Series of multi-channel microscopy

Abstract

This invention relates to a much faster way of combining multiple color channels in fluorescence microscopy for complex rapid diagnostic and research purposes. The advantage is that procedures and microscope settings can be simplified and miniaturized to such an extent that the construction of such automated systems becomes easier, and analysis is faster by omitting the erasure step, and image processing is moved to an image processing system that now operates almost in real time.

Description

Background Technology

[0001] This invention relates to a method for detecting different target portions on a biological sample by repeatedly imaging fluorescent labels and target portions without degrading the fluorescent labels between imaging steps.

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

[0003] For example, EP3037821 discloses a method for detecting and separating target portions based on, for example, a fluorescence signal using a conjugate having an enzymatically degradable spacer for reversible fluorescent labeling.

[0004] PCT / EP2019 / 060403 discloses a conjugate releasable label comprising an enzymatically degradable spacer, wherein the fluorescence quantum yield for detection is increased by inserting a linker unit comprising one or more polyethylene glycols between the releasable spacer and the fluorescent dye.

[0005] Further understanding of repeat imaging methods can be found in EP 0810428, EP1181525, EP 1136822, or EP1224472. In these methods, a biological sample is brought into contact with an antigen recognition region coupled to a fluorescent portion within a sequence period. The location of the antigen is detected through the fluorescent portion, and then the fluorescent portion is eliminated. Through this repeat label-detection-elimination process, protein networks can be mapped, different cell types can be located, and disease-related changes in the proteome can be analyzed.

[0006] All of these techniques involve a destaining step between imaging steps. Destaining can involve treating the fluorescent label with chemicals (such as oxygenating agents) or radiation to destroy it. In addition to the undesirable stress this treatment places on biological samples, destaining is a time-consuming process that significantly increases the overall method time.

[0007] It has been shown that by a two-step process of first adding a reagent and then taking a picture, and then adding a second reagent and taking a picture, an image with multiple pieces of information can be generated. Such methods are disclosed for example by the following publications: 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, Supp., May 1, 2019 (2019-05-01), p. 4048; SONG et al.: "Expression of drebrin E in migrating neuroblasts in adult rat brain: Coincidence between drebrin E disappearance from cellbody and cessation of migration", NEUROSCIENCE, NEW YORK, NY, US, Vol. 152, No. 3, January 19, 2008 (2008-01-19), pp. 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, August 2, 2019 (2019-08-02), pp. 16-20.

[0008] These publications describe methods in which a first reagent identifies a subset of molecules of a gene product and a second reagent identifies all molecules of a gene product. The publications then show how to distinguish between the various forms by simple image subtraction between the first and second staining. In these publications, the procedure for each step takes several hours due to the chosen procedure.

[0009] However, fluorescent dyes are known to have variable photostability properties and / or the conjugates used for staining have variable binding constants with their respective targets. Furthermore, some fluorescent dyes decay faster than others when photoactivated. Since "photoactivated" includes excitation radiation, the decay of emission over time cannot be avoided, and simply subtracting the emission from the image can lead to erroneous or misleading differential images. This decay of fluorescence emission over time is referred to below as "acquisition bleaching." Summary of the Invention

[0010] Therefore, the object of the present invention is to provide a method for detecting different target portions on a biological sample by repeated fluorescent labeling and imaging, the method not involving decolorization between imaging steps, and optionally considering acquisition bleaching.

[0011] It was discovered that images can be subtracted from each other's different periods, thus generating separate images for each target region while simultaneously taking into account the acquisition of bleach. Therefore, the subtractive method eliminates the need for decolorization.

[0012] Therefore, the present invention relates to a method for detecting a target portion in a biological sample, said method comprising the following cycle:

[0013] a) Contact the sample with a first conjugate comprising a first antigen recognition portion Y and a first fluorescent portion X, thereby causing at least a portion of the first conjugate to bind to the target portion recognized by the first antigen recognition portion Y.

[0014] b) Remove any of the first conjugates that are not bound to the target portion from the sample.

[0015] c) Obtain a first image of the sample, thereby detecting the target portion labeled with the first conjugate.

