Serial Multichannel Microscopy

Abstract

The present invention relates to a very fast method of combining multiple color channels in fluorescence microscopy for complex high-speed diagnostic and research purposes. The advantage is that the procedure and microscope setup can be simplified and miniaturized to the extent that it becomes very easy to build such an automated system and the speed of analysis increases by omitting the elimination step, moving to processing of images by image processing systems that now function in near real time.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting different target moieties on a sample of a biological specimen by repeated fluorescent labeling and imaging of the target moieties without degradation of the fluorescent label between imaging steps.

[0002] The current approach for repeated fluorescent labeling and imaging is a cyclic process in which the target is stained, an image is taken, and the stain is removed by oxidation or radiation before a new round of staining begins.

[0003] For example, EP 3037821 discloses a method for the detection and separation of target moieties by, for example, a fluorescent signal with conjugates having an enzymatically degradable spacer for reversible fluorescent labeling.

[0004] International application PCT / EP2019 / 060403 discloses label releasable conjugates comprising an enzymatically degradable spacer, the fluorescence quantum yield for detection of which is increased by inserting one or more polyethylene glycol-containing linker units between the releasable spacer and the fluorescent dye.

[0005] Further repetitive imaging methods are known from EP 0810428, EP 1181525, EP 1136822 or EP 1224472, in which a sample of a biological specimen is contacted in successive cycles with an antigen recognition moiety bound to a fluorescent moiety, the location of the antigen is detected by the fluorescent moiety and then the fluorescent moiety is removed.

[0006] All these techniques involve a bleaching step between imaging steps. Bleaching can involve treatment with chemicals such as oxygenating agents or radiation to destroy the fluorescent label. In addition to the undesirable stress this treatment places on the biological specimen, bleaching is a time-consuming process that significantly increases the total processing time.

[0007] It has been shown that images with multiple information can be generated by a two-step process by first adding one reagent, then taking a picture, and then adding a second reagent and taking a picture. Such an approach is described, for example, in Jennifer Pankratz et al., "REAIease Technology: Controlled release of antibody-fluorochrome conjugates for maximal flexibility in flow sorting and fluorescence microscopy applications," CANCER RESEARCH, vol. 79, no. 13, SuppL, 1 May 2019 (2019-05-01), page 4048; Song et al., "Expression of drebrin E in migrating neuroblasts in adult rat brain: Coincidence between drebrin E disappearance from cell body and cessation of migration," Neuroscience, New York, NY, US, vol. 152, no. 3, 19 January 2008 (2008-01-19), pages 670-682; and Christoph Herbel et al., "MACSima™ Imaging Platform provides new insights into cancer biology and "Target discovery by cyclic immunofluorescence-based imaging", MACS & MORE, vol. 18, no. 1, 2 August 2019 (2019-08-02), pages 16-20.

[0008] These publications describe a method in which one reagent recognizes a subset of molecules from a gene product, and a second reagent recognizes all molecules from a gene product. The publications then show how to distinguish between the various morphologies by simple image subtraction between the first and second stains. Each step in the publications takes several hours depending on the procedure chosen.

[0009] However, it is known that fluorescent dyes have variable photostability properties and / or that conjugates used for staining have variable binding constants to reflective targets. Furthermore, some fluorescent dyes decay faster when activated by light compared to others. Because "light activation" involves excitation radiation, decay of the emitted light over time is inevitable, and simple image subtraction may result in erroneous or misleading difference images. The decay of fluorescent emission over time is hereinafter referred to as "acquisition bleach."

[0010] Summary of the Invention It was therefore an object of the present invention to provide a method for detecting different target moieties on a sample of a biological specimen by repeated fluorescent labeling and imaging without bleaching between imaging steps and optionally taking into account acquisition bleach.

[0011] It has been found that images from different cycles can be subtracted from one another, thereby creating separate images for each target moiety while simultaneously accounting for acquired bleach. Thus, the subtraction process does not require bleaching.

