Methods and compositions for enhanced and selective photobleaching

Abstract

Disclosed herein are compositions, systems, kits, and methods for photo bleaching and selective photo bleaching of biological samples.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Application No. 63 / 674,750, filed July 23, 2024. The contents of the above application are incorporated herein by reference in their entirety. background

[0002] In biology and medicine, various methods can be used to observe different targets in biological samples. For example, proteins and nucleic acids in various tissue or cell products can be analyzed using histochemistry, immunohistochemistry (IHC), immunofluorescence (IF), and many nucleic acid hybridization, amplification, and visualization techniques. Analysis of proteins and nucleic acids in biological samples can also be performed using solid-state assays, such as Western blotting and RNA blotting techniques.

[0003] Many existing technologies can only detect a few targets in a single sample at a time (e.g., immunohistochemistry (IHC) or fluorescence-based Western blotting, where the number of detectable targets is limited by the fluorescence-based detection system). Further analysis of targets may require additional biological samples from the source, thus limiting the ability to determine the relative characteristics of the targets, such as the presence, absence, concentration, and / or spatial distribution of more than one biological target in the biological sample. Furthermore, in some cases, a limited amount of a single sample may be available for analysis, or a single sample may require further analysis. Therefore, there is a need for methods, reagents, and devices capable of iteratively analyzing single samples. Overview

[0004] This document discloses methods and compositions for detecting, visualizing, and / or characterizing target molecules in biological samples. The methods and compositions include PMS, PDS, salts thereof, or any combination thereof, wherein PMS and PDS are represented by the following formula: or .

[0005] In some embodiments, a method for detecting one or more targets in a biological sample is provided, the method comprising: (a) contacting the biological sample with a detectable marker and attaching the detectable marker to one or more targets; (b) detecting a signal from the detectable marker; and (c) after the detection in step (b), contacting the sample from step (b) with a PMS, a PDS, a salt thereof, or any combination thereof, wherein the PMS and PDS are represented by the following formula: or , (d) Irradiating the sample of (c), wherein irradiation includes exposing the sample to light, optionally wherein the light is light of a selected wavelength range, wherein the selected wavelength range is within the wavelength range required to detect the detectable marker; and (e) optionally, repeating steps (a)-(d).

[0006] In some embodiments, a method is provided for sequentially detecting more than one target in a biological sample, the method comprising: (a) contacting a first target probe with a sample, wherein the first target probe is specific for a first target; (b) hybridizing or binding the first target probe to the first target in the sample; (c) detecting a first fluorescently detectable label, wherein the first fluorescently detectable label is attached to the first target probe; and (d) after detection in step (c), contacting the sample from step (c) with PMS, PDS, their salts, or any combination thereof, wherein PMS and PDS are represented by the following formula: or , (e) Irradiating the sample of (d), wherein irradiation includes exposing the sample to light, optionally wherein the light is light within a selected wavelength range, wherein the selected wavelength range is within the wavelength range required to detect the first fluorescent detectable label; (f) Repeating steps (a)-(e), including an Nth target probe and an Nth fluorescent detectable label different from the first probe and the first fluorescent detectable label, optionally wherein the light within the selected wavelength range is within the wavelength range required to detect the Nth fluorescent detectable label.

[0007] In some embodiments, a method for detecting more than one target in a biological sample is provided, the method comprising: (a) contacting a sample with more than one target probe comprising N subsets of target probes, wherein each probe in the subset of target probes is specific for a different target; (b) hybridizing or binding the target probes to more than one target in the sample; (c) detecting a first fluorescently detectable label, wherein the first fluorescently detectable label is linked to a first subset of the more than one target probe; and (d) after detection in step (c), contacting the sample from step (c) with PMS, PDS, their salts, or any combination thereof, wherein PMS and PDS are represented by the following formula: or , (e) Irradiating the sample of (d), wherein irradiation includes exposing the sample to light, optionally wherein the light is light within a selected wavelength range, wherein the selected wavelength range is within the wavelength range required to detect the first fluorescent detectable tag; and (f) repeating steps (c)-(e), including an Nth fluorescent detectable tag connected to an Nth probe, wherein the Nth fluorescent detectable tag and the Nth subset of target probes are different from the first subset of target probes and the first fluorescent detectable tag, optionally wherein the selected wavelength range of light is within the wavelength range required to detect the Nth fluorescent detectable tag. Brief description of the attached diagram

[0008] Figures 1A-1B Iterative reuse strategies to overcome spectral overlap in IF applications. (A) Fluorescent reporter probes allow selective imaging of ≤5 target probes in standard IF applications. Detection of more targets is problematic due to overlap of fluorescence signals from individual probes (see Overlap of Emission Spectra). (B) Iterative imaging methods allow for a higher degree of reuse. This occurs through the deployment of iterative reporter probe binding and signal removal steps. Signal removal methods include irradiation, antibody elution, and antibody barcoding.