[0016] Steps a) to c) are repeated with at least one second conjugate comprising a second antigen recognition portion Y' and a second fluorescent portion X' to obtain at least one second image, wherein the first and second antigen recognition portions Y' are bound to different target portions, characterized in that the intensity of the first image is reduced due to a degradation function and subsequently subtracted from the at least one second image.

[0017] Figure 1 This diagram illustrates “digital” staining using eight different conjugates, including 20% ​​exposure of the acquisition bleach in each image. This means that the specificity of staining in one round is reduced by 20% in the next, and so on.

[0018] The first row shows four rounds of staining (1, 2, 3, 4) using four different conjugates. Images were taken after each round of staining. The images from the next round were then subtracted from the images from the previous round. The second row shows the resulting images, displaying only the specific signal added in the second round.

[0019] The third row shows the overlap from stains 1 and 5. Stain 1 has been attenuated by the five images taken over the five cycles and becomes invisible in subsequent cycles. As shown in the first image of the fourth row of the image rows, this partial overlap allows for the extraction of a specific signal in the fifth round.

[0020] In the method of this invention, a series of images are captured in a fluorescence microscope, and after each exposure, the staining intensity decreases depending on the stability of the corresponding fluorescent dye used. The fluorescent dye degrades over time while the fluorescence images are being captured. The degradation function of each fluorescent dye is used to enhance the accuracy of the image subtraction.

[0021] According to the method disclosed by Song et al. in Biophysical Journal, Vol. 68, June 1995, pp. 2588-2600, the degradation function can be calculated as the strength reduction.

[0022] In a simplified method, the degradation of the fluorescent dye over time is estimated to follow a linear function of time. Since the dyeing and washing processes of this invention are typically carried out within a comparable timeframe of 1-10 minutes, a simplified degradation function can be used without losing much information. In this variant of the invention, the degradation function is calculated as a reduction in intensity of 5-50%, preferably 10-30%.

[0023] In both variants, the terms "intensity reduction" or "acquisition bleaching" refer to the decrease in emission intensity of the fluorescent dye between two images. In other words, intensity reduction depends on the image processing time. The faster the processing time or the sequence of images captured, the smaller the intensity reduction and degradation function will be.

[0024] For calibration purposes, the reduction in emission intensity of the fluorescent dye between two images should be measured, depending on the fluorescent dye used. This may result in the use of different degradation functions for different fluorescent dyes.

[0025] In this implementation, for each image in a series of images at each exposure, the decay of the fluorescent dye can be predicted and subsequently used for additional image processing based on the specific photostability properties of pixels in the image, using the previous staining and decay parameters. This particularly allows for timely spacing of similar markers across multiple staining series and maximizes the difference between two images.

[0026] By using a combination of antibodies that are not typically expressed on the same cells or at the same locations, and are adjacent to each other during the process, the method of the present invention provides an image in which staining is established in the image by adding staining to areas that were not stained in the previous step. This physical separation through staining allows for further improvement in the image subtraction results.

[0027] By using fluorescent antibodies, the staining intensity decreases with each image capture due to a property known as acquisition bleaching. Consequently, the staining intensity of one staining cycle will be weaker after each subsequent cycle.

[0028] The series of methods of this invention allows for miniaturization and can be performed in a very small chamber on a single field of view. This miniaturization allows for the parallelization of the method with the analysis of multiple samples. Attached Figure Description

[0029] Figure 1 This illustrates a staining process with eight cycles according to the present invention.

[0030] Figure 1 The sample was repeatedly stained by four different conjugates: a) conjugate 1; b) conjugate 1+2; c) conjugate 1+2+3; and d) conjugate 1+2+3+4.

[0031] Figure 2 shows Figure 1 Subtraction of the image, where a) Figure 1 a) staining; b) Figure 1 staining of b-1a; c) Figure 1 c-(1b+1a) staining; and d) Figure 1 Staining of d-(1c+1b+1a).

[0032] Figure 3 The simulation shows 11 staining cycles, including the assumption that 20% of the images were bleached as a result of the image capture. Detailed Implementation

[0033] In serial multichannel microscopy, two important factors are the space occupied by the antigen-recognizing reagent and the intensity range of the antigen-recognizing region. Serial multichannel microscopy requires measuring or defining a space (region) or a fraction of the intensity range captured by the camera within a given space (region). The first parameter can be increased by increasing the resolution of the microscope, while the second parameter can be influenced by the precise intervals of the antigen-recognizing reagent concentration within the cycle.