[0012] Therefore, the present invention provides: a) contacting the sample with a first conjugate comprising a first antigen recognition moiety Y and a first fluorescent moiety X, thereby binding at least a portion of the first conjugate to the target moiety recognized by the first antigen recognition moiety Y; b) removing from the sample any first conjugate that is not bound to the target moiety; c) obtaining a first image of the sample, thereby detecting the target moiety labeled with the first conjugate; repeating steps a) to c) with at least one second conjugate comprising a second antigen recognition moiety Y' and a second fluorescent moiety X', thereby obtaining at least a second image; wherein the first and second antigen recognition moieties Y' bind to different target moieties, characterized in that the intensity of a first image is reduced by a decomposition function and then subtracted from at least one second image.

[0013] Figure 1 shows a schematic of "number" staining in eight different conjugates, with 20% acquisition bleach per image exposure, meaning that a particular staining in one round will be 20% weaker in the next round.

[0014] The first row shows four rounds of staining with four different conjugates, 1, 2, 3, and 4. After each round of staining, an image is taken, and the image from the next round is subtracted from the image from the previous round. The second row shows the resulting image, showing only the specific signal added in the second round.

[0015] The third row shows the situation where staining numbers 1 and 5 overlap. Staining number 1 is already weak through the five images taken during cycle 5 and becomes invisible in the following cycles. This partial overlap allows us to extract a specific signal in round 5, as shown in the first image in the fourth row of images.

[0016] In the method of the present invention, successive images are taken with a fluorescence microscope, and after each exposure, the intensity of the staining decreases depending on the stability of each fluorescent dye used. As the fluorescence images are taken, the fluorescent dyes decompose over time. A decomposition function for each fluorescent dye is used to improve the accuracy of the image subtraction.

[0017] The resolution function can be calculated as the decrease in intensity according to the method disclosed in Song et al., Biophysical Journal, Vol. 68, June 1995, p. 2588-2600.

[0018] In a more simplified approach, the degradation of the fluorescent dye over time is assumed to follow a linear function over time. Because the staining and washing processes of the present invention are performed over a significant time frame, typically 1-10 minutes, a simplified degradation function can be used without losing too much information. In this variation of the present invention, the degradation function is calculated as a decrease in intensity of 5-50%, preferably 10-30%.

[0019] In both variants, the term "intensity decrease" or "acquisition bleach" refers to the decrease in the intensity of the fluorescent dye emission between two images. In other words, the intensity decrease depends on the processing time of the images. The faster the processing time or sequence of the captured images, the smaller the intensity decrease and the resolution function.

[0020] For calibration purposes, depending on the fluorochrome used, the decrease in the emission intensity of the fluorochrome between the two images should be measured. Consequently, different decomposition functions may be used for different fluorochromes.

[0021] In this embodiment, the decay of the fluorescent dye can be predicted for each image in the series at each exposure and then used to perform additional image processing using the specific photostability characteristics of the pixels in the image based on previous staining and decay parameters. This allows for similar markers to be spaced apart in time, particularly in multiple staining series, to maximize the difference image between the two images.

[0022] By using a combination of antibodies that are not usually expressed in the same cells or at the same location but are adjacent to each other in the process, the method of the present invention provides images in which staining is accumulated in the image by adding stain to areas that were not stained in the previous step. This physical separation of staining achieves further improvement in the image subtraction results.

[0023] With fluorescent antibodies, the staining intensity of the fluorescent antibody decreases with each image taken due to a property called acquisition bleaching, which causes the staining intensity of one staining cycle to weaken after each subsequent cycle.

[0024] The continuous process of the present invention allows for miniaturization of the process and can be performed in very small chambers on a single field of view. This miniaturization allows for parallelization of the process for multiple sample analysis. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 illustrates the staining process according to the invention in eight cycles, showing repeated staining of a specimen with four different conjugates: a) conjugate 1; b) conjugate 1 + 2; c) conjugate 1 + 2 + 3 and d) conjugate 1 + 2 + 3 + 4. [Figure 2] Figure 2 shows the images of Figure 1 subtracted with a) staining of Figure 1a); b) staining of Figure 1b-1a); c) staining of Figure 1c-(1b+1a) and d) staining of Figure 1d-(1c+1b+1a). [Figure 3] Figure 3 shows a simulation of 11 cycles of staining, including an assumed 20% acquisition bleach as a result of capturing the image.

