi-species read volumes of interest have been decoded with a satisfactory statistical confidence level for at least each dye combination and / or affinity reagent and / or marker and / or target molecule in the user-defined subset (e.g., a set of target molecules of interest).

[0202] Secondary qualitative multi-species readout volume decoding

[0203] Alternatively or in addition to the above methods, the decoding of the readout sequence can make use of intensity information. It can be assumed that all dyes exhibit essentially equivalent brightness and an essentially linear response under the measurement conditions. In addition, differences in the brightness of the various dyes can be accounted for by performing appropriate calibrations. This is a general assumption that underlies, for example, fluorescence microscopy measurements. Under this assumption, it can be said that when higher signal intensities are observed, the probability of a false positive result is lower. For example, when the first readout sequence contains a "1" for dye A.1 and dye B.2, it means that they are both detected, but the intensities of these dyes may be different, for example, the intensity of dye A.1 may be 1AU and the intensity of dye B.2 is 10AU. In this case, the code that can be summarized under the readout sequence with a "1" in the DyeB.2 position corresponds to a marker that has a higher probability of actually being present in the readout volume, i.e. a marker with a better intensity adjustment. I value and / or other suitable statistical confidence measure.

[0204] Secondary quantitative multi-species readout volume decoding

[0205] Alternatively or in addition to the primary and secondary qualitative decoding, the multi-species read volume can also be decoded quantitatively. This can be achieved by finding a scaling of the proportions of markers in the overlapping region so that they match the observed intensity distribution in an optimal way. Since both the intensity distribution and the identity of the markers in the read spot are known after the primary and / or secondary qualitative decoding (based on a certain statistical confidence level), this becomes a well-defined system of linear equations, essentially equivalent to linear unmixing. Using the above steps, a finite number of cycles / iterations can be used to reliably decode the multi-species read volume, providing the identity of the markers in the read spot / volume (i.e., confocal volume / effective PSF) based on an appropriate statistical confidence metric, and obtaining a certain level of statistical confidence, which can be expressed as, for example, a marker-specific p-value or an intensity-adjusted p-value. I The quantitative information can be provided in the form of a value or other appropriate combination of statistical confidence measures specific to the dye combination / affinity reagent / marker / target molecule. In addition, relative or absolute quantitative information can be derived. In this case, the response of the readout device and the dye must be within the linear range. In order to obtain an absolute quantitative readout, an appropriate calibration must be performed to combine the area under the curve (AUC) of the emission spectrum of a given dye with the number of dye molecules. It is important to note that this method does not require an absolute quantitative readout.

[0206] The probability of observing the same absent marker multiple times

[0207] If the method described in this document is used for samples with very high labeling density (such as whole genome labeling (e.g., 20,000 markers)), multi-species read volumes may appear with high frequency, and each multi-species read volume may include a large number of species, i.e., different target molecules (e.g., 100-1000 different target molecule species). This raises the following question: In an iterative decoding scheme, how likely is it to detect the same absent marker multiple times? In the case of n=5 and y A =y B =y C =y D =y E= 10, ν = 20,000 markers and ψ = 100,000 unique codes available. What is the probability of observing the absence of a marker in the overlapping region (i.e. obtaining the same wrong or untrue result multiple times)? Therefore, it is useful to consider the probabilities after the second round. After obtaining the first read sequence, the first dye combination that can be summarized under the first read sequence is retrieved from the memory, where the κ1 code can be summarized under the first read sequence. In our example, κ1 may be equal to 1 for a single-species read volume and between 1 and 20,000 (the maximum number of codes used) for a multi-species read volume. Typically, for a multi-species read volume, the value of κ may be between 10-5000. Therefore, after the decoding round, there is a large uncertainty about the true content of the multi-species read volume. This leads to the following question: what is the probability p of obtaining a read sequence under which the code for κ can be summarized? κ How big is it? Since the way markers and codes are assigned to each other is random, we can assume that there is a stable overlap in the set of all inducible codes, i.e., the codes corresponding to the markers actually present in the sample, plus the randomly assigned markers, which could be inducible to the observed read sequence by chance. ν = the probability p that a given marker among the 20,000 markers can be inducible to the read sequence. κ Depends on the read sequence and is provided by κ / ψ, where ψ is the cardinality of multiple dye combinations (S1). For example, for κ=1000, the probability of observing the same non-existent marker twice in each round is 1000:100000x 1000:100000 or 1:10000. Importantly, the possibility of observing a certain κ can be estimated a priori and used as information to guide the user in choosing how many dyes to use (i.e., the cardinality of the unique code set), and how many iterations are needed to decode a certain number of markers at a certain statistical confidence level. Whether a higher or lower κ is observed depends on many parameters. For example, the κ distribution rate α=ν / ψ, corresponding to the relationship between the set of dye combinations assigned to the marker and the total number of available dye combinations. In addition, κ depends on the entropy S of the set of dye combinations assigned to the marker, and is inversely proportional to S, κ~1 / S, i.e., the higher the entropy of the set of dye combinations assigned to the marker, the better the p value can be obtained in fewer iterations.

[0208] In this particularly preferred embodiment, the method based on the iterative process of staining the sample, reading the sample information and inactivating the dye further comprises the step of inactivating at least one of the plurality of markers, at least one group of markers, at least one marker. In this document, inactivating one or more markers means preventing the bound fluorescent dye from emitting fluorescence from the sample in the future. This can be achieved by removing the fluorescent dye from the sample or bleaching the fluorescent dye. Thereby, crosstalk between fluorescent dyes bound to different groups of markers is greatly reduced. In other words, by inactivating a group of markers, the structures marked by the group will not be visible in future images / readouts. This means, for example, that fluorescent dyes with similar emission spectra can be used in subsequent images, thereby increasing the total number of markers that can be used in a single round, a single experiment and / or a single biological sample.

[0209] Preferably, the inactivation step is performed by at least one of bleaching a fluorescent dye unique to the at least one marker and removing the at least one marker from the sample, preferably by at least one of dissociating or cleaving the fluorescent dye from the affinity reagent or dissociating the affinity reagent from the target structure.

[0210] Differentiate dyes based on their properties

[0211] In another preferred embodiment, the step of generating a channel is based on at least one of spectral unmixing (also referred to as spectral imaging and linear unmixing, or channel unmixing), the fluorescence lifetime of a fluorescent dye, and the excitation fingerprint of a fluorescent dye. Spectral unmixing can be performed in a variety of ways, including but not limited to linear unmixing, principal component analysis, unsupervised learning methods for the spectrum, support vector machines, neural networks, (spectral) phasor methods, and Monte Carlo unmixing algorithms. In order to reduce the crosstalk between the fluorescent dyes combined with different markers, a variety of techniques can be used. Unmixing techniques are used to separate the contributions of different fluorescent dyes to the same detection channel, i.e., the crosstalk generated due to overlapping emission spectra. Adopting these techniques can significantly improve the sensitivity of the method because the noise is lower. In addition, the fluorescence lifetime and excitation fingerprint of the fluorescent dye can be utilized to correctly identify the fluorescent dye. For example, phasor S-FLIM (as described in Scipioni, L., Rossetta, A., Tedeschi, G. et al. Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging. Nat Methods 18, 542–550 (2021)) is a suitable method for increasing the total number of dyes that can be reliably distinguished by utilizing both emission spectra and fluorescence lifetime information. This can be used to use more sets of markers in each image, i.e., including more sets of markers in one set. In turn, this greatly increases the total number of markers that can be imaged. Both τ gating and τ unmixing are suitable strategies for increasing the number y of distinguishable dyes per set by utilizing fluorescence lifetime.

[0212] At present, fluorescence lifetime is not widely used as orthogonal contrast in microscopy and cytometry. This may be related to the fact that the fluorescence lifetime of most organic dyes (accounting for the vast majority of commercially available fluorescent dyes) is in the range of 1-5ns, which makes separation based on lifetime challenging. In addition, perhaps more importantly, the fluorescence lifetime depends to a large extent on the molecular environment, and many dyes have shortened fluorescence lifetimes in aqueous or polar environments (typical environments of biological specimens). Nevertheless, the widespread use of fluorescence lifetime as orthogonal contrast seems feasible. In a preferred embodiment of the present invention, the fluorescence lifetime of the dye is stabilized relative to environmental conditions by one of the following means: encapsulation, cage lock, doublet formation, derivatization of rotaxanes from the dye, cocrystallization of the dye into, for example, SMILE, polymerization of the dye, and incorporation of the dye into nano- or microstructures (such as polymer beads).

[0213] In another preferred embodiment, a classifier is trained using machine learning, deep learning, or other artificial intelligence methods to distinguish dyes based on a combination of at least two of the following properties: excitation fingerprint, fluorescence emission spectrum, fluorescence lifetime, fluorescence intensity, brightness. Such a trainable classifier may be similar to the "Unsupervised Learning Methods for Spectra" (LUMOS): McRae TD, Oleksyn D, Miller J, Gao YR (2019) Robust blind spectral unmixing for fluorescence microscopy using unsupervised learning. PLoS ONE 14(12): e0225410, which is based on k-means clustering.

[0214] In a particularly preferred embodiment, a learning algorithm based on machine learning, deep learning or artificial intelligence technology (including but not limited to support vector machines, classical neural networks, convolutional neural networks, recurrent neural networks, generative adversarial networks, self-organizing maps, Boltzmann machines, deep reinforcement learning, autoencoders) is trained to separate the dyes based on their emission spectra.

[0215] In a particularly preferred embodiment, a learning algorithm based on machine learning, deep learning or artificial intelligence techniques (including but not limited to support vector machines, classical neural networks, convolutional neural networks, recurrent neural networks, generative adversarial networks, self-organizing maps, Boltzmann machines, deep reinforcement learning, autoencoders) is trained to separate the dyes based on their emission spectra and fluorescence lifetimes. This can be based on simple fluorescence lifetime gating or on more complex fluorescence lifetime analysis. A particularly suitable method for deriving training data for this method can be, for example, phasor S-FLIM (as described in Scipioni, L., Rossetta, A., Tedeschi, G. et al. Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging. Nat Methods 18, 542–550 (2021)).

[0216] In another preferred embodiment, the method further comprises the step of capturing a hyperspectral image of the sample. Compared to multispectral imaging, which captures only a limited number of wavelengths (typically fewer than or approximately 10), each pixel of a hyperspectral image captures dozens or hundreds of wavelengths. In other words, hyperspectral images have very high spectral resolution. This enables finer differentiation of fluorescent dyes based on their emission spectra, thereby improving the sensitivity and reliability of the method.

[0217] Readout robustness

[0218] The robustness of the readout is an important consideration. Ideally, each spot is read out individually, which means that the points read out in parallel are spatially separated so that the optical system used for their detection can resolve them into separate spots. If two or more markers with different reactivities / specificities are too close in space, i.e., they are essentially located in the same readout volume and are read out at the same time, then it may not be possible to obtain a clear decoding of the encoded information in a single round of readout. In this case, the strategy described below for densely labeled samples can be adopted. If a densely labeled sample including a large number of markers is to be used, such as a whole genome study, where multi-species readout volumes may appear at a high frequency, this method can be applied to reliably detect multi-species readout volumes and decode the information contained therein, i.e., the identity of the markers in the spot, through an iterative process with a limited number of rounds. This is a particularly preferred embodiment of the present invention and, relative to the prior art, a breakthrough has been made in the level of "multiplexing" that can be achieved per round, which is several orders of magnitude higher than currently available methods. This has been described above and is referred to as "first qualitative iterative multi-species readout volume decoding by redistributing codes between iterations ("code swapping")". As mentioned above, relative and absolute quantitative decoding / decryption can also be performed.

[0219] Strategies for densely labeled samples

[0220] When the method disclosed in the present invention is intended to be used in combination with densely labeled samples, it may be beneficial to adapt the method.A kind of such adaptation is based on the prior knowledge of protein localization, such as nucleus, cytoplasm, nucleoplasm, secreted protein, protein located on or in an organelle or protein located on an intracellular or extracellular cell membrane, this allows multiple markers to be stratified into multiple subgroups, these subgroups are subsequently brought into the sample, and in the multiple rounds of iterative dyeing and imaging process, the chance of two different markers being co-located in the same spot in the same round is minimized. This can be combined with the expansion microscopy scheme described in Martinéz et al., Scientific Reports, Volume 10, Article number: 2917 (2020) so that sample is expanded approximately 4 times in all spatial directions, thereby further reducing the probability of two different markers being co-located in the same spot or the same read-out volume in the same round. In addition, group number n can be increased, and multiple dyes can be divided into dye subgroups, and each dye subgroup can then be used to generate a dye combination for corresponding marker subgroups. As more and more dyes become available with narrower excitation and emission spectra, higher n and / or higher y can be more easily accommodated. Similar problems exist with ultra-high resolution microscopy; random labeling of target structures or random blinking of fluorescent dyes can be used to circumvent the above problems. Blinking of fluorescent dyes can be achieved in a variety of ways, while some dyes (such as quantum dots) typically blink, other fluorophores can be photoactivated, photoconverted, or ground state depleted to cause them to blink. These techniques can be applied to the methods disclosed herein to image densely labeled samples. In a particularly preferred embodiment, DNA-PAINT is used, and the markers are read out randomly, so that the readout from 1 to n is repeated i times, thereby obtaining a first readout sequence. In a preferred embodiment, random optical reconstruction microscopy or a related blinking method is used, and the markers are read out randomly, so that the readout from A to n is repeated i times, thereby obtaining a first readout sequence.

[0221] Spot Detection

[0222] To read out dye combinations, in certain embodiments of the present invention, such as in whole-secretome profiling, it is preferable to ensure that markers of a given specificity are located at distinct locations or spots in the sample. In such cases, spot detection is very useful. Spot detection is based on image segmentation. In the sense of this document, a "spot" is a feature. In most assay formats where the readout volume is a single species readout volume, the dye combination is preferably read out per spot.

[0223] Image segmentation analysis can be performed using classical methods, artificial intelligence-based techniques (including machine learning and neural networks / deep learning), or other techniques, including thresholding techniques, dimensionality reduction techniques, clustering methods, compression-based methods, histogram-based methods, edge detection, dual clustering methods, region growing methods, partial differential equation-based methods, variational methods, graph segmentation methods (e.g., Markov random fields), watershed transforms, model-based segmentation, multi-scale segmentation, semi-automatic segmentation, trainable segmentation using various machine learning, neural network, and artificial intelligence methods (e.g., pulse coupled neural networks (PCNNs) and convolutional neural networks (U-Nets), recurrent neural networks (RNNs)), and object co-segmentation methods (e.g., Markov networks, convolutional neural networks, or long short-term memory (LSTM)). Alternatively or additionally, features such as size and / or color and / or fluorescence intensity and / or fluorescence lifetime can be used to identify components of a marker from image data. Various algorithms can be used for recognition, including the Harris Corner algorithm, Scale-Invariant Feature Transform (SIFT), Speeded Up Robust Features (SURF), Speeded Up Segment Test Features (FAST), and Oriented Rapid Rotation BRIEF (ORB) algorithms, which are all known and can be used to identify components and / or features of a marker from image data.

[0224] General considerations for applying this method

[0225] In another preferred embodiment, the method further includes applying a second excitation light at a time subsequent to the first excitation light. Preferably, the time between applying the first excitation light and the second excitation light is longer than the fluorescence lifetime of the first set of fluorescent dyes excited by the first excitation light. This ensures that only the fluorescence emitted by the second set of fluorescent dyes excited by the second excitation light is captured to generate the second image / readout. This reduces crosstalk between fluorescent dyes and further improves the sensitivity of the method.

[0226] In another preferred embodiment, at least one of the first wavelength spectrum and the second wavelength spectrum for dye excitation includes a wavelength range or a single wavelength of less than 50 nm, less than 30 nm, less than 10 nm. These wavelength bands are typical ranges of, for example, dichroic beam splitters or bandpass filters used in fluorescence microscopy. Various methods can be used to generate corresponding wavelength spectra for sample illumination or fluorescent dye excitation. For example, a bandpass filter that filters out a specific wavelength range can be used in combination with a light source that emits a broad spectrum (e.g., a mercury lamp or a xenon lamp). Alternatively or additionally, a white light laser that emits supercontinuum white light can be used in combination with an AOTF for selecting one or more single wavelength emission lights.

[0227] In another preferred embodiment, each set of unique fluorescent dyes can be excited by essentially one wavelength spectrum, or by the same wavelength spectrum. This allows the same set of fluorescent dyes to be excited by a single light source having, for example, a narrow emission spectrum. This embodiment of the method can be easily implemented using existing fluorescence microscopes, which typically include such light sources.

[0228] In another preferred embodiment, the fluorescent dyes in the dye sets A to n have emission spectra that span at least partially different wavelength ranges. Thus, the fluorescent dyes in the dye sets A to n can be easily distinguished from the other dyes based on their emission spectra. This reduces or eliminates the computational load of unmixing required to separate the channels in each image, making the method faster and more reliable.

[0229] In another preferred embodiment, at least two fluorescent dyes in the dye group of A to n each have a different fluorescence lifetime. Therefore, at least two fluorescent dyes can be distinguished by their lifetime. Specifically, this can be used to increase the number of channels per image, that is, each image captures more markers. Therefore, the total number of dyes that can be imaged in each group will be greatly increased. Specifically, the base structure of existing fluorescent dyes can be modified or placed in different molecular environments to transform them into derivative fluorescent dyes with similar excitation and / or emission spectra but different fluorescence lifetimes. Specifically, existing fluorescent dyes can be encapsulated in microcapsules or nanocapsules, embedded in polymers such as polystyrene, enveloped or co-crystallized in materials such as SMILE to stabilize their molecular environment, thereby extending their fluorescence lifetime. This strategy can also be used to generate dye groups of the same dye type with different fluorescence lifetimes. Rotaxanes, especially those derived from squaric acid, are extremely attractive dyes in this regard because the interlocking of dye molecules in the macrocycle can stabilize the molecular environment, thereby extending the fluorescence lifetime.