[0009] Figure 2 Peroxymonosulfate (PMS) and peroxydisulfate (PDS) are activated by dye excitation. After the dye (dye*) is excited by visible light, the excited dye activates PMS or PDS through an electron transfer process. PMS and PDS decompose to form sulfate radicals, which then react with dye molecules; this reaction leads to the oxidation and decolorization of the dye. In this system, the dye has a bifunctional role, acting as both a photosensitizer and a substrate for radicals derived from activated PMS and PDS.

[0010] Figure 3 A schematic overview of an iterative multiplexing system with chemically enhanced photobleaching. In step 1, a biological sample is prepared and fixed onto a carrier slide or chamber mounted on a microscope stage. Images of endogenous tissue fluorescence (background signal) are recorded using multiple detection channels. In step 2, the tissue is labeled with target probes and reporter probes targeting endogenously expressed biomarkers. In step 3, the reporter probe signal is optically detected and recorded in one or more acquisition channels. In step 4, the reporter probe signal is removed by enhanced photobleaching. Enhanced photobleaching includes a signal irradiation process in which the decolorization of the reporter probe signal is accelerated by the presence of photosensitive molecules (described herein as PMS or PDS). In step 5, another background signal is acquired. Steps 2-5 can be repeated. n Photobleaching can be performed by irradiating with white light to simultaneously photobleach all reporting probe signals, or by using one or more selected wavelengths of light for selective photobleaching.

[0011] Figures 4A-4B : Selective photobleaching of fluorophores was performed in vitro using LEDs or white light of different wavelengths. The dye was dissolved in PBS at a concentration of 5 μM with 5 g / L sodium persulfate (SPS, 21 mM). (A) Irradiation was performed using a selectively tuned LED (see illustration) set at 2.52 mW / cm². 2 The output power was maintained for up to 20 minutes. Fluorescence of the dye solution was measured on an Agilent Biotek Synergy Neo2 fluorescent plate reader after 0, 1, 2, 5, 10, 15, and 20 minutes. Selective photobleaching using an LED with an emission spectrum within the approximate excitation wavelength of the corresponding fluorophore is most effective. LEDs of other wavelengths can also be used to irradiate the fluorophore, but they are less effective. (B) For white light exposure, a setting of 1600 mW / cm² was used. 2 The results showed that the combination of SPS+ at its preferred wavelength for dye excitation is necessary and effective for the selective decolorization of organic fluorescent dyes. This result is consistent with Gao's findings. 1 The results were different, Gao 1 In the results, white light was used to decolorize the dye in a non-selective manner. All results are shown as mean ± SD, n=3.

[0012] Figures 5A-5B Selective bleaching with AF594 demonstrates in vitro wavelength-dependent SPS photobleaching. (A) A mixture of AF488 and AF594 with 5 g / L sodium persulfate (21 mM) in PBS was bleached using a 590 nm LED (2.52 mW / cm²). 2 Irradiation was performed for a maximum of 20 minutes. While AF594 showed a ~90% decrease in fluorescence signal after 20 minutes of light exposure, AF488 did not show a signal decrease. (B) The same mixture of dye and SPS was exposed to white light (LED 430-750 nm, 1600 mW / cm²). 2 This resulted in a decrease in the signals of both AF488 and AF594. These results demonstrate the selective and highly localized reactivity of certain fluorophores under wavelength-specific irradiation in buffered SPS solution. Result (A) provides strong evidence that SPS activation and subsequent decolorization require excitation of dye molecules by exposure to their specific excitation wavelength in the presence of SPS. It also shows that the decolorization reaction is locally confined to the excited dye molecules and does not affect the unexcited molecules of another dye with different spectral properties in the same solution. The results in (A) are consistent with those in Gao 1 Clearly different, Gao 1 The dyes were decolorized in a non-selective manner using only white light. All results are shown as mean ± SD, n=3.

[0013] Figure 6 Effect of SPS concentration on in vitro bleaching efficiency. Dye was added at a concentration of 5 μM to PBS containing different concentrations of sodium persulfate ranging from 0–10 g / L. The bleaching process was performed using PBS set to full power (470 nm: 13.17 mW / cm²). 2 525 nm: 8.82 mW / cm 2 590 nm: 2.52 mW / cm 2 630 nm: 7.2 mW / cm 2 The optimal LED was used to irradiate each dye for photobleaching purposes over a period of up to 20 minutes. Fluorescence intensity was measured at t=0 minutes prior to irradiation, and normalized fluorescence intensity values ​​were set to 100%. Each fluorophore tested exhibited good photostability during photobleaching without SPS (blue line), while gradually accelerated photobleaching was triggered by the addition of SPS in a concentration-dependent manner. These data indicate that SPS is responsible for the bleaching observed during selective fluorophore illumination / irradiation. Higher concentrations of SPS resulted in better decolorization. All results are shown as mean ± SD, n=3.