[0026] Detailed Description In serial multichannel microscopy, there are two important factors: the space occupied by the antigen recognition moiety reagent and the intensity range of the antigen recognition moiety. Serial multichannel microscopy requires measuring or defining the space (area) or the portion of the intensity range captured by the camera in a specific space (area). The first parameter can be increased by increasing the resolution of the microscope, and the second parameter can be influenced by the precise interval of the antigen recognition moiety reagent concentration throughout the cycle.

[0027] FIG. 1 shows the method of the present invention in a schematic diagram of a "number" staining with eight different conjugates as an example to prove the principle. The first row shows four rounds of staining with four different conjugates, 1, 2, 3, and 4. After each staining round, an image is taken, and the image of the next round is subtracted from the image of the previous round. The second row shows the resulting image showing only the specific signal added in the second round. In the schematic shown in FIG. 1, a 20% acquisition bleach is included with each image exposure. This means that the specific staining in one round becomes 20% weaker in the next round.

[0028] The third row in the diagram schematic shows the situation where stains 1 and 5 overlap. Stain 1 is already weak after five images taken during five cycles and becomes invisible in subsequent cycles. This partial overlap allows for the extraction of a specific signal in round 5, as shown in the first image in the fourth row of images. By spacing the overlapping areas in the selection of markers, it is even possible to visualize markers present in the same region of the specimen.

[0029] In a first embodiment of the present invention, steps a) to c) are repeated for at least two cycles, in which an image is acquired in each cycle, and after each cycle, the image of the previous cycle is subtracted from the image of the current cycle. In other words, steps a) to c) are repeated for at least two cycles, in which an image is acquired in each cycle, and after each cycle, a difference image between the current image and the image of the previous cycle is acquired.

[0030] In a second embodiment of the present invention, steps a) to c) are repeated for at least two cycles, where an image is acquired in each cycle, and after the final cycle, the image in each cycle is subtracted from the image in the previous cycle. In other words, steps a) to c) are repeated for at least two cycles, where an image is acquired in each cycle, and after the final cycle, a difference image between the image in each cycle and the image in the previous cycle is acquired.

[0031] Preferably, steps a) to c) are repeated 2 to 500 cycles, preferably 2 to 100 cycles.

[0032] In a third embodiment of the present invention, the images are acquired as pixel graphic images, whereby the subtraction of the images is obtained by a pixel subtraction algorithm.

[0033] Conjugate that does not bind to the target moiety may be removed from the sample by washing.

[0034] In the simplest case of multiple recognition of the same antigen or the same location, spacing the use of the second reagent in the procedure can extend the intensity range, as the fluorescence intensity of the previous reagent decays with multiple image exposures. The function underlying the decay is likely multi-exponential, as discussed, for example, by Song et al., Biophysical Journal, Vol. 68, June 1995, pp. 2588-2600. Depending on the light source and the intensity of the fluorescent dye, decay can be observed within 1 minute or up to 60 minutes. Only special light-stable semiconductor-based dyes (e.g., quantum dots) do not show decay even after several hours.

[0035] In other embodiments, the fluorescent moiety of the conjugate is degraded by exposure to radiation. The resulting luminescence decay can be calculated as disclosed in Song et al., Biophysical Journal, Vol. 68, June 1995, p. 2588-2600, and used to "space" the intensities of different staining cycles during image subtraction. Our own measurements indicate realistic radioactive decomposition of at least 10% per cycle.

[0036] By spacing out the application of the second reagent, any remaining intensity difference from the first stain is reduced, and the additional fluorescent intensity from the second reagent leads to a larger difference.

[0037] Image Processing Cyclic multichannel microscopy requires the removal of fluorescent stains by various means after each surrounding staining. This step is time-consuming. The procedure disclosed herein allows for several channel stains in a sequential manner before requiring an active removal step for the fluorescent stain. This accelerates the acquisition of multichannel microscopy.