[0230] In particularly preferred embodiments, at least one dye or label is a small molecule ion isolation lattice (SMILE), which are small crystals consisting of a cationic dye co-crystallized with a counterion, such as, for example, an anionically bound cyanostar or a displacer such as a bisamide, cyclodextrin, quaterphenyl or pyrene, as described in Benson et al., 2020 Chem 6, 1978–1997, August 6, 2020.

[0231] In particularly preferred embodiments, at least one dye or label is a polymeric microbead or nanostructure comprising a small molecule ion isolation lattice (SMILE).

[0232] In preferred embodiments, at least one dye or label is a rotaxane dye, such as a squaraine-rotaxane dye.

[0233] In a preferred embodiment, at least one of the dyes or labels is a dyad consisting of an antenna moiety and an emitter moiety.

[0234] In a particularly preferred embodiment, at least one dye or label is a FRET pair consisting of at least two dyes, formed by a donor and an acceptor connected by a linker (such as a nucleic acid). At least one fluorescent dye can be based on a FRET pair, wherein at least one fluorescent dye serves as a FRET donor, and at least one fluorescent dye serves as a FRET acceptor. FRET pairs can be physically connected by a linker comprising: DNA, RNA, peptide nucleic acid, morpholino or locked nucleic acid, ethylene glycol nucleic acid, threose nucleic acid, hexanol nucleic acid or other forms of artificial nucleic acid, DNA nanostructure and / or peptide.

[0235] In another preferred embodiment, in the first and / or second subset, at least two fluorescent dyes, each unique for a marker, have emission spectra in substantially the same wavelength range and substantially the same fluorescence lifetime under a first condition of the sample (such as a specific first pH value, a specific first solvent, a specific first redox level, a specific first temperature, or a specific first ligand concentration (e.g., a lower concentration of at least one of the following: Cu(II), Zn(II), small molecules)), and have emission spectra in substantially the same wavelength range and substantially different fluorescence lifetimes under a second condition of the sample (such as a specific second pH value, a specific second solvent, a specific second redox level, a specific second temperature, or a specific second ligand concentration (e.g., a lower concentration of one of the following: Cu(II), Zn(II), small molecules).

[0236] The present invention also relates to a device for analyzing a biological sample, which is suitable for implementing the above-described method for analyzing a biological sample. The device has the same advantages as the method and can be supplemented by the features of the dependent claims directed to the method. The device can be particularly configured to image samples in an array format, such as a microplate.

[0237] In a preferred embodiment, the device is configured to read out a sample flowing through a flow cell.

[0238] In a preferred embodiment, the apparatus includes at least one first light source configured to emit a first excitation light, and at least one second light source configured to emit a second excitation light. Alternatively or additionally, the apparatus includes a tunable light source configured to emit the first and second excitation lights. Preferably, at least one of the first excitation light and the second excitation light is coherent light.

[0239] In another preferred embodiment, the separation of the first and / or second image (readout) into at least two channels is accomplished by at least one of a spectrometer comprising a prism or a grating and at least one detector. A diffraction assembly can be used to optically or spatially separate the captured fluorescence into different channels by wavelength, for example, by directing light of different wavelengths to different parts of a single detector or to different detectors. Since these channels are created by the detector hardware, they are also referred to as detection channels hereinafter. Examples of such spectrometer devices for confocal scanning microscopes are disclosed, for example, in US 6,614,526 B1.

[0240] In another preferred embodiment, the first and / or second image (readout) is separated into at least two channels by at least one time-sensitive detector. Such detectors record not only the wavelength spectrum, but also the arrival time of the captured fluorescence. They can also be time-gated, i.e., configured to record events within discrete time periods (i.e., so-called time gates), so that lifespan information can be determined based on the arrival time of the captured fluorescence. Therefore, fluorescent dyes with significantly overlapping emission spectra but different fluorescence lifetimes can be reliably separated into different channels. This further increases the number of markers that can be grouped into a single subgroup (i.e., simultaneous imaging).

[0241] The present invention also relates to a microscope system comprising the above-mentioned apparatus for analyzing biological samples. The microscope system is preferably a lensless microscope, a light-field microscope, a wide-field microscope, a fluorescence wide-field microscope, a light-sheet microscope, a scanning microscope, or a confocal scanning microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[0242] Hereinafter, specific embodiments will be described with reference to the accompanying drawings, in which:

[0243] Figure 1 Two fluorescent dyes and commonly used affinity reagents are schematically shown;

[0244] Figure 2 is a schematic diagram of the spectrometer and dispersive optical elements;

[0245] Figure 3 Schematic illustration of how orthogonal dimensions can be used to discriminate between a group of dyes that can be excited by the same excitation light;

[0246] Figure 4 Different approaches for dye separation based on gating, unmixing, topological methods, and phasor-based methods are schematically summarized, which may or may not be combined with ML / DL / AI methods to train dye separation classifiers;

[0247] Figure 5 Schematic representation of various markers directly conjugated to dyes grouped by component;

[0248] Figure 6A Schematic diagram showing various dyes y A +y B +y C …+y n , these dyes are grouped into n dye groups (group A to group n) excited with different excitation lights;

[0249] Figure 6B Schematically shows a variety of dyes (Y D ), the relationship between multiple dye combinations (S1) and multiple affinity reagents (A=S2=T1*);

[0250] Figure 6C Schematically shows a variety of dyes (Y D ), the relationship between multiple dye combinations (S1) and multiple affinity reagents (A=S2=T1*);

[0251] Figure 6D Schematic illustration of various dyes (Y D ), multiple dye combinations (S1) and multiple affinity reagents (A=S2=T1*), and multiple markers (M);

[0252] Figures 6E to 6H Further schematically illustrates a variety of dyes (Y D ), multiple dye combinations (S1) and multiple affinity reagents (A=S2=T1*), and multiple markers (M);

[0253] Figure 7A Schematic diagram showing the use of multiple dyes (y A +y B +y C …+y n ) Different methods of generating unique codes (group-based and binary combination coding);

[0254] Figure 7B Schematic illustration of various affinity reagents (A=S2=T1*);

[0255] Figure 7C Schematic illustration of a code readout scheme using sequential excitation (based on group and binary combination coding);

[0256] Figure 8 Schematic representation of bijective mapping or pairs of dye combinations with unique oligonucleotide sequence barcodes;

[0257] Figure 9 Schematic illustration of a marker-bound target molecule consisting of an affinity reagent and a reporter;

[0258] Figure 10A Schematic illustration of how multiple copies of the same dye species can be attached to a marker;

[0259] Figure 10B Schematically illustrates how information about landmarks is stored in a database and a storage device;

[0260] Figure 11 An example of sequential readout of a marker where the affinity reagent is an antibody is schematically shown;

[0261] Figure 12 An example of sequential readout of markers in which the affinity reagents are oligonucleotide probes is schematically shown;

[0262] Figure 13 Schematic illustration of the independence of the readout from the spatial position of the dye within or on the reporter for reporters of sub-diffraction limit size;

[0263] Figure 14 The bar graph shows the achievable values ​​for different n and y values ​​using available dyes and detector technologies. A +y B +y C …+y n the number of unique codes available;

[0264] Figure 15 Schematic diagram of a cell;

[0265] Figure 16 Schematic diagram of the relative sizes of affinity reagents and dyes;

[0266] Figure 17 Schematic diagram illustrating the relative sizes of the PSFs of antibodies, 10 nm quantum dots, and a 1.4 NA objective lens;

[0267] Figure 18 A flowchart describing the iterative staining process;

[0268] Figure 19 Schematic diagram of cells in wells pre-loaded with hydrogel or in hydrogel beads, where capture reagents are coupled to the hydrogel;

[0269] Figure 20A and 20B Schematic diagram of cells in wells pre-filled with hydrogel or in hydrogel beads, where the capture reagent is coupled to the microbeads embedded in the hydrogel;

[0270] Figure 21 Schematic diagram of cells in pre-filled hydrogel wells or hydrogel beads, where multiple spots are read out, decoded, and counted to derive the secretion profile of the cell;

[0271] Figure 22 Flowchart for the blob detection and analysis pipeline;

[0272] Figure 23 Schematic diagram of cells in a sample carrier and wells of a readout device;

[0273] Figure 24 is a schematic diagram of cells in a hydrogel bead readout by a cytometer, imaging cytometer, or microscope as they flow through a flow cell;

[0274] Figure 25 Schematic diagram of a bead-based assay with cytometer readout, where capture reagents covalently attached to microbeads capture target proteins, which in turn are labeled with a marker;

[0275] Figure 26 Schematic diagram of a bead-based assay for detecting the presence of a target molecule or analyte and a bead-based assay for detecting the interaction of a molecule with a target molecule;

[0276] Figure 27 is a schematic diagram of a cell-based assay with a cytometer as a readout, wherein a marker is bound to a molecule expressed on the cell surface;

[0277] Figure 28 Schematic illustration of the difference between single-species and multi-species readout volumes;

[0278] Figure 29 schematically illustrates a multi-species readout volume including dyes excited with a first excitation light and a resulting first readout sequence;

[0279] Figure 30 Strategies for decoding multispecies readout volumes in different ways based on dye blinking and localization microscopy are schematically illustrated;

[0280] Figure 31 Schematic illustration of the A =y B =y C =y D =y E =10 and n=5 Example Different Code C i or password X i Impact on y and a;

[0281] Figure 32 Schematic illustration of the effect of different examples of the first readout sequence on κ;

[0282] Figure 33ASchematic illustration of a "multiple dye combinations (S1)" containing all unique codes or dye combinations and an unsorted first "set of dye combinations assigned to markers";

[0283] Figure 33B Schematic illustration of a "multiple dye combinations (S1)" containing all unique codes or dye combinations and an unsorted second "set of dye combinations assigned to markers";

[0284] Figure 33C Schematic illustration of a "multiple dye combinations (S1)" containing all unique codes or dye combinations and an unsorted third "set of dye combinations assigned to markers";

[0285] Figure 34A Schematic illustration of "multiple dye combinations (S1)" sorted as being inducible and inducible under the first readout sequence;

[0286] Figure 34B Schematic illustration of "multiple dye combinations (S1)" sorted as being inducible and inducible under the second readout sequence;

[0287] Figure 34C Schematic illustration of "multiple dye combinations (S1)" sorted as being inducible and inducible under the third readout sequence;

[0288] Figure 35A Schematic illustration of an exemplary marker μ2015 in the first round;

[0289] Figure 35B Schematic illustration of an exemplary marker μ2015′ in the second round;

[0290] Figure 35C Schematic illustration of an exemplary marker μ2015" in the third round;

[0291] Figure 36A is a histogram of the example probabilities of observing the same absent marker in iterations 2 to 5 with κ = 1000 and ψ = 100000 in all rounds;

[0292] Figure 36B is an example illustrating the effect of ψ and κ on the probability p of observing the same absent marker in multiple iterations;

[0293] Figure 37 It is an iterative workflow for the initial qualitative decoding of multiple spots;

[0294] Figure 38A Schematically illustrate how intensity information can be used for secondary qualitative and quantitative decoding;

[0295] Figure 38B Schematic illustration of how intensity thresholding is performed on a first readout sequence, thereby reducing the number of dye combinations that can be assigned to a marker and summarized under a readout sequence;

[0296] Figure 39 Schematic illustration of markers with directly conjugated linker oligonucleotides and indirectly oligonucleotide-coupled dyes;

[0297] Figure 40 Schematic illustration of the method for assembling combinatorial fluorescent codes on linkers containing multiple dye nonspecific coupling or attachment sites;

[0298] Figure 41 Schematic illustration of the method for assembling combinatorial fluorescent codes on linkers containing multiple dye-specific coupling or attachment sites while ensuring stoichiometry;

[0299] Figure 42 Schematic illustration of the method for assembling combinatorial fluorescent codes on micro / nanobeads containing multiple dye nonspecific coupling or attachment sites;

[0300] Figure 43 Schematic illustration of the method for encapsulating the combined fluorescent code in microbeads / nanobeads or microcapsules / nanocapsules;

[0301] Figure 44 Schematic illustration of methods for linking SMILE assemblies to affinity reagents using linker peptides, polymers, or oligonucleotides;

[0302] Figure 45 Schematic illustration of the method for linking SMILE assemblies to affinity reagents using nanostructures as attachment platforms.

[0303] Figure 46 Schematic illustration of how SMILE is integrated into micro / nanobeads;

[0304] Figure 47 Schematic illustration of how SMILE is integrated into microcapsules / nanocapsules;

[0305] Figure 48A Schematic illustration of nanostructured linkers based on oligonucleotides, polymers / peptides, and nanorulers / DNA origami;

[0306] Figure 48B Examples of peptide-linkers and oligonucleotide-bead-linkers are schematically shown;

[0307] Figure 49 Schematic representation of different options for coupling linkers to affinity reagents;

[0308] Figure 50ASchematically shows the main functional units and some optional functional units of the readout device;

[0309] Figure 50B The microscope is schematically illustrated;

[0310] Figure 51 Three dyes with significant spectral overlap are shown, which can be unmixed by excitation spectral imaging. The upper graph shows the excitation spectra of the three dyes, and the lower graph shows the emission spectra of the three dyes. The dyes shown are suitable for use in the present invention;

[0311] Figure 52 The cyclic DNA-Exchange process is schematically shown;

[0312] Figure 53A-1 、 53A-2 , 53B, and 53C schematically illustrate a readout volume and a readout trajectory, respectively;

[0313] Figure 54 further schematically illustrates the assignment of two affinity reagents to a subset of combinatorial labels, the subset of combinatorial labels comprising a subset of dye combinations;

[0314] Figure 55 、 55A , 55B and 55C are schematic diagrams showing how readout traces, progressive decoding, statistical analysis, presence calling, mathematical input / output models, and sources of prior information can be related to each other, respectively;

[0315] Figure 56 Schematic illustration of short and long residence times for unbound and bound reporters;

[0316] Figure 57 Schematically illustrates the strategy for achieving “barcode cycling” and thereby performing intra-round code exchange in a dynamic or repetitive manner;

[0317] Figure 58 The association and dissociation of guest molecule-modified oligonucleotides are shown;

[0318] Figure 59 A barcoding cycle using a barcoded antibody as an affinity reagent is shown;

[0319] Figure 60 shows a barcoding cycle using oligonucleotide probes as affinity reagents; and

[0320] Figure 61 An alternative strategy is shown that enables barcode cycling and thereby intra-round code swapping in a dynamic or repetitive manner. DETAILED DESCRIPTION

[0321] Figure 1 A fluorescent dye of the prior art (also referred to as a marker 100, represented by a circle) is schematically shown, wherein the left half 102 is pre-loaded with a pattern indicating / reflecting the excitation spectrum, so that dyes having the same left half pattern can be excited by the same excitation light, and wherein the right half 104 is pre-loaded with a pattern indicating / reflecting all properties that can be used alone or in combination to distinguish the dyes from each other by a device for reading (including, for example, fluorescence emission spectrum, fluorescence lifetime, fluorescence polarization, brightness and excitation fingerprint), so that two dyes depicted with the same right half cannot be distinguished by the device for reading. Figure 1 Two fluorescent dyes 100a and 100b are shown. Each fluorescent dye is represented by a circle 100a and 100b with a solid line border. Each circle 100a and 100b is divided into two semicircles 102a, 102b and 104a, 104b with different hatching. The hatching type of the left semicircle 102a and 102b represents the excitation light that can excite the corresponding fluorescent dyes 100a and 100b, that is, the fluorescent dyes of the left semicircle 102 with the same hatching type can be excited by the excitation light with the same wavelength spectrum or the same single wavelength. The hatching type of the right semicircle 104 represents the properties of the corresponding fluorescent dye 100, which can distinguish these dyes on the imaging system used in the data acquisition step or the readout process, including, for example, its emission spectrum, fluorescence lifetime and excitation fingerprint. This means that the fluorescent dyes 100 of the right semicircle 104 with the same type of hatching have the same or substantially the same (i.e., indistinguishable) emission characteristics.

[0322] It is worth noting that this conceptually depends on the readout, as readouts that provide orthogonal contrasts (such as emission spectrum and fluorescence lifetime) can distinguish more dyes, while readouts that provide only one contrast cannot. Figure 1 Further shown are a series of commonly used affinity reagents 106, including single domain antibodies 108, multimeric (single domain) antibodies 110, conventional antibodies 112, aptamers 114, oligonucleotides 116, toxins / drugs / drug-like molecules / small molecules (e.g., biotin) 118, which are labeled by direct conjugation to the dye 100. The same affinity reagent can also be labeled with a peptide tag, hapten, or oligonucleotide. Figure 1 A series of commonly used affinity reagents 106 are further shown, including single-domain antibodies 108, multimeric (single-domain) antibodies 110, conventional antibodies 112, aptamers 114, oligonucleotides 116, toxins / drugs / drug-like molecules / small molecules (e.g., biotin) 118, which are labeled by direct conjugation with oligonucleotides 120. In the sense of this document, 124 refers not only to dimers but also to multimeric (single-domain) antibodies. Such combinations 124 can be engineered to achieve specific affinities (bispecific reactivity) that are not otherwise obtainable or to improve affinity.

[0323] Direct and indirect immunofluorescence labeling is widely used in life science research and diagnostic applications to analyze the presence of molecular targets such as proteins, RNA, DNA, and other molecules. Typically, such fluorescence-based assays are read out using at least one of a microplate reader, high-content screening equipment, a microscope, a cell counter, or a fluorescence-activated cell sorter.

[0324] Readout devices for performing fluorescent multicolor imaging typically include at least one light source for generating excitation light, a detection system including at least one detection channel, and may also include filters and / or dispersive optical elements (such as prisms and gratings) to direct the excitation light to the sample and the emission from the sample to the detection system, and / or to an appropriate area of ​​the detector.