[0034] Figure 1The method of the present invention is illustrated by way of example, with a schematic diagram showing the coloring of a "digit" using eight different conjugates to demonstrate the principle. The first line shows four rounds of coloring 1, 2, 3, and 4 using four different conjugates. An image is taken after each round of coloring. The image of the next round is then subtracted from the image of the previous round. The second line shows the resulting image, displaying only the specific signal added in the second round. Figure 1 The diagram shown includes acquisition bleaching with 20% exposure for each image. This means that the specific staining in one round is reduced by 20% in the next round, and so on.

[0035] The third row in the schematic diagram shows the overlapping staining from samples 1 and 5. The staining of sample 1 has been attenuated by the five images taken over the five cycles and becomes invisible in subsequent cycles. As shown in the first image of the fourth row, this partial overlap allows for the extraction of specific signals in the fifth round. By separating the overlapping areas in the selection of markers, markers present in the same areas of the sample can even be observed.

[0036] In a first embodiment of the invention, steps a) to c) are repeated over at least two cycles, wherein an image is obtained in each cycle, and wherein 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 over at least two cycles, wherein an image is obtained in each cycle, and wherein after each cycle, a difference image of the current image and the image of the previous cycle is obtained.

[0037] In a second embodiment of the invention, steps a) to c) are repeated over at least two cycles, wherein an image is obtained in each cycle, and wherein after the last cycle, the image of each cycle is subtracted from the image of the corresponding previous cycle. In other words, steps a) to c) are repeated over at least two cycles, wherein an image is obtained in each cycle, and wherein after the last cycle, a difference image of each cycle and the corresponding previous cycle is obtained.

[0038] Preferably, steps a) to c) are repeated in 2-500 cycles, more preferably in 2-100 cycles.

[0039] In a third embodiment of the present invention, the image is obtained as a pixel graphic image, and the image is obtained by pixel subtraction algorithm.

[0040] Unbound conjugates can be removed from the sample by washing.

[0041] In the simplest case, the same antigen or the same location is identified multiple times. In this procedure, the interval between the use of the second reagent over time allows for an extended intensity range because the fluorescence intensity of the previous reagent will decay with repeated image exposures. For example, as discussed by Song et al. in the Biophysical Journal, Vol. 68, June 1995, pp. 2588-2600, the function underlying decay is most likely a multi-exponential function. Depending on the intensity of the light source and the fluorescent dye, decay can be observed within 1 minute or up to 60 minutes. Only special photoresistive semiconductor-based dyes (e.g., quantum dots) do not show decay even after several hours.

[0042] In another embodiment, the fluorescent portion of the conjugate is attenuated by exposure to radiation. The resulting attenuation of emission can be calculated according to the method disclosed by Song et al. in Biophysical Journal, Vol. 68, June 1995, pp. 2588-2600, and can be used to “separate” the intensity of different staining cycles during image subtraction. Our own measurements revealed an emission attenuation of at least 10% per cycle.

[0043] By using the second reagent separately, the difference between the residual intensity of the first stain and the additional fluorescence intensity of the second reagent is reduced, resulting in a greater difference.

[0044] Image processing

[0045] Cyclic multichannel microscopy requires the removal of fluorescent staining by various means after each staining step. This step is time-consuming. The procedure disclosed herein allows for serial staining of several channels because it necessitates an active removal step for the fluorescent staining. This, in turn, accelerates the acquisition process in multichannel microscopy.

[0046] When the sample is stained with a fluorescent detection reagent, the area to be detected by the reagent is marked, and the mark is detected in a microscope using the necessary light and detector system.

[0047] Using the method of this invention, an increase in staining following a second staining after a first staining and image acquisition is detected. By performing simple image processing using already disclosed software, a differential image between the images after the first and second staining reveals additional staining from the second fluorescent stain. Depending on the specificity and intensity of the reagents chosen, multiple series of staining and image acquisition steps are possible. Furthermore, by selecting small changes in the color of the fluorescent dye, additional information can be used to further improve the sensitivity and selectivity between the two staining steps, if desired.