[0014] Figure 7 Effect of bleaching light intensity on in vitro bleaching efficiency. The dye was used at a concentration of 5 μM with 5 g / L sodium persulfate (21 mM) in PBS. The setting was 2.52 mW / cm². 2 Or full power (470 nm: 13.17 mW / cm) 2 525 nm: 8.82 mW / cm 2 630 nm: 7.2 mW / cm 2 The optimal LED was used to irradiate each dye for photobleaching purposes over a period of up to 20 minutes. Fluorescence intensity was measured at t=0 minutes before irradiation, and normalized fluorescence intensity values ​​were set to 100%. As shown, increased light intensity resulted in more effective and faster bleaching. The accelerating effect gradually decreased as the fluorophore decolorized. For the absolute differences in fluorophore intensity, see the table next to each figure; these data confirm that photobleaching with stronger (i.e., higher irradiance) light initially accelerates dye decolorization. The stronger the irradiation, the better the decolorization. All results are shown as mean ± SD, n=3.

[0015] Figures 8A-8BThe effects of pH and buffer system on SPS-mediated in vitro bleaching. The dye was used at a concentration of 5 μM with 10 g / L sodium persulfate (42 mM) in different buffers (as shown). The setting was at full power (470 nm: 13.17 mW / cm). 2 525 nm: 8.82 mW / cm 2 590 nm: 2.52 mW / cm 2 630 nm: 7.2 mW / cm 2 The optimal LED was used to irradiate each dye for photobleaching purposes over a period of up to 20 minutes. Fluorescence intensity was measured at t=0 minutes prior to irradiation, and normalized fluorescence intensity values ​​were set to 100%. (A) Effect of pH in the PBS buffer system. Bleaching was performed in PBS with pH values ​​ranging from 6 to 8 (6, 7.2, and 8). Overall bleaching efficiency was not affected by buffer pH for all dyes tested. (B) Effect of different buffer systems. Bleaching efficiency was tested in PBS, TBS (both pH 7.2), and ddH2O. Overall bleaching efficiency was not affected by buffer system for all dyes tested. All results are shown as mean ± SD, n=3.

[0016] Figure 9 The in vitro bleaching efficiencies of different peroxydisulfate were compared. Dyes were used at a concentration of 5 μM with 42 mM peroxydisulfate (ammonium peroxydisulfate (APS), sodium peroxydisulfate (SPS), or potassium peroxydisulfate (PPS)) in PBS. The setting was full power (470 nm: 13.17 mW / cm). 2 525 nm: 8.82 mW / cm 2 590 nm: 2.52 mW / cm 2 630 nm: 7.2 mW / cm 2 The optimal LED was used to irradiate each dye for photobleaching purposes over a period of up to 20 minutes. Fluorescence intensity was measured at t=0 minutes prior to irradiation, and the normalized fluorescence intensity value was set to 100%. No difference in bleaching efficiency was observed among the different peroxydisulfate dyes when used at the same concentration. All results are shown as mean ± SD, n=3.

[0017] Figure 10In situ efficient photobleaching of SPS-mediated immunofluorescence signals. Formalin-fixed paraffin-embedded (FFPE) human tonsil sections were labeled with three target probes, including a nuclear staining agent (DNA-PI) and two antibodies (anti-panCK and anti-histone H3). Each target probe was chemically conjugated with a different reporter probe / fluorophore (as shown above each column). The reporter probes were excited and imaged with appropriate wavelengths to produce the images in the top row. Middle row: Irradiated with white light in PBS only (20 s, 420–750 nm, 800 mW / cm²). 2 This resulted in partial attenuation of the fluorescence signal (compared to the staining image in the top row). Bottom row: Irradiation with white light in PBS + 5 mg / mL SPS resulted in complete removal of the fluorescence signal after 10 seconds. These data indicate that SPS is essential for rapid and complete in-situ destaining of fluorophores. Scale bar represents 100 µm.

[0018] Figure 11 In-situ efficient photobleaching of SPS-mediated fluorescent RNA FISH signaling. Photobleaching + SPS was used to remove three different fluorescent molecules. FFPE human tonsils were labeled with three RNA target probes, each detected with a different fluorophore (as shown above each column). Reporter probes were excited and imaged with appropriate wavelengths to produce the images in the top row. Middle row: Irradiated with white light in PBS only (20 s, 420–750 nm, 800 mW / cm²). 2 This resulted in partial attenuation of the fluorescence signal (compared to the staining image in the top row). Bottom row: Irradiation with white light in PBS + 5 mg / mL SPS resulted in complete removal of the fluorescence signal after 10 seconds. These data indicate that SPS is essential for rapid and complete in situ destaining of RNA probe fluorophores. Scale bar represents 100 µm.