[0325] Figure 2 Schematically, two detection systems 204 and 206 with dispersive optical elements (e.g., prisms or gratings) and a detector 208 with multiple channels are shown. In the sense of this document, a detection system can consist of several detection channels and can be a spectral detector that detects multiple spectral bands in parallel or a hyperspectral detector that detects a continuous part of the spectrum. The detection system comprises at least one detector, which can be a point detector (e.g., a photomultiplier tube, an avalanche diode, a hybrid detector), an array detector, a camera or a hyperspectral camera. The detection system can record the intensity of each channel (as is usually the case in a cell counter) or can be an imaging detection system that records an image (as is the case in a microplate reader or a microscope).

[0326] The readout device allows a certain number of dyes to be analyzed from a given biological sample in a given run. This number typically depends on the number n of detection channels the readout device is configured to provide (i.e. can spectrally resolve or distinguish). In the case of microscopes, the number of detection channels is typically 4-5 for camera-based widefield detection (e.g. widefield epi-fluorescence microscopes, spinning disk microscopes, light sheet fluorescence microscopes) and 5-12 for microscopes employing spectral detection concepts (typically relying on excitation or emission fingerprinting and (spectral / linear) unmixing). In order to detect the emission of multiple fluorophores, quantum dots and / or fluorescent proteins and to assign the detected photons to the corresponding fluorophores, quantum dots and / or fluorescent proteins, the readout device can adopt various strategies. Emission filters are typically used to direct the desired spectral band to the detector. Multiple emission filters are typically mounted on a filter wheel so that the emission light band reaching the detector can be quickly changed. Alternatively or additionally, Figure 2The dispersive optical assembly schematically shown in the figure can spectrally separate the light 200 emitted by the biological sample. Both the prism 204 and the grating 206 are widely used in excitation and emission beam guidance and spectral separation in readout devices such as microscopes, cell counters and microplate readers. On the detection side, the prism 204 and the grating 206 can be arranged in a simple or more complex manner to spectrally separate the emitted light 202 and guide it to a suitable detector 208, which can be composed of multiple point detectors (such as photomultiplier tubes, avalanche diodes), array detectors, cameras or hyperspectral detectors. Depending on the configuration of the detection system, the readout device will be able to reliably separate the emissions from n dyes. According to the current use of readout devices, this number n determines the number of molecular markers that can be obtained on the corresponding readout device per round. Alternatively or in addition to separating the dyes according to their spectral properties, the fluorescence lifetime τ can also be used on devices that are configured to measure lifetime, i.e., have a pulsed laser source and a time-resolved detector.

[0327] Figure 3 A portion of the concept disclosed in PCT / EP2021 / 063310 is schematically shown, wherein the excitation and emission characteristics of one set of fluorescent dyes from a plurality of n sets of fluorescent dyes are schematically shown. Figure 6A As shown, the fluorescent dye set is constructed in such a way that each dye in the set can be excited by the same excitation light, single band, multi-band, single wavelength or multi-wavelength. The fluorescent dye set is configured in such a way that the readout device and readout software can distinguish the dyes from each other. This means that readout devices with more channels, especially readout devices that provide orthogonal contrast, allow more dyes to be unambiguously assigned to channels. Figure 3 In the example shown, a set of fluorescent dyes that can be excited by excitation line A (e.g., 390 nm) comprises 15 dyes, which are divided into three groups or gates (based on τ gating or τ unmixing) 304a, 304b, 304c according to their fluorescence lifetimes, each group containing five fluorescent dyes that can be distinguished from each other according to their emission spectra.

[0328] Figure 3 An example of the prior art is schematically shown, which utilizes the principle of forming n groups of selectively excitable dyes, i.e., dyes from group A can be excited by excitation light A, but are substantially not excited by excitation light B, up to group n, and combines the ability to distinguish a maximum of y dyes per group, or a maximum of y dyes per group if the number of dyes per group is different. A 、y B …y n Reading.

[0329] Figure 4A "topological approach" 400 is schematically shown, where, for example, spectra are plotted as curves relative to fluorescence lifetime and intensity, where lines of the same pattern correspond to different levels of (normalized) intensity of the same dye species 402a, 402b, 402c. Topological approaches are particularly suitable for machine learning, deep learning or other forms of or artificial intelligence-based dye separation, achieved by training classifiers. Such training can be supervised or unsupervised. The rich texture provided by this "topology" is an ideal input for these methods. A phasor-based approach 404 uses information such as intensity and / or emission spectrum and / or fluorescence lifetime for dye separation, and is equally applicable to classical dye separation algorithms or methods, as well as machine learning, deep learning or other forms of or artificial intelligence-based dye separation. Again, such training can be supervised or unsupervised.

[0330] Figure 5 Examples known in the art are shown schematically, which utilize a 1:1 relationship between the unique specificity of an affinity reagent and a dye that is unique to a plurality of dyes, meaning that dye 100 corresponds to a channel, in which case the channel corresponds to a marker. The present invention changes this 1:1 relationship between the unique specificity of an affinity reagent and a unique dye and conceptually replaces it with a one-shot or preferentially bishot (i.e., 1:1 correspondence) relationship between a marker and a combination of dyes in a single round, and a 1:many relationship between a marker and a combination of dyes in multiple rounds. For example, using the method described in PCT / EP2021 / 063310, using a plurality of dyes, where n=5 and y A =y B =y C =y D =y E =10, will allow 50 dyes 100 to be read out or imaged, thereby 50 markers 500 with 50 different reactivities / specificities to be read out or imaged. This illustrates the powerful advantage of the present invention in terms of the number of markers that can be read out in a single round and overall (i.e., in an experiment with a certain number of iterations), which is acceptable to most users and can be within the range of 1-25 iterations. For example, the present invention only requires 2 rounds of experiments to achieve whole-genome readout (about 20,000 proteins corresponding to about 1 molecular target / analyte for each protein-coding gene), and the p-value obtained is generally acceptable for the vast majority of users in the vast majority of application scenarios. More importantly, the p-value can be exponentially improved with the rounds, making the method more powerful. Its core lies in the exponential growth of the cardinality of the unique code set (multiple dye combinations S1), which is contrary to the large but limited number of markers required for whole-genome readout.

[0331] Figure 6AA variety of dyes are shown, according to their excitation properties by excitation light A to n and the y in each group A to y n members, grouped into multiple groups of dyes A to n 600. As shown in the figure, the multiple dyes include y A +y B +y C …+y n =y σ The amount of excitation light may be n, and the amount of dye may be different from the value of n.

[0332] Figure 6B and 6C Schematic diagram of various dyes Y D By constructing combinations of dyes 602 according to certain "rules for forming dye combinations," a plurality of dye combinations S1 can be formed. The combinations are constructed in such a way that each dye combination in the plurality of dye combinations S1 is unique, and the plurality of dye combinations S1 preferably contains the maximum number of elements (i.e., dye combinations) that can be formed by the respective "rules for forming dye combinations," or in other words, maximizes the cardinality ψ of the plurality of dye combinations S1. Preferably, the dye combinations are constructed in a unique and randomly distributed manner. Many different "rules for forming dye combinations" are compatible with this method. Figure 6B Further shown is the mapping between the various dye combinations S1 and the various affinity reagents A=S2=T1*, which use the codes C1, C2, C3, ... C n and / or password X1, X2, X3, ...X n , i.e. encoding or encryption. Encoding and / or encryption can be performed in different ways, as shown in case α and case β. In both cases, the codes C1, C2, C3, ... C n and / or password X1, X2, X3, ...X n All are total functions, preferably bijective, but can also be injective.

[0333] refer to Figure 6B

[0334] refer to Figure 6C

[0335] Figure 6D Schematic illustration of the assignment or mapping of affinity reagents to dye combinations and vice versa. The figure above shows a double arrow similar to a bijective mapping, which means that the encoding / encryption forms a i -s i Pairing, in other words, multiple affinity reagents A = S2 = element a of T1*i Combine elements s in S1 with various dyes i There is a one-to-one correspondence between them. The injective case is also allowed but is not shown in the figure for clarity. Figure 6D It further shows how to combine the affinity reagents a in the multiple affinity reagents A=S2=T1* i Assign (encode / encrypt) the dye combination s in the plurality of dye combinations S1 i , and vice versa, thereby obtaining the marker μ among the multiple markers M i As long as this assignment is purely virtual, a marker can be considered a virtual marker when a physical binder (i.e. an affinity reagent such as an antibody molecule etc.) is physically coupled to a binder (e.g. a mixture) of a dye combination, and a "physical marker" if the marker is a dye or a reporter.

[0336] Figure 6E Schematically depicts a one-to-one (1:1) assignment or mapping of affinity reagent to dye combination and vice versa. Such a mapping can be described as a "bijection."

[0337] Figure 6F A one-to-many assignment or mapping of affinity reagent to dye combinations is schematically shown. Such mapping can be described as "at least one-shot".

[0338] At least some of the affinity reagents a from the plurality of affinity reagents A are uniquely assigned to dye combinations s of the plurality of dye combinations S1. In both mappings, no dye combination s is assigned to more than one affinity reagent a in the plurality of affinity reagents a. However, in the one-to-many mapping, elements a of the plurality of affinity reagents A are uniquely assigned to dye combinations s of the plurality of dye combinations S1. j Can be assigned to more than one dye combination s in the plurality of dye combinations S1. In this case, a j Multiple unordered pairs, each unordered pair corresponds to a given marker. For example, the marker μ h ={a j ,s f},μ h′ ={a j ,s z},μ h″ ={a j ,s r} can be sharing the same affinity reagent a jIn this case, a given target will be addressed by multiple markers with different dye combinations s. In addition, the same target can be addressed with multiple markers using different affinity reagents a in the plurality of affinity reagents A that bind to the same predetermined structure or analyte. Alternatively, or in addition, ordered pairings are formed between affinity reagents a in the plurality of affinity reagents A and dye combinations s in the plurality of dye combinations S1. An exemplary illustration of markers forming bijective pairs is given in Figure 6G and 6H The pairings shown are bijective or one-to-one, as no affinity reagents or dye combinations are shared by any two markers.

[0339] Figure 7A Schematic diagram of the various dyes Y D 702 Two different examples of "rules for forming dye combinations". In a particularly preferred embodiment of the present invention, each dye corresponds to a number 700 in a binary code 704b, which is a digital code structured into code blocks / code segments 706 corresponding to dye groups A to n, so that each code block / code segment 706 contains a single "1", so that each dye combination includes / contains n dyes. In other words, for example, if the excitation lines 1-5 are 405nm, 488nm, 560nm, 630nm and 700nm, respectively, then in each block corresponding to a group of dyes A to n, the encoding will randomly select one dye from the group to be included in the dye combination. For example, 405nm - Atto930; 488nm - Atto490 LS; 560nm - Alexa Fluor 564; 630nm - Alexa 633nm; 700nm - Atto690. In this case, the encoding provides y of the multiple dye combinations S1. A xy B xy C …xy n Dye combination.

[0340] "Group-based encoding" and "binary encoding" are based on certain "rules for forming dye combinations". In "group-based encoding", the dyes Y D The dyes y are grouped into groups A to n, which can be excited by corresponding excitation lights A to n. By selecting one dye from each group A to n to form a combination, the number of dye combinations in the plurality of dyes S1 is maximized, and each dye combination is unique for the plurality of dye combinations. In group-based encoding, each dye combination is an n-tuple with n members. In "binary encoding", the "rule for forming dye combinations" makes the plurality of dyes y1, y2, y3...y σCorresponding to the specific number 700 in binary code. In "binary coding", according to this preferred embodiment, each combination of dyes includes or contains 1 to y σ members.

[0341] In another particularly preferred embodiment of the present invention, each dye corresponds to a number 700, so that each code 704b may include / contain a different number of dyes, the number being randomly selected and ranging from 1 to y. A +y B +y C …+y n =y σ This is referred to as "binary coding" in this document and results in 2 of the multiple dye combinations S1 (ya+yb+yc…+yn) Dye combination.

[0342] Although combinatorial coding has been described in the prior art, the number of codes that can be obtained is limited because the number of dyes that can be used is limited to 5. Using the methods disclosed in this document, 25 or more members of commercially available fluorescent dyes can be used to define a plurality of dyes that can be read out using commercially available readout equipment. Using available detector and dye technology, a plurality of dyes with 120 or more members can be defined based on the methods disclosed in this article from available fluorescent dyes that can be read out using dedicated readout equipment. When, for example, n = 8 excitation lines (e.g., excitation lines 1-8: 360 nm, 405 nm, 440 nm, 488 nm, 560 nm, 590 nm, 630 nm, 700 nm) are combined with y A =y B =y C …=y n This is feasible when using a set of dyes with a total of 15 dyes (divided into 3τ classes, so that each τ class contains 5 dyes with sufficient spectral separation). In this case, the cardinality (i.e., the number of dye combinations that can be encoded in the set of dye combinations) is in the range of about 2.56 billion dye combinations for group-based encoding, and about 1.33x10 in the case of binary encoding. 36. While this number is significantly higher than, for example, 20,000 to 30,000 (a rough estimate of the number of protein-coding genes in the human genome), using such a large number of available codes may still be preferable because it allows experiments to be set up in such a way that only a small fraction of the codes are actually used. If groups with n = 8 and y = 15 are used in conjunction with this approach to analyze 20,000 target molecules, then α only accounts for 0.00078% of the available codes, while there are 2.56 billion group-based coding dye combinations in our example. If binary encoding were used for the same example, the proportion of dye combinations in the set of unique codes that are actually assigned to a marker, α, would be around 0.0000000000000000000000000000000002%. Remarkably, this is already achievable based on existing dyes and readout technologies. Using a very small α means that the entropy of a set of randomly assigned dye combinations will be higher, and therefore decoding will be easier. Smaller values ​​of α also result in a lower probability of observing multiple type II false positives.

[0343] In a particularly preferred embodiment of the invention, this strategy is used to improve the readout of spots in densely labeled samples.

[0344] Figure 7B Examples of various affinity reagents are shown schematically.

[0345] Figure 7C Schematic representation of a given read volume in a sample is shown, for example, containing a single target molecule bound to a marker 708, the marker comprising a reporter comprising a linker and a combination of dyes 602c, 602d, which are read out in a single 1 to n acquisition sequence 710. N = 5 sets of dyes can be excited, for example, at 405 nm (set A), 488 nm (set B), 565 nm (set C), 630 nm (set D), 700 nm (set E), or in reverse or a different order. For these excitation wavelengths, fluorescent dyes with y can be easily constructed from commercially available fluorescent dyes. A =y B =y C =y D =y E = a dye set of 5 dye members that can be separated spectrally or by combining the spectrum with fluorescence lifetime information. Figure 7B As shown, the fluorescent dyes are preferably excited sequentially so that all dyes are excited and their fluorescence emissions are detected at least once in one readout round.

[0346] Figure 8A particularly preferred embodiment of the present invention is shown in which the combination of dyes 706a, 706b, 706c is mapped to unique oligonucleotide sequence barcodes (UOSBs) 800a, 800b, 800c in a 1:1 relationship. The advantage of this scheme is that it allows the combination of dyes 706a, 706b, 706c to be reversibly connected to a specific marker carrying the corresponding complementary sequence barcodes 800a, 800b, 800c. This means that a library of dye combinations can be constructed and connected to an oligonucleotide library. In order to make the affinity reagent compatible with this process, it is only necessary to attach the complementary sequence barcode (UOSB) to the affinity reagent. This embodiment is very suitable for iterative processes, as described below, and the encoding can be changed between multiple rounds.

[0347] like Figure 9 As shown, oligonucleotide sequence barcodes can also be used for different dye species, in which case the sequences 906a*, 906b*, 906c* direct the conjugated dye to complementary binding sites 906a, 906b, 906c on a linker, which can be a linker 902 comprising an oligonucleotide or a longer DNA, RNA, LNA, morpholino, or other artificial nucleic acid sequence. The linker 902 (which can include multiple different elements) and the dye combination constitute the reporter 908. Figure 9 A schematic example is further shown: a target molecule or analyte 900 is bound to an affinity reagent 500 having a corresponding specificity. This is schematically depicted by the pattern they share in the figure. The affinity reagent is further directly or indirectly attached to a linker 902, which includes a unique barcode of attachment sites 906a to 906e that are complementary to the sequence 906a* to 906e* of a reporter, the sequence of which includes fluorescent dyes or labels 100A to 100E. The linker may also optionally include a cleavage site 904.

[0348] Figure 10A It is shown that multiple dyes of the same kind can be added to a dendritic structure, which can be an oligonucleotide, a peptide or, in particular, a sugar. The sequence 906a* of the reporter can be linked to a single fluorescent dye or label 100A, such as Figure 9 As shown, in Figure 10A Alternatively, the reporter sequence 906a* can be attached to a dendritic structure comprising multiple fluorescent dyes or labels 100A, such as Figure 10A As shown in 1008b on the right.

[0349] Figure 10BSchematically illustrates how dye combinations and / or information related to at least one of dye combinations, affinity reagents, markers, and target molecules are stored in a database and / or storage device together with a unique identifier (unique ID) and related information (e.g., the dyes it contains, identified by Dye_ID) and information about assigning markers. This information may include information about connectors, reporters, markers, and UOSBs (unique oligonucleotide sequence barcodes) and other sequence barcodes (in the case of oligonucleotide connectors 402, where the dye is attached by a dye-specific sequence barcode). The information stored in the storage device may also include information about affinity reagents, such as validation data, suggested dilutions, source species, cross-reaction data, target molecules, and access information to commonly used gene, transcript, and protein databases. Figure 10B Further schematically illustrates where the types of information related to landmarks such as 112 can be stored. A relational database can be used to efficiently record trajectory-related information, enabling fast retrieval and storage while requiring minimal memory on a storage device.

[0350] Figure 11 The sequential readout of markers bound to a target molecule is illustrated, where the affinity reagent is an antibody and the target molecule is a protein. The readout is achieved by directing excitation light of different wavelengths into the biological sample, where the markers are specifically attached to the corresponding analyte via a readout / acquisition sequence 710 (denoted by 1 to n). Figure 12 Schematically shows the Figure 11 The same process is used, but with a nucleic acid target (e.g., DNA or RNA target) that is bound by hybridization to a complementary oligonucleotide marker, which may include DNA, RNA, PNA, LNA, morphine, or other forms of artificial nucleic acid.