[0048] This method can be used for static serial staining and image acquisition steps, or for continuous flow and continuous imaging under conditions where the addition and removal of staining reagents are controlled by adding reagents to the fluid system.

[0049] Images captured by a fluorescence microscope can be processed using known software, such as the open-source software program "FIJI". FIJI provides an "Image Calculator" module that allows adding two images using the "Add" function and subtracting them using the "Subtract" function. The results of the image operations are then displayed in a new window and saved as a new image file. Subtracting two images is a feature of many imaging processing programs, and such software is readily available.

[0050] Similar to the software, the images can be taken by a technician using any known camera, such as the Prosilica GT 6600 from AlliedVision.

[0051] The staining intensity obtained by staining a sample depends on the amount of staining reagent used. The amount of staining reagent used can be adjusted so that the presence or absence of staining is detectable by making the staining visible, rather than by introducing a strong stain that reaches the total capacity of the image detection device. In subsequent staining, additional staining can be performed on top of the first staining with a very well-adjusted amount of staining reagent, resulting in increased intensity in the case of overlapping staining patterns at the points where the two reagents combine. An image calculation program can then be used to distinguish between single-stained pixels and double-stained pixels based on a comparison of staining intensities between images.

[0052] The series of multichannel microscopy methods of the present invention can also be combined with known methods, wherein staining of fluorescent reagents is removed by known procedures (such as radiation, enzymatic oxidation to remove fluorescent dyes or staining reagents). The series of staining procedures also allows for the serial use of other labeling techniques, such as labeling staining reagents with fluorescent oligonucleotide probes. Preferably, such other labeling techniques are used after the last cycle of the method of the present invention.

[0053] In another embodiment of the invention, images of the first and second conjugates not bound to their respective target portions are used to calibrate for attenuation upon exposure to radiation. For this purpose, a sample support may be coated with a protein recognized by the conjugate. In channels not used for staining cells, a fluorescent dye attenuation factor can be determined. This attenuation factor can be used to improve the image subtraction results.

[0054] In another embodiment of the invention, first and second conjugates having slightly different absorption spectra or at least maximum absorption at slightly different wavelengths are used. When using fluorescent dyes with overlapping absorption spectra, they can be used one after another instead of simultaneously. The different spectra will again improve the image subtraction result. Therefore, the method according to the invention can include providing at least two fluorescent portions Y with a maximum absorption phase difference of at least 10 nm.

[0055] In another embodiment of the invention, the method is performed as a continuous process, and images are captured periodically regardless of the staining steps. As a result, images of staining and staining decay can be combined into a cinematic sequence. This method allows for optimization of the timing between different staining steps. The "movie" could be a slow-motion film captured at a rate of several images per minute, sufficient to extract the necessary information. Increased time available for image capture will increase the sensitivity of the images. Increasing the number of images will improve the results of differential calculations of specific staining within a given period.

[0056] Conjugates used in this invention

[0057] Generally, any conjugate known in the field of cellular fluorescent labeling can be used in the method of the present invention. This includes any conjugate providing a fluorescent portion X and an antigen recognition portion Y. As the antigen recognition portion Y, any high-affinity (e.g., antibody) or low-affinity (e.g., FAB molecule) unit can be used, either directly or via a spacer linked to the fluorescent portion X. The conjugate used in the method of the present invention can have the general formula (I) X. o -Y m Or (II) (X) o -L) n -Y m Or (III) (X) o -L) n -PY m Furthermore, the conjugate may contain a unit that allows for the removal of the label from the target cell, for example, by adding 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 bound linker unit L, and X represents a covalently bound fluorescent part X. l and x are integers between 0 and 100, and n, o, and m are integers between 1 and 100.

[0058] Target section

[0059] The target portion to be detected by the method of the present invention can be on any biological sample, such as tissue sections, cell aggregates, suspended cells, or adherent cells. The cells can be alive or dead. Preferably, the target portion is an antigen expressed intracellularly or extracellularly on a biological sample (such as whole animals, organs, tissue sections, cell aggregates, or single cells of invertebrates (e.g., Caenorhabditis elegans, Drosophila melanogaster), vertebrates (e.g., zebrafish, Xenopus laevis), and mammals (e.g., house mice and other subspecies, various subspecies of the genus Rat, humans)).