[0019] Figures 12A-12B In situ selective SPS-dependent irradiation of fluorophores under selective wavelength irradiation (single wavelength). (A) Human FFPE tonsil tissue sections were stained with antibodies against CD45 and histone H3 labeled with two different fluorophores, AF488 and AF647, respectively. The image shows the overlap of the two labels in green and magenta. (B) Tissue sites were alternately exposed to 454–497 nm (60 mW / cm²). 2 ) or 626-644 nm (40 mW / cm 2 SPS-mediated selective photobleaching of fluorophores was performed using light for 10 seconds. This resulted in residual signal at each site, with AF488 retained after irradiation with 626–644 nm light and AF647 retained after irradiation with 454–497 nm light. This demonstrates in-situ wavelength-selective SPS-mediated fluorophore removal.

[0020] Figures 13A-13D Repeated SPS-mediated photobleaching did not diminish the ability to detect biomarker signals in situ. Different 5 μm FFPE tissue sections were stained with Sytox Orange DNA dye, imaged, and subsequently exposed to 50 cycles of SPS-mediated photobleaching (532–555 nm, 100 mW / cm²). 2 DNA staining and imaging were repeated every 5 cycles (lasting 10 seconds) to assess tissue integrity. After 50 cycles of photobleaching, all sections were stained with fluorescently labeled antibodies against CD20, FoxP3, pan-CK, and CD3, and then imaged again. As a control, sequential sections of the same tissue were stained with DNA staining agent and antibodies, but without 50 repeated photobleaching cycles. (A) Tonsillar section (B) Thymus section (C) Breast cancer section (D) Head and neck cancer section. The image set shows stained sequential tissue sections that underwent 50x cycles of enhanced photobleaching in the top row, while the bottom row was untreated. The results indicate that all markers were detectable on all tested tissue sections after 50 cycles of repeated SPS-enhanced photobleaching, suggesting that epitope stability was preserved under the conditions of at least 50 cycles of SPS-enhanced photobleaching.

[0021] Figure 14A- Figure 14C SPS-mediated photobleaching did not induce tissue degradation after repeated in situ exposure. Cellular DNA in 5 μm FFPE tissue sections was stained and imaged before the first cycle and after 50 cycles of repeated enhanced photobleaching (as shown in Figure 13) to evaluate the effect of SPS-mediated photobleaching on tissue integrity. (A) Normal tissue sample; (B) Cancer tissue sample. (C) Cell number detected by cell quantification based on the StarDist segmentation algorithm before and after 50 cycles of repeated SPS photobleaching. Consistent unaltered shape, staining pattern, and nuclear intensity between the two experimental conditions indicated no qualitatively measurable damage to tissue structure. Therefore, tissue integrity remained intact even after repeated events of enhanced photobleaching. This is also reflected in the mean difference of less than 3% in cell number detected between cycle 1 and cycle 50 in all samples (C). The combined images of cycle 1 and cycle 50 data shown in the bottom rows of (A) and (B) demonstrate no difference in the fine morphology of the cell nuclei compared between different conditions, ultimately confirming the preservation of tissue morphology after SPS-enhanced photobleaching.

[0022] Figure 15A- Figure 15BSPS-mediated photobleaching resulted in a measurable reduction in endogenous tissue background autofluorescence. Tissue autofluorescence was imaged on two consecutive 5 μm pancreatic cancer FFPE sections in different photoexcitation channels to capture a broad range of autofluorescence emission spectra. One section was imaged with white light (420–750 nm, 800 mW / cm²). 2 (A) SPS-mediated photobleaching was performed for 10 s, while control sections were not bleached before autofluorescence imaging. Both sections were then stained with antibodies against Ki-67 and pan-CK, as well as Sytox Orange DNA dye. (B) The mean cell autofluorescence intensity before bleaching was measured in the segmented regions of the sections after DNA-based StarDist cell segmentation. In the green (507–527 nm) and yellow (558–586 nm) ranges of the autofluorescence emission spectrum, tissue autofluorescence decreased to approximately 1 / 4 and 1 / 4.5, respectively, after 10 s of enhanced photobleaching. In the orange (612–644 nm) and red (672–712 nm) ranges, autofluorescence decreased to ~1 / 2.8 and ~1 / 1.6, respectively. (C) The staining of both Ki-67 and pan-CK markers was more easily distinguishable from the tissue autofluorescence in sections that underwent enhanced photobleaching (some stained cells are marked with arrows for better representation). This indicates that enhanced photobleaching improves data quality by reducing background autofluorescence in fluorescence microscopy.