[0351] Figure 12 Sequential readout of markers bound to target molecules is illustrated, where the affinity reagents are oligonucleotides and the target molecules are DNA or RNA sequences.

[0352] Figure 13 Schematically shown for three spots or read volumes Figure 11The same process is followed, illustrating the independence of the combinatorial code from the spatial position of the reporter oligonucleotide on the adapter oligonucleotide barcode. Readout volumes K, M, and P contain individual target molecules, each derived from the same species and bound to a label molecule with the same specificity, but with differentially positioned attachment sites in their adapters for the same dye combination. As shown in the figure, regardless of the placement of the dyes, as long as the dye spacing is essentially within the readout volume, i.e., the detection optical system cannot spatially resolve, the same readout sequence is obtained for readout volumes K, M, and P.

[0353] like Figure 14 As shown in the histogram and discussed above, the exponential nature of the encoding is the basis of the statistical power of the method. Figure 14 Another example is shown where group-based encoding is used, where n=5 groups of dyes are excited at 405 nm (group A), 488 nm (group B), 565 nm (group C), 630 nm (group D), 700 nm (group E), and each group contains 5 dyes, i.e., y A =y B =y C =y D =y E=5. In this case, 3125 unique codes can be easily generated, which is enough to provide a uniquely labeled marker for each protein in the human secretome, thereby enabling the profiling of the secretome to be completed in a single round of analysis, i.e., without the iterative process of staining, readout, and dye inactivation. In addition, this means that only 1-10 rounds of iterative processes of staining, readout, and dye inactivation are required to uniquely label the entire human genome and its major gene products. In contrast, modern multiplexing solutions can image approximately 60+ biomarkers based on fluorescence imaging in 12 rounds of imaging. Similarly, modern cytokine profiling solutions allow the readout of approximately 40 cytokines (a special class of secreted molecules) in a single round of imaging. Therefore, the method disclosed in this article has significantly improved over the existing technology and can characterize immune cells, etc. based on full secretome analysis. This is particularly important in immunology, infection biology, and immuno-oncology to analyze the function and effectiveness of immune cells and derive predictive biomarkers that can be used to stratify patient populations. For example, based on such analyses, we might be able to predict whether a person infected with a virus (such as SARS-CoV-2) will have a mild or severe illness based on a full secretome profile of immune cells. Similarly, we might be able to predict whether a cancer patient will have a good or poor prognosis without treatment, and whether the immune cells are still effective or have entered a state of immunosenescence or become ineffective for other reasons. Similarly, full secretome profile analysis can help predict the effectiveness of genetically modified immune cells such as CAR-T cells and stratify patients into those who respond and those who do not respond to various immunotherapies (cell-based or non-cell-based). In addition, based on such analyses, we might be able to predict which interventions (such as drug combinations) are likely to activate less effective immune cell populations.

[0354] Figure 15 is a schematic diagram of a whole cell 1500 as an example of a biological sample. Figure 15 , for the sake of clarity, only the marker and its linker 902 are shown, and the dye 100 is omitted (i.e., not shown). The cell includes a nucleus 1502 and a cytoplasm 1504, and each cell includes a structure called an epitope 1506a-c, to which antibodies can attach. The figure illustrates two markers 112 as an example, which include a single domain antibody 1518 that binds to an epitope 1506c located in the nucleus 1502 and the cytoplasm 1504, respectively. An affinity reagent can be connected to the linker 902. Another marker includes a first antibody 1512 and a second antibody 1514 conjugated to the linker 902. The first antibody 1512 is attached to the epitope 1506b in the cytoplasm 1504, and the second antibody 1514 is attached to the first antibody 1512. In addition, Figure 15Two markers 1520a and 1520b are shown, which include labeled oligonucleotide sequences as their affinity reagents. The two markers 1520a and 1520b are attached to complementary sequences 1508 and 1510, respectively. The first of the two markers 1520a is attached to an RNA sequence 1510 in the cytoplasm 1504. The second of the two markers 1520b is attached to a DNA sequence 1508 in the nucleus 1502. The marker including a toxin 1522 (e.g., phalloidin) as its affinity reagent is attached to actin filaments 1524.

[0355] Figure 16 The schematic diagram shows the size range of relevant structures in immunoassays and immunohistochemistry experiments. For ease of illustration, the figure shows atoms 1600, small molecules 1602, single domain antibodies or nanobodies 1604, GFP molecules 1606, antibodies 1608, quantum dots / polymer dots / graphene-based dots / other nanostructures in the range of 2-10 nm 1610, roughly to scale.

[0356] Figure 17 Schematically, an antibody 1608 coupled to a 10 nm diameter quantum dot 1610 is placed in the PSF 1700 of a 1.4 NA objective lens. Figure 11 and Figure 12 showed that the optical resolution was insufficient to read out the reporter oligonucleotide located on the linker oligonucleotide barcode, which is why despite Figure 13 In the example shown, the positions are different but the readout of spots K, M, P is exactly the same.

[0357] Figure 18 An iterative staining and imaging process is schematically shown, which is known in the art and is used for multiplex imaging. The process begins in step S1800, and in step S1802, the sample is stained. In this step, a marker is introduced into the sample, brought into contact with its predetermined structure, and labeled. Typically, in the methods disclosed in this document, these markers are directly conjugated to a dye. Alternatively, the marker is typically indirectly conjugated to a dye and can enter the sample with or without a reporter attached. In the latter case, the reporter can enter the sample but dynamically binds and dissociates from the marker. The reporter can enter the sample in step S1802. In step S1804, the reporter is sequentially excited and read out. In step S1806, the reporter is inactivated by bleaching or removal from the sample, and the process can be repeated or terminated in step S1808. Alternatively, after the readout step S1804, the sample can be sent to a downstream analysis process step S1810.

[0358] Optionally, after image acquisition or non-image-based readout, the fluorescent dye 100 is inactivated in step S1806. Inactivation is performed to prevent the fluorescent dye 100 from emitting fluorescence in the future. Methods for inactivating the fluorescent dye 100 include bleaching the fluorescent dye 100, i.e., chemically inactivating the fluorescent dye 100 or photophysically bleaching the fluorescent dye 100; or removing the fluorescent dye 100 from the sample. To remove the fluorescent dye 100 from the sample, the connection between the primary affinity reagent 108 to 118 and the predetermined structure, target molecule, or analyte 900 must be severed. For example, if the affinity reagent is an antibody 108 to 118, this can be achieved by antibody elution. Alternatively, the fluorescent dye 100 can be removed from the primary affinity reagent 108 to 118 or the secondary affinity reagent (not shown for clarity). For example, this can be achieved by enzymatic cleavage at the binding site 904 of the peptide 4802 or oligonucleotide 402 that connects the fluorescent dye 100 to the affinity reagent 108 to 118. Alternatively, the fluorescent dye 100 can be reversibly bound to the affinity reagents 108 to 118, for example, by oligonucleotide hybridization and the use of barcoded antibodies. For hybridization-based oligonucleotides 116, standard in situ hybridization or fluorescence in situ hybridization (FISH) protocols can be used to hybridize and dehybridize the oligonucleotides. In this case, the fluorescent dye can be hybridized to the linker and the intact marker can be introduced into the sample. Alternatively, the sample can be stained using affinity reagents 108 to 118 with barcoded linkers 902 and the dye can be added at a later time point. Figure 1 Unlabeled primary affinity reagents 122 to 132 are also shown.

[0359] Figure 19 A cell-based secretory protein detection method is schematically illustrated. A region 1900 is shown around a cell 1500. This region can be a virtual volume, where the detected secretory proteins are assigned to the included cells 1500, or a physical volume, such as when the cells 1500 are encapsulated in discrete entities such as hydrogel beads. In both cases, the cells 1500 are embedded in a matrix, such as a matrix used for 3D cell culture, including but not limited to hydrogels, agarose, cellulose, alginate, Matrigel, or the like. TM , collagen, bio-ink and similar biocompatible materials. The matrix can be monophasic, i.e. the area for cell culture and the area for capturing and detecting secreted molecules can be the same, or it can be multiphasic, i.e. there can be different culture areas, capture areas and detection areas, which can include different materials. In the latter case, polymers (e.g. polyacrylamide, polyethylene glycol, polylactic acid, polyvinyl alcohol, polyoxazoline, polystyrene) can be used as capture and detection areas. Figure 19In either case, the capture affinity reagents 1902a, 1902b can be covalently attached to the matrix. After the secreted proteins 1524a, 1524b, 1524c are secreted, they can be released into the matrix. Figure 19 In the example shown, secreted proteins 1524a, 1524b are then immobilized by capture affinity reagents 1902a, 1902b, as shown in the magnified inset. This immobilization allows detection of the secreted molecules even under harsh washing conditions via appropriate markers 1526a, 1526b, which are identified by their respective partitioning dye combinations 706a and 706b.

[0360] Figure 20A A particularly preferred embodiment of the present invention is schematically shown, wherein Figure 19 The same process is shown using a different immobilization strategy. In this case, capture affinity reagents 1902a, 1902b are immobilized on microbeads 2000, such as latex or polystyrene beads. Immobilization can be achieved through direct covalent coupling using a variety of chemical methods (e.g., NHS, maleimide coupling), or through indirect coupling using oligonucleotide barcode linkers, biotin-(strept)avidin interactions, or protein A or protein G-mediated coupling. For example, human cells 1500 can be embedded using hydrogel beads with a diameter of 50-250 μm (typical size is 5-50 μm), and a mixture of microbeads 2000 with a size of 50-500 nm, carrying multiple capture affinity reagents 1902a, 1902b, can be added during the embedding step. Due to the size of the microbeads, they are immobilized in the hydrogel, meaning that they are in a substantially stable position relative to each other and the circumference of the hydrogel bead. Therefore, they are well-suited for defining detection spots or detection areas, as their size can be adjusted and selected based on the numerical aperture of the readout device. A high local concentration of the capture affinity reagent on the beads is particularly desirable because it results in higher intensities per unit area, better signal-to-noise ratios, and eases segmentation and analysis. Furthermore, this high local concentration of the capture affinity reagent on the beads can produce a significant avidity effect, allowing even weak and transient interactions to be detected by the method—in other words, significantly improving assay sensitivity. Larger libraries of beads loaded with capture affinity reagents can be prepared and flexibly applied to a variety of assays and different assay formats, such as microtiter plates or flow-through assays.

[0361] Figure 20B A particularly preferred embodiment of the present invention is schematically shown, wherein the Figure 19 and Figure 20A The same process as shown in , where the capture affinity reagent is immobilized on a nanostructure 2002, such as graphene, carbon tubes, nanorulers or other DNA origami based nanostructures.

[0362] Figure 21 Schematically illustrates how microbeads and similarly sized nanostructures in the ranges of 1-5 nm, 5-10 nm, 50-100 nm, 100-200 nm, 250-350 nm, and 350 nm-1000 nm, as well as 1000 nm-5000 nm, are particularly preferred for use in various assays involving secreted proteins, such as Figure 19 、 20A , as shown in 20B, because the random placement of the beads facilitates the different placement of the detection spots and thus facilitates the readout. Figure 21 Schematically shown are a combination of two spots identified as protein C by their unique combination codes 800c and the dyes 706c attached thereto, and a combination of three spots identified as protein D by their unique combination codes 800d and the dyes 706d attached thereto. Figure 21 It further explains how to decode and count the spots. Figure 21 The bottom bar graph shows a schematic representation of spot counting results for several proteins.Alternatively or additionally, DNA origami-based nanostructures are well suited for such assays because they can be produced in relevant sizes and can be easily modified to include attachment sites for fluorescently labeled oligonucleotides or other tags and functionalizations.

[0363] Figure 22 Shown for Figures 19 to 21 Flowchart of a workflow for finding and reading out a single species readout volume in an assay shown, starting at step S2200. This may also be referred to as an image processing pipeline. Such an image processing process may include at least one of the following: background removal, compression, filtering, denoising, enhancement, reconstruction, correction, deconvolution, multi-view deconvolution, multi-view registration, multi-view fusion, and other tools familiar to those skilled in the art of digital image processing, and typically includes an image segmentation step and a feature classification step, wherein the image segmentation step generates a set of segmented objects, which may also be referred to as features. For many of the above-mentioned image processing tools, there are both classic software algorithms and (modern) machine / deep learning or artificial intelligence-based algorithms. In particular, for pixel classification, image denoising, deconvolution, enhancement, segmentation, and classification of segmented objects / features, machine / deep learning or artificial intelligence-based algorithms exist and are known to outperform most classic algorithms. Performing image analysis, image improvement, and / or image deconvolution on the images generated by the optical readout may be more advantageous.

[0364] Some steps can be omitted or repeated. Figure 22Other steps not shown in the figure can be included in the image processing pipeline. Substeps of the pipeline can be performed using classical image processing algorithms and / or machine learning or deep learning algorithms. The output of feature extraction can be a set of features that includes features from the hydrogel bead (which may be embedded in the biological sample), such as its outer boundary, volume, or center of mass; a subset of features from the external structure of the sample (including the read volume of a single species); and a subset of features from the biological sample.

[0365] In step S2202, the image data is preprocessed using various feature detection methods, including segmentation and filtering, which may include background removal. The components of the marker can be identified by their keypoint features, edges, and points of interest / features. A feature point or point of interest can be any detectable object, such as a microbead 2000. Keypoint features can be any object with a specific neighborhood. For example, a microbead 2000 can be segmented and its center of mass determined.

[0366] The image segmentation analysis in step S2204 can be performed using at least one of the following methods: classical methods, artificial intelligence-based techniques (including machine learning and neural networks / deep learning), or other techniques, including thresholding techniques, clustering methods, compression-based methods, histogram-based methods, edge detection, biclustering methods, region growing methods, partial differential equation-based methods, variational methods, graph segmentation methods (e.g., Markov random fields), watershed transforms, model-based segmentation, multi-scale segmentation, semi-automatic segmentation, trainable segmentation using various machine learning, neural network, and artificial intelligence methods, such as pulse coupled neural networks (PCNNs), convolutional neural networks (U-Nets), recurrent neural networks (RNNs), and object co-segmentation methods (e.g., Markov networks), convolutional neural networks, or long short-term memory networks (LSTMs). Alternatively or additionally, features such as size and / or color and / or fluorescence intensity and / or fluorescence lifetime can be used to identify components of the marker from the image data. Various algorithms can be used for identification, including the Harris Corner algorithm, Scale-Invariant Feature Transform (SIFT), Speeded Up Robust Features (SURF), Speeded Up Segment Test Features (FAST), and Oriented Rapid Rotation BRIEF (ORB), which are all known and can be used to identify components and / or features of landmarks from image data.

[0367] Particularly preferred is the use of machine learning and deep learning methods (such as content-aware feature enhancement) for blob detection and / or feature extraction and / or feature classification analysis S2206. This is particularly advantageous when fluorescent microbeads, fluorescent nanorulers, or similar structures are used to generate the markers, as this allows the neural network to be pre-trained to enhance these features based on content-aware features. Similarly, in this case, extensive pre-training can be easily performed on real data (i.e., image data of hydrogel microbeads containing only the markers). Importantly, the real data can be generated on different imaging systems. Specifically, the real data can be generated on imaging systems with high optical performance (e.g., numerical aperture, resolution, light collection efficiency, light flux, signal-to-noise ratio, chromatic or spherical aberration, and any other imaging aberrations). Importantly, the method can be implemented in such a way that each run generates corresponding training data, which can potentially improve the quality of the generated image data by improving methods such as denoising, background removal, image correction, deconvolution, and feature extraction performance. Similarly, the network can be pre-trained using suitable reference samples to classify features. Specifically, the network can be pre-trained to classify features as hydrogel beads, fluorescent microbeads, fluorescent nanorulers, or the like, as well as cells, cell populations, or other types of biological samples.

[0368] After feature extraction and classification, the spots are read out and their dye combinations are decoded in step S2208 by looking up the corresponding markers and their target molecules / predetermined structures in a storage device. Each spot marked with a specific dye combination is then counted and the results, along with the intensity information, are stored in a storage device. The process ends in step S2210.

[0369] Figure 23 Schematically shows how to perform the sample transfer in a standard microscopic sample carrier (e.g., a microplate 2300 with 96 wells or sample containers 2302, each with a transparent window or bottom 2304). Figures 19 to 21 In this case, the sample (e.g., cell 1500a) is segmented and its centroid is found 2308, and a virtual read volume 2310 assigned to the cell 1500a is relative to this reference point. The sample and virtual read volume 2310 are imaged or read using a microscope or microplate reader 2312 having a light source 2314, a beam path with a beam routing and beam splitting assembly 2316, an illumination / detection optical assembly 2318 (which may be the same optical assembly or a different optical assembly), and a detector 2320 for reading the fluorescence intensity. The detector 2320 may also be a Figure 2 The spectrometer shown.

[0370] In a particularly preferred embodiment of the invention, the detector and light source are configured to perform spectral fluorescence lifetime imaging, which can be used, for example, with spectroscopic FLIM phasors and provides high dye separation capabilities for the readout device.

[0371] Figure 24 Schematic illustration of how flow cytometry can be performed in an imaging cytometer, cell counter, or microscope configured to perform flow-based imaging. Figures 19 to 21 In this case, Figure 23 The imaging system 2312 described in the embodiment is used in conjunction with a flow cell 2400 having an inflow 2402 and an outflow 2404, and at least one fluid channel in which an immersion medium or flow medium 2406 flows. In this case, the sample 1500 is embedded in hydrogel beads 2408, which is particularly advantageous because it protects the enclosed sample 1500 and allows Figure 19-21 The assays described in can be performed in flow-through at very high throughput. For example, PCT / EP2021 / 061754, the entire contents of which are incorporated herein by reference, describes methods for embedding and identifying biological samples in hydrogel beads.