[0060] Fluorescent part

[0061] The terms X and X' refer to different fluorescent portions, and all the characteristics disclosed for X also apply to X', and vice versa.

[0062] Suitable fluorescent moieties are known in the field of immunofluorescence techniques, such as flow cytometry or fluorescence microscopy. In these embodiments of the invention, the target moieties labeled with conjugates are detected by exciting the fluorescent moieties X and detecting the resulting emission (photoluminescence). Useful fluorescent moieties can be small organic molecular dyes, such as xanthan dyes (e.g., fluorescein), or rhodamine dyes, coumarin dyes, anthocyanin dyes, pyrene dyes, oxazine dyes, pyridyloxazole dyes, pyromethene dyes, acridine dyes, oxadiazole dyes, carbopyronine dyes, benzopyran dyes, fluorene dyes, or organometallic complexes (e.g., Ru, Eu, Pt complexes). In addition to monomolecules, clusters of small organic molecular dyes, fluorescent oligomers, or fluorescent polymers (e.g., polyfluorene) can also be used as fluorescent moieties. Furthermore, fluorescent moieties can be protein-based (e.g., phycobiliproteins), nanoparticles (e.g., quantum dots, upconversion nanoparticles, gold nanoparticles, stained polymer nanoparticles).

[0063] The fluorescent moiety X can be covalently coupled to the linker unit L. Methods of covalent coupling are known to those skilled in the art. It is possible for the activating group on the fluorescent moiety X or the linker unit L to react directly with the functional group on the linker unit L or the fluorescent moiety X, or via a heterobifunctional linker molecule that first reacts with one and then with another binding partner.

[0064] For example, fluorescent dyes can have groups reactive to amino or thiol groups, such as reactive esters that react with the amino group on the linking unit, such as N-hydroxysuccinimide (NHS), sulfodichlorophenyl ester (SDP), tetrafluorophenyl ester (TFP), and pentafluorophenyl ester (PFP), or Michael acceptors or haloacetyl groups that react with the thiol group on the linking unit, such as maleimide, iodoacetamide, and bromomaleimide groups. A large number of heterobifunctional compounds can be used for linking with entities. Illustrative entities include: azidobenzoylhydrazide, N-[4-(p-azidosalicylic acid)butyl]-3'-[2'-pyridyldithio]propionamide, bis-sulfosuccinimide octanoate, dimethyl hexadiamide, disuccinimide tartrate, Ny-maleiminobutyryloxysuccinimide ester, N-hydroxysulfosuccinimide-4-azidobenzoate, N-succinimide[4-azidophenyl]-1,3'-dithiopropionate, N-succinimide[4-iodoacetyl]aminobenzoate, glutaraldehyde, succinimide-[(N-maleiminopropionamido)polyethylene glycol] ester (NHS-PEG-MAL), and succinimide-4-[N-maleiminomethyl]cyclohexane-1-carboxylic acid ester. The preferred linking group is 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) or 4-(N-maleiminomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC), wherein a reactive thiol group is present on the fluorescent moiety and a reactive amino group is present on the linking group unit.

[0065] The conjugate used in the method of the present invention may contain 1-100, preferably 2-30, fluorescent portions X.

[0066] Antigen recognition part Y

[0067] The term "antigen recognition moiety Y" refers to any kind of molecule that binds with sufficiently high affinity to a target moiety expressed intracellularly or extracellularly on a biological sample so as to remain on the target after washing away any unbound antigen recognition moiety. The terms Y and Y' refer to distinct antigen recognition moieties, and all characteristics disclosed in Y also apply to Y', and vice versa.

[0068] The term “antigen recognition part Y” specifically refers to antibodies, fragmented antibodies, fragmented antibody derivatives, peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors of co-stimulatory molecules or artificially engineered binding molecules, peptides, lectins or aptamers, RNA, DNA, oligonucleotides and their analogues.

[0069] Fragmented antibody derivatives include, for example, Fab, Fab', F(ab')2, sdAb, scFv, di-scFv, and nanobodies. Such fragmented antibody derivatives can be synthesized through recombinant processes, including covalent and non-covalent conjugates containing these types of molecules.