[0038] When the sample is stained with a fluorescent detection reagent, the area to be detected by the reagent is labeled, and the label is detected with a microscope equipped with the necessary light and detector systems.

[0039] With the method of the present invention, an increase in staining after a second staining following a first staining and image acquisition is detected. Through simple image processing with previously disclosed software, a difference image between the images after the first and second staining shows additional staining due to the second fluorescent stain. Depending on the selection of reagent specificity and intensity, multiple successive staining and image acquisition steps are possible. Furthermore, by selecting a small change in the color of the fluorescent dye, additional information can be used, if desired, to further improve sensitivity and selectivity between the two staining steps.

[0040] The method can be used in both static sequential staining and image acquisition steps, or under continuous flow and sequential imaging conditions where the addition and removal of staining reagents is controlled by the addition of reagents to the fluidic system.

[0041] Images captured by a fluorescence microscope can be processed with known software, for example the open-source software program "FIJI." "FIJI" provides a module "Image Calculator" that can be used to add two images with the function "Add" and to subtract two images with the function "Subtract." The result of the image manipulation is displayed in a new window and saved to a new image file. Subtraction of two images is a feature of many image processing programs, and such software is readily available.

[0042] As with the software, images can be taken by any camera known to those skilled in the art, such as a Prosilica GT 6600 from Allied Vision.

[0043] The staining intensity obtained by staining a sample depends on the amount of staining reagent used. The amount of staining reagent used in the staining visualization method can be adjusted to detect the presence or absence of staining without introducing a strong stain that reaches the overall capacity of the image detection device. In subsequent staining, additional staining can be performed in a well-adjusted amount of staining reagent in addition to the initial stain, resulting in an increase in intensity when the staining patterns overlap at the location where both reagents bind. Image calculation procedures can then be used to distinguish between single-stained and double-stained pixels based on a comparison of staining intensity between images.

[0044] The serial multichannel microscopy method of the present invention may be combined with known methods in which fluorescent staining is removed by known procedures such as radiation, oxidation of the fluorescent dye, enzymatic removal of the staining reagent, or other labeling techniques. Sequential staining procedures may also employ other labeling techniques in succession, such as labeling of the staining reagent with a fluorescent oligoprobe. Preferably, such other labeling techniques are utilized after the final cycle of the method of the present invention.

[0045] In another embodiment of the invention, images of the first and second conjugates not bound to their respective target moieties are used to calibrate decay due to exposure to radiation. For this purpose, the sample support may be coated with a protein recognized by the conjugate. The extinction coefficient of the fluorescent dye may be determined in a channel not used to stain the cells. The extinction coefficient can improve the image subtraction results.

[0046] In other embodiments of the invention, first and second conjugates are used that have slightly different absorption spectra or at least absorption maxima at slightly different wavelengths. When fluorescent dyes with overlapping absorption spectra are used, they can be used sequentially rather than simultaneously. Again, the different spectra improve image subtraction results. Thus, the method according to the invention may comprise providing at least two fluorescent moieties Y that have absorption maxima that differ by at least 10 nm.

[0047] In another embodiment of the invention, the method is performed as a continuous process, with images taken periodically, regardless of the staining step. As a result, images of staining and stain decay can be combined in a cinematic sequence. This approach allows for optimizing the timing between different stains. This "movie" can be a slow-motion movie, with several images taken per minute, sufficient to extract the necessary information. The longer the time available for taking images, the higher the sensitivity of the images. Increasing the number of images improves the results of the difference calculation for a particular stain in a given cycle.

[0048] Conjugates used in the present invention Generally, any conjugate known in the art of fluorescent labeling of cells may be used in the methods of the present invention. This includes conjugates provided with a fluorescent moiety, X, and an antigen recognition moiety, Y. The antigen recognition moiety, Y, may be either a high affinity (such as an antibody) or a low affinity (such as a FAB molecule) unit, either directly or via a spacer linked to the fluorescent moiety, X. Conjugates for use in the methods of the present invention have the general formula (I) X o -Y m , or (II)(X o -L) n -Y m , or (III) (X o -L)nPY m Furthermore, the conjugate may contain a unit that allows removal of the label from the target cell, for example, by addition of an enzyme and / or a linker unit that increases the fluorescence quantum yield. Such conjugates may have the general formula (IV) (X o -L) n -P(L) l (X) x -Y m In general formulas (I) to (IV), P represents an enzymatically degradable spacer, L represents a covalently bonded linker unit L, and X represents a covalently bonded fluorescent moiety X. l and x are integers from 0 to 100, and n, o, and m are integers from 1 to 100.