[0372] Figure 25 An assay of a bead-based cytometer 2500 is shown, wherein microbeads 2000a carrying capture reagents 1902a are excited by light emitted by a light source 2314, and the emitted fluorescence is detected by a detector 2320. Figure 25 As shown, each microbead 2000a can carry an affinity reagent 1902a having a particular specificity, effectively making it a readout volume for a single species. Such capture beads 2000a can be incubated with cells in, for example, a well for a given time (see Figure 23 ), followed by cytometry analysis. In a particularly preferred embodiment of the method, the method is used to analyze the entire secretome using the bead-based format and cytometry as readout.

[0373] Alternatively or additionally, the capture microbeads 2000a can be incubated with a cell lysate, a sample lysate, or an environmental sample lysate to detect the presence of an analyte in the sample. In a particularly preferred embodiment of the method, the method is used to detect the presence of a large number of analytes in the same experiment using a bead-based format and cell counting as a readout.

[0374] Optionally, the captured beads 2000a can be sorted using a fluorescence activated cell sorter (FACS) 2502 that directs the beads to corresponding collection tubes 2506 using guide plates 2504 .

[0375] Figure 26 Shown Figure 25The assay described in can be used to detect the presence of an analyte in the capture reagent-target molecule-marker structure 2600 shown at the top, which can also be as shown in FIG. Figure 19 、 20A Or the structure shown in 20B. Alternatively or additionally, the format can also be adjusted to the capture reagent-target molecule-interaction molecule-marker structure 2602 shown below. The former allows the analysis of the presence of the analyte, while the latter is able to detect the interaction between one or more analytes captured by the capture reagent and the interaction molecules (i.e. other analytes, such as small molecules, proteins, nucleic acids), which in turn can be read out by marking the markers of the bound interaction molecules. The method disclosed herein is used together with such microbead-based detection methods, combining the advantages of microbead-based detection methods (e.g., the significant affinity effect produced by concentrating the capture reagent on the microbeads) with the advantages of the method disclosed herein (the method provides a method for detecting a large number of analytes and / or evaluating a large number of interaction molecules). The interaction molecules may be present in solution or may be molecules expressed on the cell surface. In this way, microbead detection based on capture reagent-target molecule-interaction molecule-marker structure 2602 can be used to detect the presence of cells based on a combination of multiple cell surface molecules. In this way, the method can be used to detect the presence of certain cell types, which can be rare cell types, such as circulating tumor cells or immune cells that respond to specific antigens. Both detection methods are well suited for situations where a large number of events, a large number of analytes, high throughput or a short time to result is required.

[0376] Figure 27 Shown with Figure 25 Similar cytometer 2500 and FACS 2502. In this case, the samples in the sample stream are single cells 1500. Figure 27 The analytical method schematically depicted in FIG illustrates a particularly preferred embodiment of the present invention, wherein a plurality of markers are used to label proteins or other target molecules (e.g., carbohydrates) bound to the cell surface of cells 1500. These markers may be, in particular, cell surface receptors and "cluster of differentiation" proteins (CD proteins). These cell surface-bound target molecules 2700 are bound to corresponding markers, which are composed of a combination of affinity reagents 1526d and dyes 706.

[0377] In a particularly preferred embodiment of the present invention, Figure 19-27 The assay described in is configured to generate primarily a single species readout volume, see e.g. Figure 28 2802a, 2802b, these readout volumes can be read directly, that is, the readout data therein directly decodes the identity of the marker.

[0378] In a further particularly preferred embodiment of the present invention, Figure 25-27 The assay described in is used to perform ultra-high multiplexed cytometry, e.g., cytometry with 100-1000, 1000-2000, 2000-10000, 10000-30000, 30000-100000, and more than 100000 markers read out in the same experiment, preferably in a single round (multi-species readout volume), see e.g. Figure 28 2804 of them.

[0379] In another particularly preferred embodiment of the present invention, Figure 23 and 24 The detection methods described in the present invention are used for ultra-high-density imaging in standard sample carriers or flow cells, for example, imaging of 100-1000, 1000-2000, 2000-10000, 10000-30000, 30000-100000 and more than 100000 markers, which can be read out in the same experiment and preferably read out in a single round or multiple rounds of staining, imaging, and dye inactivation (multi-species read volume).

[0380] Figure 28 The difference between single species read volumes 2802a and 2802b and multi-species read volume 2804 is shown. Figure 28 As shown, a single-species read volume contains markers with the same reactivity or specificity 500, which are present in either a single copy 2802a or multiple copies 2802b. Therefore, the read sequence of a single-species read volume 2802a or 2802b is the same as one of the dye combinations in the set of marker-assigned dye combinations. This means that to decode a single-species read volume, i.e., to identify the marker species contained therein, it is sufficient to read the single-species read volume and retrieve the corresponding marker ID by comparing the read sequence with the dye combinations in the set of marker-assigned dye combinations stored in a storage device. Figure 28 To further illustrate, the multi-species read volume 2804 includes at least two labels 500 having different reactivities or specificities.

[0381] In contrast to single-species read volumes, multi-species read volumes cannot be decoded simply by acquiring the read sequences and retrieving the underlying tags. Figure 29 As shown, a multi-species readout volume may contain many different markers. Figure 29As shown, the confocal readout volume or effective point spread function 1700 and the number of dyes, for example 100, of a first set of dyes are shown. For the sake of clarity, each dye corresponds to one marker molecule, and the rest is omitted (i.e., the affinity reagent and the other dyes in the corresponding dye combination). For example, if a plurality of different marker molecules are excited with a first excitation light, the first dye in the corresponding dye combination will be excited and emit fluorescence. This light will be detected and separated into the corresponding dye channels, which is schematically represented by the column of circles on the right, which intuitively represents the readout sequence 2900. The circle on the right has only a single pattern, reflecting all the properties used for dye separation, while the circle in the PSF 1700 corresponds to dye 100, the left half of which corresponds to the excitation spectrum, and the right half of which corresponds to the dye properties that can be used for dye separation (e.g., emission spectrum, fluorescence lifetime, excitation fingerprint). In Figure 29 In the example shown, after directing a first excitation light onto the sample, the emitted fluorescence is detected and the method records the presence of 10 dyes, which are represented by ten circles with ten different patterns. In addition, the method also records the intensities of these dyes. Now, all other excitation lights are applied in a similar manner and the fluorescence is detected as described above. In this way, a first readout sequence is obtained (e.g., the first iteration from the staining-imaging-readout iterative process). Depending on the number of different markers in the spot, this may result in a large number of codes that can be summarized under this readout sequence, and also in a large uncertainty, because many of the summable codes may be false positives, i.e., the markers to which they are assigned may not be present in the confocal volume or the effective point spread function.

[0382] The following discussion first discusses strategies to mitigate this problem and then moves on to robust solutions to this problem.

[0383] To reduce the difficulty of decoding multi-species readout volumes, dye scintillation techniques and methods for local microscopy can be used to effectively convert the multi-species readout volume into multiple temporally separated, sub-diffraction localized single-species readout volumes. Figure 30 As shown, only one dye lights up in each PSF.

[0384] In a particularly preferred embodiment of the present invention, the difficulty of multi-species readout volume decoding is solved in principle. Figure 31 As shown, for y A =y B =y C =y D =y E = 10 and n = 5, the group-based encoding generates ψ = 100,000 combinations, and the binary encoding generates ψ = about 10 15 This means that α is 20%, and is about 1.8x10-11 In other words, as the histogram shows, only a small or minimal portion of the available codes are actually assigned to markers (grey rectangles).

[0385] Figure 32 It illustrates how different initial readout sequences affect the number of dye combinations that κ can be summarized under a given initial readout sequence. In "Example 1", a readout sequence is observed in which each number corresponding to dyes A1...n. y is displayed as "1", i.e., all dyes are observed. In this case, all multiple dye combinations S1 3200 can necessarily be summarized under the readout sequence of "Example 1". This is schematically represented by the large circle 3200. In "Example 3", the readout sequence can clearly identify a single dye combination 602, thereby identifying a single marker (single species readout volume). In "Example 2", a readout sequence is obtained under which a large number of dye combinations can be summarized, forming a "set of dye combinations that can be summarized under the first readout sequence" 3202. The latter case is the actual situation of the multi-species readout volume, which will be discussed further below.

[0386] Figure 33A This is illustrated in schematic form. In this case, we have a first readout sequence and all the various dye combinations S1 are shown, a total of 3200. Each circle in the figure corresponds to a dye combination 602. Most circles are filled in white, corresponding to dye combinations that are not assigned to a marker. Some circles are filled with patterns (large confetti patterns or diagonal stripes), corresponding to dye combinations assigned to markers. Most circles are drawn with thin outlines, similar to all the dye combinations that cannot be summarized under the first readout sequence. Some circles are drawn with only solid black thicker outlines and represent dye combinations that can be summarized under the first readout sequence. Figure 33A similar, Figure 33B and 33C The situation under the second and third readout sequence after reassigning the dye combination to the affinity reagent or vice versa and thereby reassigning the dye combination to the marker (and vice versa) is shown. In this sense, Figure 33A The first iteration in the experiment is shown, Figure 33B is shown a second time, and Figure 33C , where iteration refers to Figure 18 and 37 The iterative staining, imaging, and dye inactivation process shown in FIG. Between these steps, the plurality of dye combinations S1 3200 may remain the same, as shown, but their assignments may be randomly changed. Alternatively or additionally, the assignments may be deterministically changed. Alternatively or additionally, a second plurality of dye combinations S1 3200 may be formed based on a suitable second "dye combination forming rule." 1.2and is used in the second iteration (not shown in the diagram for clarity). By quickly changing Figure 33A This can be best observed with -C (which animates the example.) This can be compared to repeated drawing with put / replace.

[0387] Figure 34A -C should be understood as Figure 33A -C, wherein the "set of dye combinations that cannot be assigned to the first, second, and third readout sequences" 3300a, 3300b, 3300c and the "set of dye combinations that can be assigned to the first, second, and third readout sequences" 3302a, 3302b, 3302c are shown. The "set of dye combinations that can be assigned to the first, second, and third readout sequences" 3302a, 3302b, 3302c are further divided into the set of markers that are actually present (true positives) 3400a, 3400b, 3400c and false positives (which can be either Class I (dye combinations that can be assigned to the readout sequence are not assigned to a marker) or Class II (dye combinations that can be assigned to the readout sequence are assigned to markers that are not physically present in the readout volume / effective PSF). Figure 34A The best way to observe -C is to quickly switch Figure 34A -C, presents this example in animated form. As the example shows, the assignment of dye combinations changes in a random manner in each iteration (similar to random draw and replacement). In addition, the composition of 3400a, 3400b, 3400c changes according to the dye combination, but not according to the markers they encode. Since 3400a, 3400b, 3400c are the true positive groups, their associated dye combinations can be summarized under the corresponding readout sequence in all cases. In other words, the affinity reagents and their respective labels will be elements of 3400 in all iterations. However, since the assignment of dye combinations to affinity reagents changed in the three iterations shown, the true positives read out on the first occasion will be in the same order. Figure 34A In the figure, the markers μ23, μ2015, and μ434 are expressed. The second true positive reading is Figure 34B In the figure, the markers μ23', μ2015', and μ434' are represented. The third true positive is Figure 34C It is indicated as markers μ23", μ2015", μ434". It should be understood that markers μ23, μ23' and μ23" share the same affinity reagent but include different dye combinations. The composition of the set of false positives 3402a, 3402b, 3402c will change significantly in each iteration, both for the dye combination and the included markers. Figure 34A and Figure 34BAs shown, from one iteration to the next, the set of false positive results may accidentally contain one or more identical markers, such as μ4124, μ4124'. This means that an iterative approach can be used to decode a multi-species read volume, and the probability of observing a false positive depends on the proportion of dye combinations that can be attributed to the read sequence. In other words, if 3202 accounts for a large portion of 3200, it is more likely that false positives (type I and type II) will be observed, and more iterations must be performed to obtain the statistical confidence level required by the user. If 3202 accounts for a small or extremely small portion of 3200 (which can easily happen) (compare Figure 31 ), then a few iterations will yield excellent p-values ​​and statistical confidence in detecting the marker within the readout volume / effective point spread function. In other words, smaller values ​​of α lead to higher confidence with fewer iterations. Therefore, the cardinality of 3,200 dye combinations (also referred to as multiple dye combinations S1) fundamentally limits this approach. Because the cardinality grows exponentially while the number of target molecules is essentially limited (e.g., approximately 20,000 protein-coding genes), this approach is extremely powerful for solving a wide range of problems in microscopy and cytometry, both in life science research and diagnostic applications.

[0388] Figures 35A to 35C A given marker 112 with a specific reactivity 500 is shown with a target molecule 900. Figure 35A In the first iteration shown, the first dye combination is attached to the marker μ2015; Figure 35B In the second iteration shown, a second dye combination is attached to the marker μ2015′; Figure 35B In the third iteration shown, a third dye combination is attached to the marker μ2015".

[0389] Figure 36A is the probability p of observing an absent marker with the same probability multiple times (type II false positive) i The probability is calculated by κ i / ψ is given by, where κ i is κ in round i. For simplicity, κ i is set to 1000 in all rounds, corresponding to 1% of ψ in this example. i It is an important factor affecting the marker-specific p-value, which itself is strongly dependent on and proportional to α.

[0390] Figure 36B shows that n=4 and y A =y B =y C =y D=5 and ψ=625 and an exemplary readout sequence and a list of dyes and corresponding marker combinations that can be summarized under the readout sequence. Dye combinations that can be summarized but not assigned to a marker (i.e., class I false positives) are directly excluded from the list and are therefore not shown in this example. In the example, κ=180, which corresponds to an α= of 28.8%. This means that in order to observe a relatively small list of markers that include true positives and class II false positives, it can be expected that only a small fraction of the α available codes can be used. If, as in this example, ψ is small, then this is an important practical limitation. For achievable ψs, the range can easily reach >10 4 、>10 6 、>10 10 、>10 15 、>10 20 and above, but this is becoming less and less relevant in practice. Figure 36B In the example shown, more than 10 iterations are required to reduce the probability p of observing the same absent marker multiple times (a type II false positive) to i At 10 -6 If α is small enough, this can be achieved after the second iteration.

[0391] Figure 37 A workflow for a first qualitative iterative multi-species read volume decoding is shown. The workflow begins at step S3700. In step S3702, the sample is stained with at least one set of markers labeled with a first set of dye combinations. In step S3704, a first read sequence is acquired, and in step S3706, a first set of all dye combinations that can be grouped under the first read sequence is retrieved from a storage device and stored in the storage device. Now, in step S3708, the dyes from the first iteration introduced in step S3702 are inactivated. Next, in step S3710, the sample is stained with at least the affinity reagent subset used in step S3702, now with a second set of dye combinations (i.e., a second marker subset with the same reactivity as the first marker subset but with a different labeling scheme). In step S3712, a second read sequence is acquired, and in step S3714, a second set of all dye combinations that can be grouped under the second read sequence is retrieved from the storage device and stored in the storage device.

[0392] In step S3716, the first and second dye combinations that can be summarized under the first and second readout sequences are compared, an overlap region is established, and the overlap region is stored in a storage device. In step S3720, the statistical confidence of the decoding result is evaluated, and a p-value and / or other suitable statistical confidence metric is calculated for each marker in the overlap region in step S3720. This may include the use of intensity information and other information including multiple measurements in the confocal volume / effective point spread function of the overlap region. If the p-value is acceptable to the user in step S3722, the process can be terminated in step S3724. Alternatively, further iterations can be performed as indicated by the arrow pointing back to step S3708.

[0393] Figure 38A An example of a readout sequence and the intensity distribution corresponding to the intensity of the corresponding dye channel are shown. The dye detected in the confocal volume / effective point spread function is represented by a "1" in the corresponding number of the readout sequence. At the same time, the intensity information of each dye is stored in the storage device. Figure 38A As shown, the initial qualitative decoding 3800 uses the multiple readout sequences obtained by the above-mentioned iterative process and processes the set 3200 of dye combinations to find multiple dye combinations that can be summarized under the corresponding readout sequence, thereby establishing overlapping areas and statistical confidence in the decoding results. Secondary qualitative decoding 3802 is itself a decoding strategy, or it can be an additional optional step after the initial qualitative decoding 3800. Compared with the initial qualitative decoding 3800, the secondary qualitative decoding 3802 takes into account intensity information, such as intensity distribution. This can be achieved in a simple way by performing intensity thresholding on the readout sequence, which can significantly improve the statistical confidence of the overall result, as shown in FIG. Figure 38B As shown. Alternatively or in addition, it can be used for a special form of linear unmixing. Essentially, this is because the initial qualitative decoding 3800 qualifies the identity of the markers included in the confocal volume / effective PSF according to a certain level of statistical significance. If this list of markers is taken as input, the problem will be a set of just-determined or over-determined linear equations that can be solved. This is similar to the way a linear decomposition is performed, where the intensity of a single dye is separated from the emission spectrum of the overlapping region. In this case, the relative amount of dye that can be attributed to all readout sequences can be found and the shape of the intensity distribution explained. The result of this process is a relative qualitative decoding, that is, it is found that a particular set of readout sequences, a particular set of marker combinations found in the confocal volume / effective PSF based on a certain level of statistical significance are consistent with certain relative amounts of markers and target molecules, such as the following: μ14 is 4 times higher than μ9 or μ548, and μ9 is 1 / 10 lower than μ255, as shown in Figure 2. Figure 38BIf the readout device is properly calibrated using suitable standards, this method can also provide the absolute number of marker and target molecules within the confocal readout volume and the effective PSF.