[0070] The conjugate used in the method of the present invention may comprise 1-100, preferably 1-20, antigen recognition moieties Y. The interaction between the antigen recognition moieties and the target moieties can be high or low affinity. The binding interaction of a single low-affinity antigen recognition moieties is too weak to provide stable binding to the antigen. Low-affinity antigen recognition moieties can be polymerized by conjugation with an enzymatically degradable spacer P to provide high activity.

[0071] Preferably, the term "antigen recognition portion Y" refers to an antibody or Fab against an antigen expressed intracellularly (e.g., FoxP3, CD154, Ki67) or extracellularly (e.g., CD3, CD14, CD4, CD8, CD25, CD34, CD56, and CD133) of a biological sample (target cell).

[0072] The antigen recognition portion Y (especially antibodies) can be coupled to the spacer P via side-chain amino or thiol groups. In some cases, the glycosidic side chain of the antibody can be oxidized by periodate to produce an aldehyde functional group.

[0073] The antigen recognition portion Y can be covalently or non-covalently coupled to the spacer P. Methods of covalent or non-covalent conjugation are known to those skilled in the art and are the same as those mentioned for conjugation of the fluorescent portion X.

[0074] Uses of the method

[0075] The method of this invention can be used in a variety of applications in research, diagnosis and cell therapy.

[0076] In a first use of the invention, the detection or separation of biological samples (such as cells) is used for counting purposes, i.e., to establish the number of cells from a sample having a specific group of antigens recognized by the antigen recognition portion of the conjugate.

[0077] In the second application, one or more populations of biological samples are isolated for the purification of target cells. These isolated and purified cells can be used for a variety of downstream applications, such as molecular diagnostics, cell culture, or immunotherapy. Example

[0078] Comparative Examples

[0079] Mouse spleen sections were stained using four different monoclonal antibodies conjugated to fluorescent dyes. One antibody was used for staining each section, and an image was captured. The images were then superimposed on top of each other using computer calculations to simulate the staining of the sections without erasing the stain. In the next step, individual staining images were revealed again through image processing by subtracting the first monostained image from a second virtual double-stained image, and so on.

[0080] The antibodies used were NK1.1, which can be obtained from Miltenyi Biotec BV & Co. KG as REA 1162 antibody (1); KLRG1, which can be obtained from Miltenyi Biotec BV & Co. KG as REA 1016 antibody (2); GR-1 antibody, which can be obtained from Miltenyi Biotec BV & Co. KG as a monoclonal antibody (with the clonal name RB6-8C5); and CD38 antibody, which can be obtained from Miltenyi Biotec BV & Co. KG as Rea 616 (4).

[0081] Figure 2a and b shows that the samples were repeatedly stained with four different conjugates without bleaching, namely a) conjugate 1; b) conjugate 1+2; c) conjugate 1+2+3; and d) conjugate 1+2+3+4.

[0082] As a result of the method of the present invention Figure 2b The subtraction of the image in Figure 2a is shown, where a) a) coloring; b) coloring of image ba; c) coloring of c-(b+a); and d) coloring of d-(c+b+a).

[0083] According to embodiments of the present invention

[0084] In this embodiment, the same tissue sections as in Figure 2 are used, but this time... Figure 1The study demonstrates that bleaching is included in the simulation of the image. The following 11 antibodies were used for staining: Ki67 (A1, A2, cycle 1) obtained from Miltenyi Biotec BV & Co. KG as REA 183 antibody; KLRG1 (B1, B2, cycle 2) obtained from Miltenyi Biotec BV & Co. KG as REA 1016 antibody; NK1.1 (C1, C2, cycle 3) obtained from Miltenyi Biotec BV & Co. KG as REA 1162 antibody; CD8a (D1, D2, cycle 4) obtained from Miltenyi Biotec BV & Co. KG as REA601 antibody; CD11b (A3, A4, cycle 5) obtained from Miltenyi Biotec BV & Co. KG as REA593; F4 / 80 (B3, B4, cycle 6) obtained from Miltenyi Biotec BV & Co. KG as REA126; and antibodies obtained from Miltenyi Biotec BV & Co. KG. CD184 (C3, C4, cycle 7) obtained from Miltenyi Biotec BV & Co. KG as REA107, GR-1 (D3, D4, cycle 8) obtained from Miltenyi Biotec BV & Co. KG as a monoclonal antibody (clonal name RB6-8C5), CD15 anti-mouse clone MC-480 antibody obtained from Biolegend (A5, A6, cycle 9), LY-49A (B5, B6, cycle 10) obtained from Miltenyi Biotec BV & Co. KG as REAL436, and IgM (C5, C6, cycle 11) obtained from Miltenyi Biotec BV & Co. KG as REA979.