[0023] Figure 16A- Figure 16C Chemical bleaching of fluorophores with LiBH4 and H2O2 instead of SPS resulted in bubble formation in a closed microfluidic system. (A) A typical microfluidic sample chamber for multiplex imaging is shown after a 15-minute bleaching reaction with 5 mg / ml SPS in ddH2O; no bubbles formed in or near the tissue sample fixed in the center of the fluid chamber (arrows). (B) The release of hydrogen during 15-minute incubation of the sample with 1 mg / ml LiBH4 in ddH2O resulted in excessive bubble formation in the closed microfluidic chamber. The formed bubbles were difficult to remove from the chamber, hindering optical imaging and potentially damaging the sample and / or impairing chamber integrity due to pressure buildup and liquid displacement. (C) A microfluidic sample chamber is shown after a 30-minute bleaching reaction with 3% H2O2 in 20 mM NaOH. Oxygen generation resulted in ubiquitous bubbles within the closed microfluidic system, affecting optical imaging and chamber integrity.

[0024] Figure 17Storage stability of SPS solutions. SPS was dissolved in PBS buffer to a final concentration of 5 mg / ml and stored at 4°C for 12 months. AF488 dye was mixed with stored and freshly prepared SPS solutions to a final dye concentration of 5 μM and exposed to low-intensity white light (LED 420-700 nm; 25.2 mW / cm²). 2 10 minutes. Fluorescence of the SPS / dye solution was measured on a DeNovix DS-11 with an FX module before and after white light exposure to determine bleaching effectiveness. Under these conditions, freshly prepared SPS solution reduced the fluorescence of AF488 dye by 65% ​​compared to the unexposed solution. Similarly, SPS solution stored at 4°C for 12 months reduced dye fluorescence by 63% compared to the unexposed solution. This indicates comparable bleaching efficiency to the fresh solution, with no significant loss of SPS reactivity during storage under these conditions. Results are shown as mean ± SD, n=3. Detailed Explanation

[0025] Introduction Various methods are used in biomedical research to label biomarker targets in biological samples. For example, protein targets can be detected in tissue sections using histochemistry, immunohistochemistry (IHC), or immunofluorescence imaging (IF). RNA and other gene targets can be detected using in situ hybridization (ISH), fluorescence in situ hybridization, or single-molecule imaging. Most of these methods were developed around optical microscopy and are designed to image the location of biomarker targets at high optical resolution.

[0026] Imaging-based biomarker detection has traditionally been limited by the number of biomarkers that can be imaged simultaneously from a single sample. For example, 3,3'-diaminobenzidine (DAB) is a widely used chromophore in IHC, and it produces a brown precipitate (reporter signal) that can be imaged using conventional optical microscopy. DAB has excellent detection sensitivity, but its use for multiplexing is limited, and it is rarely used to detect more than one biomarker (at most 2-3). In contrast, IF staining methods offer greater flexibility for in-situ multiplexing. These methods employ organic fluorescent dyes (reporter probes) that can be selectively excited to emit a fluorescent reporter signal within a defined spectrum (e.g., ...). Figure 1A (The colors are red, green, and blue). Each signal can be used to selectively identify the target probe, and each signal is collected separately using conventional epifluorescence optics. These devices allow optical signals to be collected from ≤5 biomarkers in a single tissue without modifying standard workflows.

[0027] In recent years, the demand for imaging-based in situ detection and even more biomarkers has surged. For the aforementioned IHC and IF methods, this would require experimenters to use additional biological samples from the same source (e.g., serial histological sections) and repeat multiple staining several times. However, this approach limits the experimenter's ability to accurately determine the co-expression and interactions of all targets at the single-cell level. Added complexity arises due to the incompatibility of some widely used methods with each other. Specifically, chromogenic DAB staining and IF use the same target probes (antibodies) to detect the same biomarkers; however, the detection probes are different, and there are few practical workflows that allow for the integration of these methods onto a single sample.

[0028] Figure 1B A schematic overview of multiplex imaging techniques developed to increase biomarker complexity (i.e., the number of target probes that can be selectively imaged from a single sample) is provided. In their basic configuration, these techniques are similar to standard epifluorescence microscopy: they deploy 2–5 target probes, each labeled with a reporter probe of a different spectrum. Each probe is then selectively excited and imaged to represent the biomarker of interest. Target complexity is then increased through an iterative process, wherein (1) the biomarker is labeled with the target probe – (2) the target probe is labeled with the reporter probe – (3) the reporter probe is imaged using an optical microscope – (4) the target or reporter probe is removed and / or inactivated. This process is repeated. n This process continues until all biomarkers of interest have been imaged (e.g., see Figures 13-14, showing the results of n=50 cycles, each using 4 target probes (antibodies), ultimately yielding total readings from 200 biomarkers). The main differences between iterative workflows involve how reporter probes are designed and how they are removed from the tissue and / or inactivated while remaining on the tissue. These strategies are discussed in more detail below.