[0394] Figure 9 、 Figure 10A and Figures 39 to 47 Various particularly preferred marker embodiments are shown. These markers generally comprise an affinity reagent 112 that binds to a target molecule / predetermined structure / analyte 900 and its specificity-determining region, portion, or sequence 500. For example, in the case of an antibody or oligonucleotide, this region, portion, or sequence can be a paratope or complementary sequence. The affinity reagent can also be a small molecule / drug / drug-like molecule / toxin 118, i.e., an affinity ligand, which binds to an affinity receptor in the sample. The marker is directly linked to a linker 902 via covalent conjugation or indirectly via oligonucleotides and other methods. The linker is further linked to a dye combination, i.e., a specific dye combination 600. For the purposes of this document, the combination of a linker and a fluorescent dye is referred to as a reporter 908. The dyes in the dye combination can be present in stoichiometric or non-stoichiometric amounts, depending on the specific requirements of the application, the type of linker, and the coupling or linking strategy used. The linker can have other functionalities, in particular, at least one cleavage site 904, which allows for particularly easy and rapid removal of the linker containing the dye. This is particularly preferred as a means of dye inactivation. The cleavage site 904 can be a nucleotide sequence, such as a restriction site, a target for CRISPR / Cas or similar enzymes, a photocleavable linker, or a proteolytically addressable site, such as a caspase cleavage site.

[0395] Alternatively or additionally, the linker can carry a unique oligonucleotide sequence barcode (UOSB) that identifies a specific dye combination and can be used to flexibly attach the reporter to any affinity reagent carrying a sequence complementary to the corresponding UOSB. Using this strategy, or simply using a universal oligonucleotide sequence to attach the reporter to an oligonucleotide conjugated to an affinity reagent, is advantageous because it allows for flexible attachment and removal of dyes by hybridization and melting. Using oligonucleotides to connect antibodies and linkers by hybridization is particularly preferred because it allows the affinity reagent to enter and remain in the sample independently of the reporter. In addition, once the affinity reagent is bound to its target structure, this strategy allows for particularly easy switching of dye combinations during iterative multispot decoding because it does not require removal of the affinity reagent from the bound target.

[0396] Figure 39 A marker is shown with a linker 902 comprising an oligonucleotide backbone, which may also be a peptide, nanoruler or other DNA origami based structure (cf. Figure 48A and48B ) non-specific fluorescent dye binding sites 3900, which can be NHS or maleimide coupling sites, or click chemistry coupling sites such as alkyne-azide coupling. Figure 39 In the case of , the dye can be coupled to the linker simply by adding the dye mixture to the reaction mixture, which will result in a certain distribution (non-stoichiometric coupling).

[0397] Figure 40 Shown Figure 39 The state of the linker shown in FIG after the dye coupling step, wherein all coupling sites or binding sites 3900 are occupied by dyes 100C and 100E.

[0398] Figure 41 A marker is shown with a linker 902 comprising an oligonucleotide backbone, which may also be a peptide, nanoruler or other DNA origami-based structure (cf. Figure 48A and 48B ), and dye selective / specific binding sites 4100e, 4100h, 4100j, which have specificity determining moieties or functionalized fluorescent dyes 4102e, 4102h, 4102j, which can be oligonucleotides. This is a particularly preferred embodiment, as Figure 41 As shown, the dyes can be coupled to the linker simply by adding the dye mixture to the reaction mixture, which results in stoichiometric coupling, which is particularly advantageous for this method because it facilitates quantitative decoding.

[0399] exist Figure 42 In a particularly preferred embodiment of the invention shown, a two-part linker consisting of a UOSB and conjugated microbeads 2000 functionalized with nonspecific coupling sites 3900 is used. Figure 39 This is a particularly advantageous embodiment for assays requiring greater sensitivity, as a large number of dye structures or molecules can be coupled to the beads, which can serve as part of the three-dimensional support and linker.

[0400] exist Figure 43In another particularly preferred embodiment of the present invention, a dye mixture corresponding to a dye combination is encapsulated in nano / microcapsules 4300 or embedded in nano / microbeads 4302. Such structures can be efficiently synthesized using nano / microfluidics, electrospray, acoustic droplet jetting, or emulsification techniques, and have the advantage of protecting the encapsulated dyes from environmental influences. Relevant environmental influences include, among others, ozone and other reactive oxygen species, pH, solvents, buffers, and the like. Shielding the dyes is particularly advantageous in the disclosed methods because it stabilizes their fluorescence lifetime, thereby facilitating dye separation on a readout device used to record the fluorescence lifetime information (which may include other information).

[0401] exist Figure 44 In another particularly preferred embodiment of the present invention, the dye used is a SMILE or Small Molecule Ion Isolation Lattice as described in Benson et al., 2020, Chem 6, 1978–1997. The main advantage of SMILE is that they can achieve extremely high dye concentrations in a very small volume (about 100 nm per 4 nm). 3 There is one dye in the SMILE) while avoiding quenching, resulting in extremely high brightness. In addition, the co-crystallization of the cationic dye with the anion-bound cyanostar macrocycle can protect the incorporated dye from environmental influences, thereby stabilizing the fluorescence lifetime, which makes SMILE an ideal choice for dye separation. The readout device can be used to record information such as fluorescence lifetime. In addition, SMILE exhibits excellent brightness, making it very suitable for various applications requiring high sensitivity. Figure 44 As shown, SMILE 100M, 100N, 100O, 100P can be coupled with a linker to generate a SMILE-based reporter 4400.

[0402] exist Figure 45 In another particularly preferred embodiment of the present invention, SMILEs 100M, 100N, 100O, 100P are used in conjunction with nanostructure 4500, which serves as part of a linker, e.g., a platform or support to which the SMILE can be coupled. Nanostructure 4500 can specifically be a DNA origami-based nanostructure (e.g., a nanoruler having a substantially elongated shape, or any other geometric shape, such as a pyramid, grid, sphere, or complex structure), a graphene-based nanostructure, or another form of nanostructure.

[0403] exist Figure 46 In another preferred embodiment of the present invention, SMILE 100M, 100N, 100O, 100P are embedded in microbeads, generating another form of SMILE-based reporter 4600.

[0404] exist Figure 47In another embodiment of the present invention shown, SMILE 100M, 100N, 100O, 100P are encapsulated into microcapsules, generating another form of SMILE-based reporter 4700.

[0405] As shown in the above embodiments, the reporter can be linked to the affinity reagent in a variety of ways. Figure 48A and Figure 48B The set of particularly preferred joints is schematically shown, for example single part oligonucleotide sequence joint 4800, wherein DNA, RNA, LNA, peptide nucleic acid, morpholino or other artificial nucleic acids can be used. This is particularly advantageous because oligonucleotide sequence libraries can be manufactured at low cost. In addition, it is particularly advantageous because complementary sequences can be used to utilize hybridization and unzipping principle to reversibly connect dyestuff or affinity reagent to joints. Such as polymerase chain reaction, in situ hybridization, fluorescence in situ hybridization, Sanger sequencing and next generation sequencing and with restriction enzyme digestion and with a series of commonly used schemes of targeting endonuclease (such as CRIPR / Cas) shearing can be used in combination with the joint based on oligonucleotide.

[0406] In another embodiment of the present invention, the linker includes at least a combination of oligonucleotide and peptide sequences and can be referred to as an oligonucleotide-peptide-based linker 4802.

[0407] In another particularly preferred embodiment, the linker is a nanostructure 4500 , particularly a DNA origami-based structure or a nanoruler, and can be referred to as a nanostructure- or nanoruler-based linker 4804 .

[0408] In another preferred embodiment of the present invention, a peptide-based linker 4806 is used.

[0409] In a particularly preferred embodiment of the present invention, a linker 4808 comprising at least one nano / micro bead is used.

[0410] The reporter can be conjugated directly to the affinity reagent via standard coupling chemistries (e.g., NHS, maleimide, or various "click chemistries" (e.g., azide-alkyne couplings)), or it can be non-covalently linked, such as by hybridization of nucleic acids, UOSBs to complementary sequences, or high-affinity interactions between affinity ligand 4900 and affinity tag 4902 (e.g., Figure 49 Alternatively or additionally, a secondary antibody 4904 or other secondary antibody can be used to bind the reporter to the primary affinity reagent. Similarly, an aptamer-bound linker 4906 can also be used to bind the reporter to the primary affinity reagent.

[0411] Figure 50AA schematic diagram of a device 5000 for analyzing a (biological) sample is shown. Specifically, the device 5000 is capable of performing a reference Figures 1 to 49 The method for analyzing biological samples described above. The device includes at least one of the following: a light source unit 5002 (preferably comprising an LED light source), a coherent light source (e.g., a continuous wave laser or a pulsed laser with a fixed or adjustable wavelength, a white light laser), a staining unit 5004 (configured to perform Figure 18 The iterative process and Figure 37 relevant parts of processes S3702 and S3710), an imaging unit 5006 (in particular, an imaging unit for recording multiple views and / or optical slices), a flow cell / sample carrier 5008, a sample positioning unit 5010, a detection unit 5012 and a control unit 5014.

[0412] Figure 50B A device 5000 is shown, which is a microscope or imaging system. The device 5000 may include a staining unit 5004 for introducing a plurality of markers 112 into a sample. To this end, the staining unit 5004 may include one or more pipettes, which may or may not be automated. The staining unit 5004 may also include microfluidics and / or microfluidic chips. The device 5000 also includes excitation units 2314, 5002a, 5002b for exciting the fluorescent dye 100. The excitation units 2314, 5002a, 5002b include at least one light source, preferably a coherent light source. The at least one light source is configured to emit excitation light associated with each group of dyes. In order to emit excitation light of different wavelengths or wavelength spectra, the light source may be an adjustable light source. Alternatively, the device 5000 may include two or more light sources emitting different wavelengths or wavelength spectra. In Figure 23 and 24 In the illustrated embodiment, the excitation light emitted by the excitation unit 2314 is directed onto the sample 1500 via the beam splitting unit 2316 .

[0413] The imaging unit 5006 of the optional apparatus 5000 is configured to generate readouts from the fluorescence emitted by the excited dye 100, which can be image readouts or non-image-based readouts. The imaging unit 5006 includes an objective lens for aligning the sample for capturing the fluorescence. The captured fluorescence is then directed to the detection units 2320, 5012a, 5012b via the beam splitting unit 2316. The detection units 2320, 5012a, 5012b include at least one detection component and a diffraction component 204, 206 or filter for separating the fluorescence into different detection channels, such as Figure 2 shown.

[0414] After imaging the sample, it may be necessary to inactivate the fluorescent dye 100. For example, this can be achieved by photobleaching the fluorescent dye 100 using coherent light emitted by at least one of the light sources in the excitation units 2314, 5002a, and 5002b. Alternatively, a bleaching agent for chemically inactivating the fluorescent dye 1320 can be introduced into the sample 1002 using the staining unit 5004. In addition, the fluorescent dye 100 can also be removed from the primary or secondary affinity reagent. For example, this can be achieved by introducing an enzymatic cleavage agent into the sample using the staining unit 5004. Alternatively or additionally, the fluorescent dye 100 can be inactivated by antibody elution or dehybridization (i.e., melting) and elution of fluorescent-labeled oligonucleotides. Therefore, the excitation units 5002a, 5002b and / or the staining unit 5004 constitute a marker inactivation unit configured to inactivate at least one set of markers present in the sample.

[0415] Figure 52 The cyclic DNA-Exchange process is schematically shown. In this case, an antibody labeled with an oligonucleotide barcode is contacted with the analyte. In the first cycle of the staining, imaging, inactivation process, a first label including dye k is bound to the antibody. Using DNA-Exchange technology, this first label is removed after readout, and a second label including dye f is attached in the second cycle. The process is repeated n times, and dye n is read out in the nth round to generate a dye readout sequence k, f...n of the affinity reagent, which can be used to encode the identity of the affinity reagent. In other words, inter-round dye exchange is performed to encode the affinity reagent and its target analyte. Therefore, the process relies on repeated rounds of staining, imaging and label removal. DNA-Exchange, DNA-PAINT and Exchange-PAINT are related methods that can be used to Figure 52 The same analyte is repeatedly labeled with different imager strands containing different dyes in a cyclic or random manner, where the random manner may or may not require external stimulation to separate the bound imager strands from the cognate docking strand conjugated to the affinity reagent. These methods are described in Agasti et al., 2017 (DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging Chem. Sci., 2017, 8, 3080).

[0416] Figure 53A-1 Different markers comprising linked analytes and combined labels are schematically shown.

[0417] Figure 53A-2 The readout volume and readout trajectory are schematically shown.

[0418] and Figure 52 In contrast, the strategy shown here shows that "combinatorial tag subsets" 5300a and 5300b, comprising "dye combination subsets," are assigned to specific affinity reagents 5302a and 5302b. The "combinatorial tag" of "combinatorial tag subset" 5300a includes an "oligonucleotide attachment portion" 5306a / "A'," which is configured to bind to the "oligonucleotide barcode" 5304a / "A" of affinity reagent 5302a. The "combinatorial tag" of "combinatorial tag subset" 5300b includes an "oligonucleotide attachment portion" 5306b / "B'," which is configured to bind to the "oligonucleotide barcode 5304b" / "B" of affinity reagent 530ba. The configuration of A and A', and B and B', allows for both transient binding to achieve dynamic intra-round code exchange, and stimulated binding / dissociation to achieve repeated intra-round code exchange. This means that the "combinatorial tag" comprising "oligonucleotide attachment portion" 5306a "A'" in subset 5300a can dynamically or repeatedly bind to "oligonucleotide barcode" 5304a "A" in a random manner (e.g., stimulated by changing parameters such as temperature, light, host molecule concentration, or NCDA). Similarly, the "combinatorial tag" comprising "oligonucleotide attachment portion" 5306a "B'" in subset 5300b can dynamically or repeatedly bind to "oligonucleotide barcode" 5304b "B" in a random manner (e.g., stimulated by changing parameters such as temperature, light, host molecule concentration, or NCDA).

[0419] Thus, A' and B' are fully or partially complementary to A and B, respectively, but may include mismatches or hairpin structures, i.e., fragments that do not hybridize. A' has an affinity for A and, under at least one condition, binds transiently to A, i.e., for at least a period of time long enough to enable detection of the bound reporter, and then dissociates again. Dissociation may rely on additional chemical reactions, such as host-guest mediated ligand invasion, strand displacement, or the addition of specific unnatural bases and other chemicals that can be photoconverted to allow reversible photochemical control of the hybridization of A' to A. Thus, the oligonucleotides A, A' and B, B' may include unnatural bases and / or additional substances added to the natural or unnatural bases, such as the addition of guest molecules that are attached to the nucleobases and configured to complex with the host molecule (e.g., CB[7]). Oligonucleotides A, A' and B, B' may additionally or alternatively contain GG mismatches that allow A and A' or B and B' to be combined only in the presence of sufficient concentrations of CIS-NCDA, which can be obtained from trans-NCDA by photoconversion using 360nm and 430nm light to undergo cis-trans isomerization.

[0420] It is important to note that no washing is required between the acquisition of the readout sequence at time points t1, t2, ..., tn, which collectively represent the readout trace. Binding can be detected because the reporter or combined tag will bind to the respective affinity reagent for a time sufficient to allow detection. The exposure time / pixel dwell time and the dwell time of the reporter on the affinity reagent barcode can be adjusted in various ways.

[0421] Then Figure 53A-1 、 53A-2 53A-B illustrate the variation in binding between affinity reagents 5302a and 5302b and the combined labels of their assigned subsets 5300a and 5300b. The circles filled with different patterns in Figures 53A-B reflect dyes that are different from the multiple dyes.

[0422] refer to Figure 53B and 53C : A' and B' are complementary to A and B, respectively, but may contain mismatches or hairpin structures, meaning they prevent hybridization. A' has affinity for A and, under at least one condition, transiently binds to A—at least long enough to allow detection of the bound reporter—before dissociating again. Dissociation can rely on other chemical reactions, such as host-guest-mediated ligand invasion, strand displacement, or the addition of specialized unnatural bases and other chemicals that can be photochemically controlled using photoswitches, thereby reversibly controlling the hybridization of A' to A.

[0423] Figure 54 Further schematically shown are two affinity reagents assigned to a subset of dye combinations in a plurality of dye combinations, wherein each dye combination in the subset can be bound to an affinity reagent or to a corresponding oligonucleotide barcode A or B on the affinity reagent via a corresponding complementary linker A' or B', wherein A' and B' can be fully or partially complementary (i.e., contain mismatches, hairpins, etc., as long as they remain properly configured to bind to their respective attachment sites or oligonucleotide barcodes attached to the affinity reagent).

[0424] Figure 55 Shown on the following page Figure 55A 、 55B and an overview of 55C, Figure 55A 、 55B and the contents of 55C are arranged as Figure 55 The complete diagram shown. Figure 55It is a suitability overview diagram that contains the most important entities and steps in the process / workflow and illustrates the concept of collecting readout traces, which are then used to progressively decode the sample by statistically analyzing the dye combinations that can be attributed to at least a portion of the readout trace (i.e., that can be present in the readout volume), using parameters such as frequency, dye intensity and dye-dye intensity correlation, frequency sum, and average intensity value, which are compared with a mathematical model of the sample to calculate a measure of statistical confidence in the presence of a specific affinity reagent / analyte in the readout volume, such as a p-value. Presence calls can then be easily performed using a user- or system-defined global or dynamically adjusted p-value cutoff, where affinity reagents / analytes that are "called present" are assigned a value of "1" and affinity reagents / analytes that do not pass the threshold are assigned a value of "0". For example, the grayscale value in an image can be derived by taking the average or weighted sum of all intensities for all combinations of dye readouts assigned to a particular affinity reagent and then simply multiplying it by a presence call value of "1" or "0," which effectively eliminates the noise caused by false positives or true positives that were not detected with sufficient confidence. Importantly, Figure 55B The APRIORI ENGINE shown can be used to load inputs from various pre-existing data into a mathematical model, as described in European Patent Application No. EP 22179854.9, the entire contents of which are incorporated herein by reference, which can greatly improve statistical analysis in the Bayesian statistical sense. Similarly, the results of previous time points and / or neighboring regions in the sample can be regarded as prior information that can be input into the mathematical input model. Finally, a mathematical output model is generated, which can be, for example, an image-based or graphical representation of the biological sample.