[0049] target area The target moiety detected by the methods of the present invention can be present on any biological specimen, such as a tissue slice, a cell aggregate, a suspension cell, or an adherent cell. The cells can be living or dead. Preferably, the target moiety is an antigen expressed intracellularly or extracellularly on a biological specimen, such as a whole animal, an organ, a tissue slice, a cell aggregate, or a single cell of an invertebrate (e.g., Caenorhabditis elegans, Drosophila melanogaster), a vertebrate (e.g., zebrafish, Danio rerio, Xenopus laevis), or a mammal (e.g., Mus musculus and other subspecies, various subspecies of Rattus, Homo sapiens).

[0050] Fluorescent part The terms X and X' refer to different fluorescent moieties, and all functionality disclosed for X is also applicable to X', and vice versa.

[0051] Suitable fluorescent moieties are known from immunofluorescence techniques, such as flow cytometry or fluorescence microscopy. In these embodiments of the present invention, the target moiety labeled with the conjugate is detected by exciting the fluorescent moiety X and detecting the resulting light emission (photoluminescence). Useful fluorescent moieties can be organic small molecule dyes, such as xanthene dyes like fluorescein, or rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyloxazole dyes, pyrromethene dyes, acridine dyes, oxadiazole dyes, carbopyronine dyes, benzpyrylium dyes, fluorescein dyes, or organometallic complexes, such as Ru, Eu, or Pt complexes. In addition to single molecular entities, clusters of organic small molecule dyes, fluorescent oligomers, or fluorescent polymers, such as polyfluorenes, can also be used as fluorescent moieties. Furthermore, the fluorescent moiety can be protein-based, such as phycobiliproteins, nanoparticles, such as quantum dots, upconversion nanoparticles, gold nanoparticles, or dyed polymer nanoparticles.

[0052] The fluorescent moiety X can be covalently attached to the linker unit L. Methods of covalent attachment are known to those skilled in the art. Direct reaction of an activating group on either the fluorescent moiety X or the linker unit L with a functional group on either the linker unit L or the fluorescent moiety X is possible, or via a heterobifunctional linker molecule that can react first with one and then with the other binding partner.

[0053] For example, fluorescent dyes are available with groups reactive to amino or thiol groups, such as activated esters, e.g., N-hydroxysuccinimide esters (NHS), sulfodichlorophenyl esters (SDP), tetrafluorophenyl esters (TFP), and pentafluorophenyl esters (PFP), that react with amino groups on linker units, or Michael acceptors or haloacetyl groups that react with thiol groups, e.g., maleimide, iodoacetamide, and bromomaleimide groups, on linker units. Numerous heterobifunctional compounds are available for conjugating to entities. Numerous heterobifunctional compounds are available for conjugating to entities. Illustrative entities include azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartrate, N-maleimidobutyloxysuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-succinimidyl[4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, succinimidyl-[(N-maleimidopropionamido)polyethyleneglycol]ester (NHS-PEG-MAL), and succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate. Preferred linking groups are 3-(2-pyridyldithiopropionic acid N-hydroxysuccinimide ester) (SPDP) or 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC), which have a reactive sulfhydryl group on the fluorescent moiety and a reactive amino group on the linker unit.

[0054] The conjugates used in the methods of the invention may contain 1 to 100, preferably 2 to 30, fluorescent moieties X.

[0055] Antigen recognition part Y The term "antigen recognition moiety Y" refers to any type of molecule that binds to a target moiety expressed intracellularly or extracellularly on a biological specimen with sufficiently high affinity to remain on the target after washing away unbound antigen recognition moiety. The terms Y and Y' refer to different antigen recognition moieties, and all functions disclosed for Y are also applicable to Y', and vice versa.