[0029] Report the illumination (photobleaching) of the probe signal. The irradiation or photobleaching of the reporting probe can be described as iterative multiple imaging (...). Figure 1B The simplest probe removal method used in [the study] is as follows. In this method, more than one target probe is directly or indirectly labeled with a fluorescent reporter probe. After imaging, the reporter probe is decolorized by irradiation; this results in the loss of the reporter probe signal, but the reporter probe itself remains in the tissue, as do the target probes. Therefore, irradiation leads to the inactivation of the reporter probe rather than probe removal.

[0030] Photobleaching can be performed in normal physiological buffers (such as PBS) without any chemical additives. This strategy is advantageous because it causes minimal damage to tissues (depending on the wavelength and intensity of the light used). Furthermore, fluorescent antibodies are readily available from commercial suppliers, and the reagents required for multiplex imaging experiments are relatively easy to obtain. However, photobleaching can be time-consuming because many fluorophores are quite photostable, and it may not continue until completion; this ultimately leads to reduced signal-to-noise ratios and non-selective imaging data. 2 Furthermore, prolonged exposure of tissue samples to long irradiation steps may cause cumulative damage to biological samples and hinder further detection of the target.

[0031] Therefore, various strategies have been developed to improve photobleaching efficiency by adding chemical reagents. For example, the Cyclic Immunofluorescence (CyCIF) workflow combines H2O2 with irradiation to oxidize / inactivate fluorescent probes and reduce tissue background fluorescence in multiplex imaging experiments. This method is well-established and produces good results, but it is slow and can take several days to complete. 3 Reactions associated with tissue and probe exposure to H2O2 further lead to oxygen formation, which is found in closed, automated experimental systems deployed in many research laboratories today (e.g., CellScape). TM PhenoCycler TM CosMx ® Merscope TM The system has problems. Furthermore, the oxidation of the H2O2-mediated reporter probe is not sterically restricted and may have side effects such as damaging / altering certain epitopes.

[0032] Another chemical photobleaching method has been disclosed in WO 2014 / 093455. In this system, organoborates are activated by visible light to chemically inactivate fluorescent probes. While the removal of the fluorescence signal is effective, several drawbacks limit the usability of the system. First, the protocol requires the introduction of reactive oxygen species scavengers, as severe epitope damage occurs without them, preventing iterative detection of biomarkers. Second, due to the low water solubility of organoborates, more complex borate compounds with higher water solubility must be designed. Although these compounds offer better performance, they are not readily available or commercially available. Finally, the reagents required to complete this workflow have poor storage properties, all of which make the commercial development and deployment of this system impractical.

[0033] Chemical removal of the report probe signal or the target / report probe complex Determination of the use of chemical and / or thermal methods to remove reporter probe signals and / or the entire target-reporter probe complex is widespread in the multiplex imaging community. Figure 1BThese methods, collectively referred to as "stripping" methods, share the common general purpose of removing target probes or reporter signals, but their efficiencies vary. A significant advantage of stripping methods is their flexibility in use with a variety of histological techniques, and they typically do not require prior modification of the target probe, reporter probe, or tissue specimen. A disadvantage of all or most of these techniques is that they cause some degree of damage to the tissue sample, which ultimately limits the level of complexity that can be achieved. Several methods that appear to cause only slight / moderate damage have been reviewed below and have therefore been widely used in studies aiming for medium to high complexity in multiplex imaging.

[0034] Antibody elution leads to the removal of the target-reporter probe complex, achieved by applying a low pH (2.0) dissociation buffer containing sodium dodecyl sulfate (SDS) and a reducing agent (DTT, TCEP, etc.). Removal of the target / reporter probe complex is rapid and can be achieved within 2 minutes at 50°C. This allows for efficient iterative multiplexing experiments. 4 However, repeated exposure to acidic, ionizing buffers, and high temperatures can affect tissue integrity, thereby impacting the ability to acquire highly multiplexed imaging data. 5 .