[0425] like Figures 55A-55C As shown, the readout trajectory is actually composed of n -t n+mA series of readout sequences are collected, and a set of dye combinations are retrieved from a database, or these dye combinations are calculated using a hash function to generate a set of all dye combinations that can be summarized under the readout track. This represents all possible dye combinations of combination labels that may exist in the readout volume. The group is then statistically analyzed. However, some dye combinations that can be summarized under the readout track may be easily excluded because they, for example, do not point to the affinity reagents assigned in the database. There may be some false positive results that cannot be directly excluded. The detection frequency of certain affinity reagents and analytes, the frequency of a single dye combination pointing to the analyte, the intensity of the dye and its correlation, and the degree of fit of their correlation with the dye combination can all be used for statistical analysis. Aggregate observation frequency, raw intensity value (such as the average, sum or weighted average of a specific analyte and affinity reagent) and p-value can be calculated. The p-value or other statistical confidence measure represents the probability (false positive) that the method detects the analyte when the corresponding analyte is not actually present in the readout volume. A looser or stricter cutoff or threshold value can be applied to the p-value or other measure of statistical confidence for presence calls, for example, by assigning a "1" to accept presence and a "0" to reject presence. The intensity value (grayscale value) displayed in the image can be simply calculated by multiplying the raw intensity by the presence call value, effectively filtering out all statistically insignificant analyte contributions, thereby reducing background noise.

[0426] Figure 56 Schematic illustration of the fact that the unbound reporter and its dye combination do not remain in the readout volume long enough to be detected, as evidenced by the bar lengths being similar to the exposure / dwell time and the different dwell times for the unbound and bound reporters.

[0427] Figure 57 and Figure 61 Two strategies to achieve reversible / repeated binding / dissociation or dynamic binding / dissociation are schematically illustrated.

[0428] Figure 57Another strategy is shown, in which a modified DNA oligonucleotide is used, which carries a modified nucleoside ("N") 5700, which carries a guest molecule 5702, which can be complexed with a host molecule 5704 (such as cucurbit[7]uril (CB[7])), thereby triggering ligand invasion and DNA duplex dissociation. For a 15 bp duplex, according to the study of Xiao et al., 1 μM CB[7] is sufficient to reduce the melting temperature Tm by about 9.7°C (Xiao, L., Wang, LL., Wu, CQ. et al., Controllable DNA hybridization by host–guest complexation-mediated ligand invasion. Nat Commun 13, 5936 (2022)). A suitable concentration of CB[7] or another suitable guest molecule can be used in combination with an "oligonucleotide attachment portion" 5306a / "A'" with at least one N-guest nucleobase 5700 and an "oligonucleotide barcode" 5304a comprising at least one complementary N'nucleobase. Alternatively, a suitable concentration of CB[7] or another suitable guest molecule can be used in combination with an "oligonucleotide attachment portion" 5306a "A'" having at least one complementary N'nucleobase and an "oligonucleotide barcode" 5304a comprising at least one N-guest nucleobase. In this way, dynamic binding / dissociation (binding / dissociation or removal) can be achieved at low temperatures by maintaining the concentration of the host molecule within a suitable range so that the ratio of binding and dissociation is approximately 50:50. By oscillating the concentration of the host molecule, cycles between stable binding and dissociation or label removal can be achieved. This can be achieved by continuously feeding from two buffer reservoirs, one with a high concentration of the host molecule and the other with a low concentration of the host molecule or no host molecule.

[0429] like Figure 57 As shown, the oligonucleotide attachment portion 5306a / A' is part of a linker 5310 that is configured to bind to the affinity reagent 112. The linker 5310 includes at least one binding site that is configured to bind to at least one dye. Figure 57 In the example of FIG. 5 , two dyes 5312 and 5314 are bound to a linker 5310. The linker 5310 and at least one of the dyes 5312 and 5314 form a reporter 5316. The reporter 5316 and the affinity reagent 112 form a marker 5318. Figure 57 It is collectively referred to as 5318 in China.

[0430] Figure 57 Include a diagram illustrating how binding / dissociation depends on the concentration c of the host molecule. Figure 57In the example shown in Figure 2, binding is low in the range of high host molecule concentration c. Conversely, binding is high in the range of low host molecule concentration c. Between the low and high concentration ranges lies a transition zone, where binding and dissociation are essentially balanced. Thus, the transition zone separates the low-concentration region, where label removal occurs, from the high-concentration region, where stable binding occurs.

[0431] like Figure 58 In more detail, the host-guest complexation-mediated ligand invasion described above unfolds the secondary structure, thereby eliminating the labeled target binding. Figure 58 As shown on the left side of FIG, at high host molecule concentrations, after the secondary structure unfolds due to host-guest complexation, base pairs 5706 of duplex 5708 actually dissociate, thereby preventing duplex formation at high host molecule concentrations. As a result, unzipping occurs at high host molecule concentrations. In contrast, as Figure 58 As shown on the right side of the figure, at low host molecule concentrations, host-guest complexation does not prevent hybridization-induced duplex formation.

[0432] As mentioned above, the oligonucleotide modified using guest molecule can have relatively large oligonucleotide length, significantly reduce melting temperature simultaneously.Therefore, this solution has advantage over the other methods (such as DNA-Exchange, DNA-PAINT or Exchange-PAINT) of the oligonucleotide with less length.It can be approximated that the use of the oligonucleotide modified by guest molecule proposed herein can reduce melting temperature when oligonucleotide length is 20 to 24 aggressiveness, and other methods work under the significantly smaller length of 9 aggressiveness to 13 aggressiveness.On the one hand, due to its length, the oligonucleotide modified by guest molecule allows to have high-specific target molecule dyeing.On the other hand, because melting temperature is lower, they can deactivate mark easily without making background noise become too intense.

[0433] Therefore, host-guest complexation-mediated ligand invasion is a suitable strategy for implementing "barcode cycling" and "internal code exchange". In Xiao, L., Wang, LL., Wu, CQ. et al., Controllable DNA hybridization by host–guest complexation-mediated ligand invasion. Nat Commun 13, 5936 (2022). https: / / doi.org / 10.1038 / s41467-022-33738-3, the use of host-guest complexation-mediated ligand invasion as a strategy for controlling DNA hybridization is described.

[0434] Figure 59An example of multiplexing using barcoded antibodies is shown. Figure 57 According to the markers described in Figure 59 In the example shown, it is assumed that each marker 5318a / b includes only one dye 5312a / b at a given time. In addition, the marker 5318a / b includes an affinity reagent 5320 formed of an antibody that is configured to attach to the analyte 900.

[0435] In a first step S1, the sample is stained with a marker 5318a formed by a reporter 5316a comprising a linker 5310a and a dye 5312a bound thereto. After staining, the sample is imaged to create a first readout. While performing step S1, the concentration of the host molecule 5704 (e.g., CB[7]) is kept at a low level, e.g., Figure 59 As shown in the lower half, hybridization / duplex formation occurs and the analyte 900 is labeled with the dye 5312a.

[0436] In step S2, the concentration of host molecule 5704 increases, and the double strands are dissociated due to breakage. As a result, reporter 5316a including dye 5312a is removed from analyte 900.

[0437] In step S3, the sample is stained again. However, the marker 5318b used includes a reporter 5316b containing a different dye 5312b than the dye 5312a used in step S1. After staining, the sample is imaged to create another readout. During step S3, the concentration of host molecule 5704 is reduced again. As a result, hybridization / duplex formation occurs, and analyte 900 is labeled with dye 5312b.

[0438] The steps of staining the sample, imaging the sample, and removing the label can be repeated to achieve a cyclic process.

[0439] Figure 60 A modified cyclic process is shown in which oligonucleotide probes are used as affinity reagents rather than antibodies. Each oligonucleotide probe can be formed by a reporter corresponding to the above-mentioned reporter 5316. More specifically, in this case, the reporter 5316 can be adapted to bind to a nucleic acid analyte 5350, such as an mRNA gene.

[0440] In a first step S1', the sample is stained with an oligonucleotide probe formed by a reporter 5316a conjugated to a dye 5312a. After staining, the sample is imaged to create a readout. When performing step S1, the concentration of the host molecule 5704 (e.g., CB[7]) is low, e.g. Figure 60 As shown in the figure legend below, hybridization occurs and the analyte 5350 is labeled with dye 5312a.

[0441] In step S2', the concentration of host molecule 5704 is increased, causing dissociation to occur. As a result, reporter 5316a including dye 5312a is removed from analyte 5350.

[0442] In step S3', the sample is stained again. However, the oligonucleotide probe used is formed from a reporter 5316b with a dye 5312b, which is different from the dye 5312a used in step S1'. After staining, the sample is imaged to create another readout. While executing step S3', the concentration of host molecule 5704 is reduced again. As a result, hybridization occurs, and analyte 5350 is labeled with dye 5312b.

[0443] The steps of staining the sample, imaging the sample, and removing the label can be repeated to achieve a cyclic process.

[0444] Figure 61 Schematic diagram showing how an unmodified DNA oligonucleotide containing at least one GG mismatch can be used with a photoconvertible molecular glue called NCDA and 360nm and / or 430nm illumination to achieve repeated or dynamic binding / dissociation (binding / dissociation) between an "oligonucleotide attachment moiety" 5306a / "A" conjugated to a combination label and an "oligonucleotide barcode" 5304a / "A" conjugated to a dispensed affinity reagent. For nucleic acid analytes such as DNA or RNA, the "oligonucleotide barcode" 5304a / "A" may simply be the cognate nucleic acid target.

[0445] like Figure 61As shown, in the absence of cis-NCDA and / or in the presence of trans-NCDA, at least one GG mismatch inhibits stable binding between the "oligonucleotide attachment moiety" 5306a / "A'" conjugated to the combinatorial tag and the "oligonucleotide barcode" 5304a / "A'" conjugated to the assigned affinity reagent. However, photoconversion of trans-NCDA to cis-NCDA allows bridging of at least one GG mismatch with the naphthyridine moiety that recognizes guanine, allowing hybridization despite the GG mismatch. The cis / trans isomerization of NCDA is reversibly controlled by photoconversion, using 360 nm light to generate the cis isoform and 430 nm light to generate the trans isoform. Therefore, binding kinetics can be indirectly photocontrolled by varying the concentrations of cis- and trans-NCDA or by externally supplying cis-NCDA and / or trans-NCDA using buffer exchange. A particular advantage of the NCDA approach is that the "oligonucleotide attachment moiety" 5306a / "A'" and the "oligonucleotide barcode" 5304a can be composed solely of natural bases, thus eliminating the need for potentially labor-intensive and cost-intensive modifications on the barcode side. Similarly, rapid photoconversion by alternating 360nm / 430nm illumination can be used in conjunction with a buffer having NCDA, placing the NCDA in a state where the oligonucleotide barcode of the affinity reagent dynamically associates and dissociates from the attachment moiety of the reporter / combination tag linker. Photoconvertible gels for DNA are described in Dohno et al. 2007 (J. Am. Chem. Soc. 2007, 129, 39, 11898–11899).

[0446] Rapid buffer exchange can be performed within a round to increase or decrease the concentration of salts or small molecules, because the much larger dissociation constants of affinity reagents and combination labels / reporters avoid their rapid elution. Similarly, the combination label can be continuously replenished with buffers containing different concentrations of salts, small molecules, or other additives. In this way, the concentration profile of salts, small molecules, and other components of the buffer can be run within the round, that is, in the first incomplete round consisting of staining-imaging. This consideration applies to all strategies cited in this document that can be used for intra-round code exchange.

[0447] Thus, A' and B' are fully or partially complementary to A and B, respectively (i.e., A is configured to bind to A' and B to B' in a stable but reversible or transient manner). A 5304a, B 5304b, A 5306a, and B 5360b may include mismatches, particularly GG mismatches or hairpins, i.e., non-hybridizing segments, and may be composed in whole or in part of natural or non-natural nucleobases or nucleosides. Nucleobases may be modified with other moieties, such as the following guest molecules, among others: 1-adamantanemethane (AD), Ferrocenemethylamine (FC), 1,4-phenylenediamine (BA), 4-tert-Butylbenzylamine (TB).

[0448] In particularly preferred embodiments, the "oligonucleotide barcode" 5304a and / or "oligonucleotide attachment moiety" 5306a used comprises one of the following structures: ·5′-XXXXXX C*XXXXXX C*XXXXX-3′ 5′-X..XC*X..XC*X..X-3′ Where X can be any natural or unnatural nucleobase or nucleoside, and C* refers to the guest-modified cytosine molecule nucleoside. C* can also refer to any other guest-modified nucleoside.

[0449] As used herein, the term "and / or" includes any and all combinations of one or more of the following associated listed items and may be abbreviated as " / ".

[0450] Although some aspects are described in the context of an apparatus, it is clear that these aspects also represent a description of a corresponding method, where a block or apparatus corresponds to a method step or a feature of a method step. Similarly, aspects described in the context of a method step also represent a description of a corresponding block or a corresponding block or item or feature of a corresponding apparatus.

[0451] When with Figures 1 to 51 Some embodiments relate to a microscope comprising a system when one or more of the foregoing are described in connection with Figures 1 to 51 When one or more of the related descriptions are used, the microscope can be part of or associated with the system. Figure 50A and 50B as well as Figure 23-27A schematic illustration of a readout device or system 5000 configured to implement the methods described herein is shown. The readout device or system 5000 includes a microscope or cell counter 2500 and a computer system 5016 connected via a suitable communication protocol and connection 5018 (e.g., USB, TCP / IP, Ethernet, Ethernet, FibreChannel, CAN-Bus). Microscope 5014 can, for example, be used to generate an optical readout of a marker and is configured to capture an image and connected to computer system 5016. Computer system 5016 is configured to perform at least a portion of the methods described herein. Computer system 5016 can be configured to execute a machine learning algorithm. Computer system 5016 and microscope or cell counter 2500 can be separate entities, but they can also be integrated. Computer system 5016 can be part of the central processing system of microscope or cell counter 2500 and / or computer system 5016 can be part of a subcomponent of microscope or cell counter 2500, such as a sensor, actuator, camera, or lighting unit of microscope or cell counter 2500.

[0452] The computer system 5016 can be a local computer device (e.g., a personal computer, laptop, tablet, or mobile phone) having one or more processors and one or more storage devices, or it can be a distributed computer system (e.g., a cloud computing system having one or more processors and one or more storage devices that are distributed across different locations, e.g., at a local client and / or one or more remote server farms and / or data centers). The computer system 5016 can include any circuit or combination of circuits. In one embodiment, the computer system 5016 can include one or more processors of any type. As used herein, "processor" can refer to any type of computing circuit, such as, but not limited to, a microprocessor, a microcontroller, a complex instruction set computing (CISC) microprocessor, a reduced instruction set computing (RISC) microprocessor, a very long instruction word (VLIW) microprocessor, a graphics processor, a digital signal processor (DSP), a multi-core processor, a field programmable gate array (FPGA), such as a microprocessor of a microscope or a microscope component (e.g., a camera), or any other type of processor or processing circuit. Other types of circuits that may be included in the computer system 5016 may be custom circuits, application-specific integrated circuits (ASICs), and the like, such as one or more circuits (e.g., communication circuits) used in wireless devices such as mobile phones, tablet computers, laptop computers, two-way radios, and similar electronic systems. The computer system 5016 may include one or more storage devices, which may include one or more memory components suitable for a particular application, such as main memory in the form of random access memory (RAM), one or more hard disk drives, and / or one or more drives for handling removable media (e.g., compact disks (CDs), flash memory cards, digital video disks (DVDs), etc.). The computer system 5016 may also include a display device, one or more speakers, and a keyboard and / or a controller, where the controller may include a mouse, trackball, touch screen, voice recognition device, or any other device that allows a system user to input information to and receive information from the computer system 5016.

[0453] Some or all of the method steps can be performed by (or using) hardware devices, such as processors, microprocessors, programmable computers or electronic circuits. In some embodiments, one or more of the most important method steps can be performed by such devices.

[0454] Depending on the specific implementation requirements, embodiments of the present invention can be implemented in hardware or software. They can be implemented using non-transient storage media (e.g., digital storage media, such as floppy disks, DVDs, Blu-ray discs, CDs, ROMs, PROMs, EPROMs, EEPROMs, or flash memory) that store electronically readable control signals that can (or are capable of) cooperating with a programmable computer system to perform the corresponding method. Thus, the digital storage medium can be computer-readable.

[0455] Some embodiments according to the invention comprise a data carrier having electronically readable control signals, which are capable of cooperating with a programmable computer system, such that one of the methods described herein is performed.

[0456] Generally, embodiments of the present invention can be implemented as a computer program product with a program code, wherein when the computer program product runs on a computer, the program code is operative to perform one of the methods. For example, the program code can be stored on a machine-readable carrier.

[0457] Other embodiments comprise the computer program for performing one of the methods described herein, stored on a machine readable carrier.

[0458] In other words, one embodiment of the present invention is, therefore, a computer program having a program code for performing one of the methods described herein when it runs on a computer.

[0459] Therefore, another embodiment of the present invention is a storage medium (or data carrier, or computer-readable medium) comprising a computer program for performing one of the methods described herein when executed by a processor. The data carrier, digital storage medium or recorded medium is typically tangible and / or non-transitory. Another embodiment of the present invention is a device as described herein, comprising a processor and a storage medium.

[0460] Therefore, another embodiment of the present invention is a data stream or a sequence of signals representing a computer program for executing one of the methods described herein.For example, the data stream or the sequence of signals may be configured to be transmitted via a data communication connection (for example, via the Internet).

[0461] A further embodiment comprises a processing device, for example a computer or a programmable logic device, configured to or adapted to perform one of the methods described herein.

[0462] A further embodiment comprises a computer comprising the computer program installed thereon for performing one of the methods described herein.