[0056] The term "antigen recognition moiety Y" particularly relates to antibodies, antibody fragments, antibody fragment derivatives, peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules, or artificially engineered binding molecules, peptides, lectins or aptamers, RNA, DNA, oligonucleotides and analogs thereof.

[0057] Fragmented antibody derivatives are, for example, Fab, Fab', F(ab')2, sdAb, scFv, di-scFv, nanobodies, etc. Such fragmented antibody derivatives may be synthesized by recombinant methods, including covalent and non-covalent conjugates involving these types of molecules.

[0058] The conjugate used in the method of the present invention may contain 1 to 100, preferably 1 to 20, antigen recognition moieties Y. The interaction between the antigen recognition moiety and the targeting moiety can be high affinity or low affinity. The binding interaction of a single low affinity antigen recognition moiety is too low to provide stable binding to the antigen. Low affinity antigen recognition moieties can be multimerized by conjugating them to an enzymatically degradable spacer P to provide high binding avidity.

[0059] Preferably, the term "antigen recognition moiety Y" refers to an antibody or Fab directed against an antigen expressed by an intracellular biological specimen (target cell), such as FoxP3, CD154, Ki67, or an extracellular biological specimen (target cell), such as CD3, CD14, CD4, CD8, CD25, CD34, CD56, and CD133.

[0060] The antigen-recognizing moiety Y, particularly an antibody, can be attached to the spacer P via a side chain amino or sulfhydryl group. Optionally, the glycidic side chains of the antibody can be oxidized with periodate to generate aldehyde functional groups.

[0061] The antigen recognition moiety Y can be covalently or non-covalently attached to the spacer P. Methods for covalent or non-covalent attachment are known to those skilled in the art and are the same as those listed for the attachment of the fluorescent moiety X.

[0062] Use of the method The methods of the present invention can be used in a variety of applications in research, diagnostics and cell therapy.

[0063] In a first use of the present invention, biological specimens, e.g., cells, are detected or isolated for enumeration purposes, i.e., to establish the number of cells from a sample that have a particular set of antigens recognized by the antigen recognition portion of the conjugate.

[0064] In a second use, one or more populations of biological specimens are separated for the purification of target cells, which can then be used in multiple downstream applications, such as molecular diagnostics, cell culture, or immunotherapy.

[0065] Example Comparative Example Mouse spleen sections were stained with four different monoclonal antibodies conjugated to fluorescent dyes. Images were taken using one antibody per section staining. The images were then computer-generated to overlay each other, simulating staining of the sections without eliminating the staining. In a second step, the individual stained images were displayed by image processing, and the individual stained images were redisplayed by subtracting the first single-stained image from the second virtual double-stained image.

[0066] The antibodies used were NK1.1 available from Miltenyi Biotec BV&Co.KG as REA 1162 antibody (1), KLRG1 available from Miltenyi Biotec BV&Co.KG as REA 1016 antibody (2), GR-1 antibody, a monoclonal antibody with the clone name RB6-8C5 available from Miltenyi Biotec BV&Co.KG, and CD38 antibody available from Miltenyi Biotec BV&Co.KG as Rea 616 (4).

[0067] Figures 2a and 2b show replicate staining of the specimen with four different conjugates without acquisition bleach: a) conjugate 1; b) conjugate 1+2; c) conjugate 1+2+3 and d) conjugate 1+2+3+4.

[0068] As a result of the method of the present invention, Figure 2b shows the subtraction of the image of Figure 2a with a) staining of a), b) staining of image ba; c) staining of c-(b+a) and d) staining of d-(c+b+a).