[0035] Another chemical signal removal method, iterative bleaching extends multiplexity (IBEX), uses lithium borohydride (LiBH4) to achieve multiplexing in both frozen and formalin-fixed tissue samples. IBEX typically yields good results, and its open concept allows it to be used for both direct and indirect immunofluorescence applications. 6 However, borohydrides are highly toxic compounds requiring special handling. Furthermore, LiBH4 reacts with water upon dissolution, producing hydrogen gas (e.g., 1 g of LiBH4 produces >4 liters of hydrogen gas); this leads to excessive bubble formation, making the deployment of IBEX in closed systems difficult and potentially hazardous. Finally, the low stability of borohydrides in solution limits the commercial production, storage, and deployment of reagents required for the IBEX protocol.

[0036] Another recently disclosed method for generating and removing cyclic signals, spatial photo-inactivation enhanced cyclic target resolved multiplexing (SPECTRE-Plex), utilizes m-chloroperoxybenzoic acid (m-CPBA) as a chemical oxidant to inactivate fluorescent reporter probes on the sample. 7This method is faster than LiBH4 or H2O2-based signal removal and does not result in gas formation during the process, allowing it to be deployed in closed fluid systems. However, like most chemical oxidation processes, this method is non-selective and will react indiscriminately with all probes and biological target tissues, potentially leading to damage and alteration of the biological substrate. It is also ineffective against certain reporter dyes, such as Alexa Fluor 594, which is commonly used for immunofluorescence imaging. The compound is more stable than LiBH4 or H2O2 in aqueous working solutions, but its shelf life is less than 36 hours, still requiring periodic preparation of fresh working solutions, which can lead to experimenter errors and certainly requires more hands-on time.

[0037] Antibody barcoding Antibody barcoding has been developed as another method for achieving in situ high-impact biomarker imaging. For example, Co-Detection by IndEXing (CODEX) employs an oligonucleotide barcoding strategy, in which different oligonucleotide barcodes are attached to target antibodies (target probes). After labeling biomarker targets in tissues, each type of antibody can be tagged with a complementary oligonucleotide-barcoded fluorescent reporter probe. After imaging, the probe is removed by dehybridization or dissolution from the antibody-oligonucleotide complex, and a new probe labeling cycle can be started. Using this method, probes with spectral compatibility can be delivered in small, iterative groups on microscopes with conventional epifluorescence optics. 8 This method is isothermal and non-destructive to tissues, and is currently considered the most suitable system for scaling up the complexity of biomarker imaging experiments to >100 targets in a sample. 9 .

[0038] Disadvantages of elution and barcode technology While powerful and sophisticated, elution and barcoding methods have limitations that increasingly hinder the design and adoption of multiplexing experiments. For example, conjugating oligonucleotide barcodes to antibodies can disrupt cysteine ​​bridges in the antibody, thereby affecting its ability to detect appropriate target epitopes. 8 Oligonucleotide barcoding can also nonspecifically hybridize with genomic DNA in tissue samples, leading to increased detection of nonspecific nuclear staining signals and an overall decrease in the signal-to-noise ratio. Furthermore, reporter oligonucleotides themselves are variable, and they do not always produce sufficient optical signals. 10 On the other hand, elution-based multiplexing methods require buffers with acidic pH and repeated heating of tissue samples, which has been shown to affect tissue integrity. Deen et al. (2023) reported nuclear deformation and membrane marker failure in freshly frozen tissue samples multiplexed using a well-developed elution method.11 Even if successful, the elution protocols require extensive optimization, and they are rarely used or successful if researchers are attempting to image more than a dozen biomarker targets. 10 Clearly, both of these methods limit the extent to which multiple biomarker imaging can be performed, and it is reasonable to develop new systems that do not have these drawbacks. This disclosure describes a combined system of irradiation and chemical reporter probe inactivation, referred to herein as enhanced photobleaching.

[0039] The technology of this invention is based on the principle of photobleaching by irradiation with a reporter probe, but it achieves excellent results through a chemically enhanced signal removal process, while avoiding the drawbacks of well-known photobleaching methods.

[0040] Enhanced photobleaching methods and compositions This article discloses methods, systems, platforms, kits, and compositions for processing biological samples to image and visualize targets in the samples, and to perform iterative, multiplexed, or repeated imaging and visualization of targets more efficiently and accurately.