[0463] Another embodiment according to the present invention includes a device or system configured to transmit (e.g., electronically or optically) a computer program for performing one of the methods described herein to a receiver. The receiver may be, for example, a computer, a mobile device, a storage device, etc. The device or system may, for example, include a file server for transmitting the computer program to the receiver.

[0464] In some embodiments, a programmable logic device (e.g., a field programmable gate array) can be used to perform some or all of the functions of the methods described herein. In some embodiments, a field programmable gate array can collaborate with a microprocessor to perform one of the methods described herein. Generally, these methods are preferably performed by any hardware device.

[0465] Reference Mark List 100, 100a, 100b, 100A to 100E fluorescent dyes 100M, 100N, 100O, 100P SMILE 102, 102a The left half of the pattern is similar to the excitation property 104, 104a The right half of the pattern is similar to the emission properties 106 Dye-conjugated or oligonucleotide-labeled primary affinity reagents 108 Single domain antibody 110 Multimeric (single domain) antibodies 112 Antibodies 114 aptamers 116 oligonucleotides 118 Toxins, drugs, small molecules 120 oligonucleotide barcodes 124 without attached dye or oligonucleotide (108-118) 200 Fluorescence 202 Spectrally separated fluorescence 204 Prism 206 grating 208 Detector having multiple detector elements More than 300 excitation spectra 302 multiple emission spectra 304a, 304b, 304c τ gating of τ class 306 Dye groups with similar emission spectra Emission spectra of 308a, 308b, and 308c Fluorescence lifetime-emission intensity plot of 400 topology 402a, 402b, 402c Dyes A1, A2, A3 404 Spectral phasor, FLIM phasor, or Spectral-FLIM phasor 500 Reactivity or specificity determining region of affinity reagent (e.g., paratope) Dye group 600A to n Dye combinations 602, 602a, 602b, 602c, 602d, 706, 706a, 706b, 706c, 706d 604 Dye Combination(s i ) and affinity reagent (a i ) pairing (bijection; one-to-one correspondence) 606a, 606b dye combination (s i ) in affinity reagent (a i ) and vice versa (injective) Number of digits in 700 (704a, 704b) 702 Dye combination (Y D ) 704a, 704b Rules for forming dye combinations / binary codes 706 Code blocks / code snippets 708 marker, μ 710 Get Sequence 800a, 800b, 800c oligonucleotide barcode sequences 900 target molecules or analytes 902 connector 904 restriction site 906a, 906b, 906c, 906d, 906e, complementary site / sequence 906a * 、906b * 、906c * 、906d * 、906e * Attachment site / sequence 908, 1008a, 1008b reports 1500, 1500a cells 1502 Cell Nucleus 1504 Cytoplasm 1506a, 1506b, 1506c target proteins 1508 DNA target sequence 1510 RNA target sequences 1512 Primary antibody (unlabeled) 1514 Secondary Antibody (labeled) 1518 First single domain antibody (unlabeled) 1520a, 1520b Oligonucleotide-based reporters 1522 Capture antibody immobilized in the extracellular space 1524a, 1524b, 1524c Secreted target proteins 1526a, 1526b, 1526c, 1526d detection antibodies 1600 atoms 1602 Small molecules (e.g. cholesterol, drugs) 1604 Nanobodies 1606 Green Fluorescent Protein 1608 Antibody 1610 Quantum Dots, Polymer Dots, Nanostructures 1700 point spread function (diagram) 1900 A discrete entity (e.g., a hydrogel bead) or virtual space (e.g., a well in a microplate) surrounding a cell of interest; a virtual or physical volume 1902a, 1902b capture reagents 1904 Polymers (e.g., hydrogels) 2000, 2000a Micro / nano beads (e.g., latex or polystyrene beads) 2002 Nanostructures (e.g., nanorulers, DNA origami, carbon nanotubes) 2300 Microplate or Sample Carrier 2302 Well or sample container 2304 Transparent Window 2306 Hydrogel or transparent polymer 2308 Centroid of the cell or structure of interest 2310 Virtual volume around the center of mass 2312 Microscopes, imaging systems, image-based readout devices 2314 Light Source 2316 Beam Paths, Beam Splitters, Beam Routing 2320 Detector 2318 Imaging optics or objectives 2400 flow cell 2402 Inflow 2404 outflow 2406 Flowing Medium 2408 Discrete entities (e.g., hydrogel beads) 2500 Flow Cytometer 2502 Flow Cytometer 2504 Deflector Plate 2506 Collection Tube 2600 Bead-Based Analyte Detection Assay 2602 Bead-Based Analyte Interaction Assays 2700 cell surface-bound targets 2800 spots 2802a, 2802b Single species readout volume 2804 Multi-species Spots 2900 read sequence More than 3,200 dye combinations (S1) 3202 Set of dye combinations that can be summarized under the first readout sequence 3300a, 3300b, 3300c Set of dye combinations that cannot be summarized under the first readout sequence 3302a, 3302b, 3302c can be summarized as a set of dye combinations under the first readout sequence 3400a, 3400b, 3400c are sets of dye combinations corresponding to markers present in the readout spot or readout volume. 3402a, 3402b, 3402c are sets of dye combinations that can be summarized in the first read sequence but correspond to markers that are not present in the read spot or read volume. 3800 Initial Qualitative Decoding 3802 Secondary Qualitative Decoding 3804 Secondary Quantitative Decoding 3850 Skeleton 3900 Nonspecific covalent coupling or high affinity binding site 4100e, 4100h, 4100j Dye-specific or dye-conjugated affinity tags / ligand-specific covalent coupling or high-affinity binding sites 4102e, 4102h, 4102j Dye-conjugated affinity tags / ligands 4300 Micro / Nanocapsules pre-filled with dye mixture 4302 Micro / Nanobeads Preloaded with Dye Mixture 4400 SMILE combination on the report 4500 Nanostructure-based (e.g., nanoruler, DNA origami, carbon tube) nanoplatforms carrying dyes (e.g., SMILE) 4600 SMILE combination embedded in polymer microbeads 4700 SMILE combination encapsulated in microcapsules 4800 Oligonucleotide-Based Adapters 4802 Combination of oligonucleotide-based and peptide-based linkers 4804 Oligonucleotide-based and Nanoruler / DNA Origami-based Combined Linkers 4806 Peptide Linker 4808 Oligonucleotide-bead-based combinatorial adapter 4900 Affinity Ligand 4902 Affinity Tag 4904 Secondary Antibody Nanobody 4906 aptamer Queue of 5100 strongly overlapping excitation spectra 5102 Queue of strongly overlapping emission spectra 5700 Modified Nucleosides 5702 guest molecules 5704 main molecules 5312 Dye 5314 Dye 5316 Report 5318 Marker

Claims

1. A method for analyzing a sample, the sample comprising: a plurality of affinity reagents (5302a), each affinity reagent (5302a) being configured to attach to an analyte (900), at least one of the affinity reagents (5302a) being attached to the analyte (900); and a first plurality of dye combinations, each dye combination within the first plurality of dye combinations being unique and each dye combination comprising at least two dyes having different properties with respect to at least one of excitation and emission, wherein at least one of the unique dye combinations is attached to an associated affinity reagent (5302a) according to a first mapping, wherein at least another of said unique dye combinations is attached, preferably randomly attached, to said associated affinity reagent (5302a) according to a second mapping, wherein the first mapping is different from the second mapping, For the first mapping and for the second mapping, the method comprises in a time-sequential manner: iii) directing excitation light to the sample, the excitation light having characteristics for exciting at least the at least two dyes, the at least two dyes having different characteristics with respect to at least one of excitation and emission; and iv) generating at least one first readout of emission light emitted by the excited dyes.

2. A method for analyzing a sample, the sample comprising: a plurality of affinity reagents (5302a, 5302b), each affinity reagent being configured to attach to an analyte (900), at least one of the affinity reagents being attached to the analyte (900); and a first plurality of dye combinations, each dye combination within the first plurality of dye combinations being unique and each dye combination comprising at least two dyes having different properties with respect to at least one of excitation and emission, a second plurality of dye combinations, each dye combination within the second plurality of dye combinations being unique and each dye combination comprising at least two dyes having different properties with respect to at least one of excitation and emission; wherein the second plurality of dye combinations is different from the first plurality of dye combinations; wherein at least one of the unique dye combinations of the first plurality of dye combinations, in particular a first subset of dye combinations (5300a), is attached to an associated affinity reagent (5302a) according to a first mapping, wherein at least one of the unique dye combinations of said second plurality of dye combinations, in particular a second subset of dye combinations (5300b) is attached, preferably randomly attached, to said associated affinity reagents (5302a) according to a second mapping, wherein the first mapping is different from the second mapping, For the first mapping and for the second mapping, the method comprises, in a time-sequential manner: iii) directing excitation light towards the sample, the excitation light having properties for exciting at least the at least two dyes, the at least two dyes having different properties for at least one of excitation and emission; iv) generating at least one first readout of emission light emitted by the excited dye.

3. The method of any one of the preceding claims, wherein each unique dye combination in the first plurality of dye combinations is attached to only one affinity reagent, such that no unique dye combination is bound to more than one affinity reagent in the first mapping.

4. The method according to any one of the preceding claims, wherein said generating at least one first readout comprises: The emission light emitted by the excited dye is separated into detection channels, wherein the detection channels correspond to the emission characteristics of the dye.

5. The method of claim 4, wherein each dye combination is selected so that each detection channel includes one dye.

6. The method according to any one of the preceding claims, wherein the at least two dyes have different excitation characteristics, and wherein the excitation light having each excitation characteristic is directed to the sample at different times or simultaneously.

7. The method according to any one of the preceding claims, further comprising: providing the plurality of affinity reagents (5302a); providing the first plurality of dye combinations; providing said sample; and III) attaching the plurality of affinity reagents (5302a) to the first plurality of dye combinations to form a plurality of markers; and introducing the plurality of markers into the sample to allow attachment to the analyte in the sample (900); or IV) introducing the plurality of affinity reagents (5302a) into the sample to allow attachment to the analyte (900) in the sample; and attaching the plurality of affinity reagents (5302a) to the first plurality of dye combinations to form a plurality of markers attached to the analyte (900).

8. The method of claim 7, wherein attaching the plurality of affinity reagents to the first plurality of dye combinations comprises: providing a plurality of linkers (A', B'), each linker (A', B') comprising a plurality of binding sites, each configured to bind to a dye; and For each affinity reagent (112), one linker (A', B') is attached to the affinity reagent (112 / A, 112 / B), and one of the dye combinations is bound to the linker (A', B'), each dye in the combination being bound to a binding site.

9. The method according to any one of the preceding claims, further comprising at least one of the following: deactivating at least one of the dyes in the first plurality of dyes; removing said attachment between at least one affinity reagent and at least one of said dye combinations; removing said attachment between at least one affinity reagent and at least one of said analytes; and waiting for a time longer than a fluorescence lifetime of at least one of the dyes in the first plurality of dye combinations; as well as Repeat steps i) and ii) of claim 1 for the first plurality of dye combinations according to different second mappings, or repeat steps i) and ii) of claim 2 for different second plurality of dye combinations according to different second mappings.

10. The method according to claim 9, further comprising: Based on the at least one first readout, at least one dye of the second plurality of dye combinations and / or a combination of dyes and / or a rule for the second mapping is suggested by a computer processor.

11. The method of claim 9 or claim 10, further comprising iteratively repeating the steps of claim 9 and / or claim 10 for at least one of a plurality of dye combinations and a plurality of mappings until all affinity reagents attached to the analyte in the sample are determined.

12. The method of any of the preceding claims, wherein stochastic labeling is achieved by at least one of DNA-PAINT, DNA-Exchange, Exchange-PAINT, and by using oligonucleotide barcodes (5304a / A, 5304b / B) attached to the affinity reagents (5302a, 5302b) and complementary attachment oligonucleotide moieties (5306a / A', 5306b / B') attached to the dye combinations, which are configured to allow dynamic association and dissociation of the dye combinations from their assigned affinity reagents (5302a, 5304b).

13. The method of any of the preceding claims, wherein the stochastic labeling is achieved by at least one of DNA-PAINT, DNA-Exchange, Exchange-PAINT, and by using an oligonucleotide barcode (5304a / A, 5304b / B) attached to the affinity reagent (5302a, 5302b) and a complementary attachment oligonucleotide portion (5306a / A', 5306b / B') attached to the dye combination, which are configured to allow repeated association and dissociation of the dye combination and its assigned affinity reagent (5302a, 5302b) by at least one of: changes in the concentration of host molecules; changes in temperature; changes in lighting conditions; a change in the concentration of at least one capture strand; and Changes in enzyme concentration and / or activity.

14. The method of any one of the preceding claims, wherein determining, by at least one computer processor, the presence of at least one affinity reagent in the sample comprises: comparing at least two reads selected from the group consisting of: the at least one first read, the at least one second read, and any other reads generated in step ii) of claim 1 or claim 2; and The presence of at least one affinity agent is determined based at least in part on the comparison.

15. The method according to any one of the preceding claims, wherein determining the presence of at least one affinity agent is based on at least one measure of statistical confidence.

16. The method of any preceding claim, wherein the characteristic of the dye with respect to at least one of excitation and emission comprises at least one of: excitation wavelength; emission wavelength; fluorescence intensity; and fluorescence lifetime.

17. The method of any one of the preceding claims, wherein determining, by at least one computer processor, the presence of at least one affinity reagent in the sample based on the readout comprises: converting the readout into a well-determined system of linear equations or an overdetermined system of linear equations; and Solve the system of linear equations.

18. An apparatus for analysing a sample, the apparatus being configured to perform the method according to any one of the preceding claims.

19. A linker (5310) configured to bind to an affinity reagent (112, 500), the linker (5310) comprising: at least one binding site configured to bind at least one dye (5312, 5314); and An oligonucleotide attachment portion (5306a; A', B') configured to bind to the oligonucleotide barcode (5304a; A, B) of the affinity reagent (112).

20. The connector (5310) of claim 19, comprising: A plurality of binding sites, wherein at least two of the binding sites are configured to bind to dyes (5312, 5314) having different properties with respect to at least one of excitation and emission.

21. A reporter (5316), comprising: The connector (5310) according to claim 19; and At least one dye (5312, 5314), the at least one dye being bound to the at least one binding site of the linker (5310).

22. A reporter (5316) comprising: The connector (5310) according to claim 20; and A dye combination (5310, 5312), each dye being bound to one of the plurality of binding sites of the linker (5310), wherein at least two of the dyes have different properties with respect to at least one of excitation and emission.

23. A marker (5318), comprising: an affinity reagent (112) configured to attach to the analyte (900); and A reporter (5316) according to claim 21 or 22, which is attached to the affinity reagent.

24. The marker (5318) of claim 23, wherein the oligonucleotide attachment portion (5306a) of the linker (5310) is fully complementary or partially complementary to the oligonucleotide barcode (5304a) of the affinity reagent (112).

25. The marker (5318) of claim 23 or 24, wherein the oligonucleotide attachment portion (5306a) of the linker (5310) is configured to have affinity for the oligonucleotide barcode (5304a) of the affinity reagent (112), e.g., to transiently bind to the oligonucleotide barcode (5304a) under at least one condition before again dissociating from the oligonucleotide barcode (5304a), wherein the at least one condition is provided for dissociation of the oligonucleotide attachment portion (5306a) based on at least one of host-guest mediated ligand invasion, strand displacement, and photochemical control of hybridization.

26. The marker (5318) of any one of claims 23 to 25, wherein one of the oligonucleotide attachment portion (5306a) of the linker (5310) and the oligonucleotide barcode (5304a) of the affinity reagent (112) comprises at least one guest molecule (5702) attached to a nucleobase (N) thereof and configured to complex with a host molecule (5704), and wherein the other of the oligonucleotide attachment portion (5306a) of the linker (5310) and the oligonucleotide barcode (5304a) of the affinity reagent (112) comprises at least one nucleobase (N') which is complementary to the nucleobase (N) to which the guest molecule (5702) is attached.

27. The marker (5318) according to claim 25 or 26, wherein the host molecule (5704) is cucurbituril.

28. The marker according to any one of claims 23 to 27, comprising at least one base mismatch between the oligonucleotide attachment portion (5306a) of the linker (A') and the oligonucleotide barcode (5304a) of the affinity reagent (112), the at least one base mismatch preventing stable binding therebetween, wherein the base mismatches are configured to be bridged by photoconversion.

29. The marker of claim 28, wherein the base mismatch is a GG mismatch configured to be bridged by photoconversion of trans-NCDA to cis-NCDA.

30. The marker of any one of claims 23 to 29, wherein the affinity reagent is an oligonucleotide probe configured to bind to a nucleic acid analyte.

31. The marker (5318) of any one of claims 23 or 30, wherein the oligonucleotide attachment portion (5306a) of the linker (5310) and the oligonucleotide barcode (5304a) of the affinity reagent (112) are each 9 to 13 nucleotides in length.

32. A plurality of markers according to any one of claims 23 to 31, wherein each reporter comprises a unique dye combination, and wherein each reporter is attached to an affinity reagent configured for attachment to an analyte such that no unique dye combination is bound to more than one affinity reagent.

33. A solution comprising the linker of claim 19 or 20, the reporter of claim 21 or 22, the marker of any one of claims 23 to 31, or a plurality of markers of claim 32.

34. A lyophilized solid comprising the linker of claim 19 or 20, the reporter of claim 21 or 22, the marker of any one of claims 23 to 31, or the plurality of markers of claim 32.

35. A computer program having a program code for performing the method according to claims 1 to 17 when said computer program is run on a processor.

36. A computer-readable storage medium storing the computer program according to claim 35.

37. A database comprising information corresponding to: Various affinity reagents; a first plurality of dye combinations; and First mapping, and optionally, further comprising information corresponding to at least one of: a second plurality of dye combinations and / or any other plurality of dye combinations; each dye combination having a characteristic regarding at least one of excitation and emission of said dyes; a second map or any other map; at least one first readout; at least one second readout and / or any other readout; The database is used to perform at least one of steps i) to ii) in claim 1 or 2.

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