[0069] Examples according to the present invention In this example, the same tissue section as in Figure 2 was used, but this time the acquisition bleach shown in Figure 1 was included in the image simulation. Staining was performed using the following 11 antibodies: Ki67 (A1, A2, cycle 1) available from Miltenyi Biotec BV&Co.KG as REA 183 antibody, KLRG1 (B1, B2, cycle 2) available from Miltenyi Biotec BV&Co.KG as REA 1016 antibody, NK1.1 (C1, C2, cycle 3) available from Miltenyi Biotec BV&Co.KG as REA 1162 antibody, CD8a (D1, D2, cycle 4) available from Miltenyi Biotec BV&Co.KG as REA601 antibody, CDllb (A3, A4, cycle 5) available from Miltenyi Biotec BV&Co.KG as REA593, and REA126 available from Miltenyi Biotec BV&Co.KG. F4 / 80 (B3, B4, cycle 6) available from Miltenyi Biotec BV&Co.KG, CD184 (C3, C4, cycle 7) available from Miltenyi Biotec BV&Co.KG as REA107, GR-1 antibody (D3, D4, cycle 8) as a monoclonal antibody with the clone name RB6-8C5 available from Miltenyi Biotec BV&Co.KG, CD15 anti-mouse clone MC-480 antibody (A5, A6, cycle 9) from Biolegend, LY-49A (B5, B6, cycle 10) available from Miltenyi Biotec BV&Co.KG as REAL436, and IgM (C5, C6, cycle 11) available from Miltenyi Biotec BV&Co.KG as REA979.

[0070] After each staining, an image is taken, and then the next staining is performed and an image is taken again. After each round, the staining from the previous round is reduced by 20% using acquisition bleach (rows A and C). Image substraction allows the individual stainings to be revealed (rows C and D). Due to the effect of acquisition bleach, the number of subsequent stainings that can be performed is significantly increased compared to the example shown in Figure 1. The antibodies used stain various cell types present in the tissue. This allows antibodies to be selected so that they do not stain the same cell types in the immediately following round.

[0071] Figure 3 shows 11 cycles of staining, including an assumed 20% acquisition bleach as a result of which the image was taken.

[0072] The images are arranged as a grid. Rows 1, 3, and 5 are composite images, while rows 2, 4, and 6 show the calculated difference images illustrated in Figure 1. The specimens were stained with a fluorescent dye conjugate. For example, in cycle 4, the images show strong staining in the upper right portion of the image. This strong staining weakens in subsequent cycles 5 (field 3A), 6 (field 3B), 7 (field 3C), and 8 (field 3D), and is no longer visible in cycle 8, shown in field 3D.

Claims

1. below, a) contacting the sample with a first conjugate comprising a first antigen recognition moiety Y and a first fluorescent moiety X, thereby binding at least a portion of the first conjugate to the target moiety recognized by the first antigen recognition moiety Y; b) removing from the sample any first conjugate that is not bound to the target moiety; c) obtaining a first image of the sample, thereby detecting the target moiety labeled with the first conjugate; repeating steps a) to c) with at least one second conjugate comprising a second antigen recognition moiety Y' and a second fluorescent moiety X', thereby obtaining at least a second image; 1. A method for detecting a target moiety in a sample of a biological specimen by: the first antigen recognition moiety Y and the second antigen recognition moiety Y′ bind to different target moieties; the target moiety is an antigen expressed intracellularly or extracellularly in the biological specimen; 1. A method for detecting a target moiety in a sample of a biological specimen, comprising: reducing the emission intensity of a fluorescent dye in a first image by a resolution function; and then subtracting the emission intensity of the fluorescent dye in the first image from at least one second image, the resolution function being calculated as a reduction in the emission intensity of the fluorescent dye of 5 to 50%.

2. 2. The method of claim 1, wherein steps a) to c) are repeated for at least two cycles, wherein an image is acquired in each cycle and, after each cycle, the image of the previous cycle is subtracted from the image of the current cycle.

3. 2. The method of claim 1, wherein steps a) to c) are repeated for at least two cycles, acquiring an image in each cycle and subtracting the image of each cycle from the image of the previous cycle after the last cycle.

4. 2. A method according to claim 1, characterized in that the images are obtained as pixel graphic images and the subtraction of the images is obtained by a pixel subtraction algorithm.

5. 5. The method according to claim 1, wherein the fluorescent moiety of the conjugate is degraded by exposure to radiation.

6. 6. The method of claim 5, wherein images of the first and second conjugates not bound to their respective target moieties are used to calibrate degradation due to exposure to radiation.

Images