[0085] After each staining, an image was taken, and then the next staining was performed, followed by another image taking. After each round, due to acquisition bleaching, the staining from the previous round was reduced by 20% (rows A and C). Individual stainings were revealed through image subtraction (rows C and D). The number of subsequent stainings that could be performed was increased due to the acquisition bleaching effect. Figure 1 The embodiments shown are significantly enhanced. The antibodies used are present in different cell types within the tissue. This makes it possible to select antibodies in a manner that do not stain the same cell type in the next direct subsequent round.

[0086] Figure 3 The diagram shows 11 staining cycles, including the assumption that 20% of the images were collected as a result of bleaching.

[0087] The images are organized into a grid. Rows 1, 3, and 5 are composite images, and rows 2, 4, and 6 are displayed as follows: Figure 1 The calculated difference image is illustrated schematically. The sample was stained with a fluorescent dye conjugate. Strong staining can be seen in the image, for example, in period 4, 1D, in the upper right portion of the image. This strong staining diminishes in subsequent periods 5 (region 3A), 6 (region 3B), 7 (region 3C), and 8 (region 3D), and is no longer visible in period 8, as shown in the region.

Claims

1. A method for detecting a target portion in a biological sample, the method comprising: a) Contact the sample with a first conjugate comprising a first antigen recognition portion Y and a first fluorescent portion X, thereby binding at least a portion of the first conjugate to the target portion recognized by the first antigen recognition portion Y; b) Remove any of the first conjugates that are not bound to the target portion from the sample; c) Obtain a first image of the sample to detect the target portion labeled with the first conjugate, wherein the intensity of the first fluorescent portion X is reduced due to acquisition bleaching; Steps a) to c) are repeated with at least one second conjugate comprising a second antigen recognition portion Y' and a second fluorescent portion X' to obtain at least one second image, wherein the first antigen recognition portion Y and the second antigen recognition portion Y' are bound to different target portions, characterized in that the intensity of the first image is reduced by a degradation function and subsequently subtracted from the at least one second image.

2. The method according to claim 1, characterized in that... The degradation function was calculated as a 5-50% reduction in strength.

3. The method according to claim 1, characterized in that... Steps a) to c) are repeated over at least two cycles, wherein an image is obtained in each cycle, and wherein after each cycle, the image of the previous cycle is subtracted from the image of the current cycle.

4. The method according to claim 1, characterized in that... Steps a) to c) are repeated over at least two cycles, wherein an image is obtained in each cycle, and wherein after the last cycle, the image of each cycle is subtracted from the image of the corresponding previous cycle.

5. The method according to any one of claims 1-2, characterized in that... The image is obtained as a pixel graphic image, and the subtraction of the image is obtained through the pixel subtraction algorithm.

6. The method according to any one of claims 1-3, characterized in that... The first and second conjugates that were not bound to the target portion were removed from the sample by washing.

7. The method according to any one of claims 1-4, characterized in that... After the final cycle, the first and second conjugates bound to the target portion are removed from the target portion at least partially by radiation, oxidation, or enzymatic treatment.

8. The method according to any one of claims 1-4, characterized in that... The first fluorescent portion or the second fluorescent portion is selected from xaton dyes, rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyloxazole dyes, pyromethene dyes, acridine dyes, oxadiazole dyes, carbopyronine dyes, benzopyranium dyes, fluorene dyes, or fluorescent polymers.

9. The method according to any one of claims 1-4, characterized in that... The first fluorescent portion and the second fluorescent portion of the first conjugate and the second conjugate are attenuated upon exposure to radiation.

10. The method according to claim 9, characterized in that... Images of the first and second conjugates not bound to the target portion are used to calibrate the attenuation by exposure to radiation.

11. The method according to any one of claims 1-4, characterized in that... It provides at least two fluorescent portions with a maximum absorption wavelength difference of at least 10 nm.

Images