[0041] The disclosed techniques, referred to herein as "photobleaching" or "enhanced photobleaching," Figure 3 The illustration is schematic because it relates to use in multiplex imaging experiments. The techniques disclosed herein include general photobleaching (e.g., illuminating a labeled sample with white light) and selective photobleaching. As used herein, “selective photobleaching” or “selective bleaching” refers to targeted enhanced photobleaching that illuminates a sample using a selected wavelength range of light (excluding white light). In some embodiments, the selected wavelength range of light includes the entire wavelength range that can be used to detect detectable labels (e.g., fluorescent labels or dyes). In some embodiments, the selected wavelength range is within the wavelength range required to detect the detectable label (e.g., this range is narrower than the full range that can be used to detect the detectable label). In some embodiments, the selected wavelength range includes overlapping wavelength ranges or different wavelength ranges, for example, to selectively photobleach more than one detectable label simultaneously and / or sequentially. In some embodiments, a sample may be labeled and selectively photobleached (e.g., with a selected wavelength range, once or multiple times), and then typically photobleached with white light to eliminate any remaining signal. In some implementations, the sample can be photobleached with white light to reduce or remove any signal (e.g., residual signal from previous experiments on the same sample, or to reduce background, etc.), and then it can be labeled, imaged, and then selectively photobleached.

[0042] Peroxydisulfate (PDS, or interchangeably sodium persulfate, SPS) and peroxymonosulfate (PMS) are free radical initiators that can be activated to produce strongly oxidizing SO4. ●-Free radicals, and they are widely used in water and soil remediation by degrading organic pollutants. 12-15 The structures of PMS and PDS are provided below in their salt form. or

[0043] M+ can be an organic or inorganic cation. Although sodium persulfate (also known as "sodium persulfate" (SPS)) is used in the examples, other cationic forms of PDS or PMS will also serve to decolorize organic dyes, such as... Figure 9 As shown in the figures (e.g., potassium peroxydisulfate and ammonium peroxydisulfate).

[0044] Activation of PDS / PMS is mainly achieved through heating, UV irradiation (<300 nm), and inorganic catalysts (e.g., transition metals), but activation by visible light has received little attention to date. Gao et al. (2017) reported that visible light-excited organic fluorescent dyes can act as photosensitizers to activate PDS / PMS. 1 This mechanism involves exciting the dye from its ground state using visible light, followed by electron transfer from the excited dye to the PDS / PMS, ultimately leading to molecular activation and the generation of sulfate radicals. In this system, the dye acts not only as a photosensitizer but also as a substrate for sulfate radicals, which are first activated and then oxidized, causing the dye to decolorize. Figure 2 Ultimately, this process allows for selective decolorization of fluorescent dyes by PDS / PMS under visible light irradiation, thus providing an attractive probe removal solution for multiplex imaging assays.

[0045] This invention relates to an irradiation / photobleaching method utilizing PDS and PMS as enhancers in the photobleaching process. Unlike Gao et al. (2017), which disclosed general photobleaching of several potentially hazardous compounds in environmental waste, this invention provides specific targeted photobleaching of detectable markers in biological samples. The photobleaching process of this invention provides accuracy and specificity for the process, allowing for sample preservation and cost-effective, efficient multiplexing with iterative and sequential target detection. Therefore, this invention will offer numerous benefits and find wide applicability in multiplexing applications, overcoming the shortcomings of other currently available methods.

[0046] The methods, systems, platforms, kits, and compositions disclosed herein include one or more of the following features: 1) Enhanced photobleaching, such as Figure 2 As shown; 2) Only commercially available, stable, non-toxic reagents with good solubility (e.g., PMS and PDS) are required; 3) Does not affect tissue integrity (e.g., by repeatedly exposing the tissue to acidic pH and elevated temperatures); 4) No need to modify the target probe (e.g., by conjugating the antibody to an oligonucleotide) or the tissue (e.g., by eluting the tissue by repeatedly exposing it to acidic pH and elevated temperatures); 5) Quite fast; 6) Compatible with a variety of reporter probes (e.g., rhodamine, cyanide, and BODIPY dyes) and other common tissue staining agents (eosin and other dyes); 7) Compatible with and allows integration of different workflows based on optical imaging for signal detection; 8) Compatible with common hardware designs (e.g., closed microfluidic systems); and 9) This can be achieved through the illumination settings provided by standard epifluorescence imaging technology.

[0047] The photobleaching technique disclosed in this article involves multiple imaging experiments. Figure 3 The diagram is illustrated below and discussed in more detail.

[0048] This document discloses methods and compositions for detecting targets in biological samples. In embodiments, the biological sample is processed for imaging to simultaneously detect a single target, a single set of targets, or sequentially detect multiple targets or sets of targets.

[0049] Biological samples are typically derived from multicellular subjects, such as plants, fungi, or animals (e.g., mammals, birds, reptiles, fish, amphibians), but can also come from single-celled organisms, such as bacteria, yeast, or protists.

[0050] Biological samples suitable for use in this invention include, but are not limited to, tissue samples, liquid samples containing one or more types of cells (e.g., blood, urine, bone marrow samples), and cultured cells or tissues (e.g., organoids or tissue explants).

[0051] Biological samples, such as tissue samples, can be fresh or preserved (e.g., frozen, formalin-fixed, paraffin-embedded), and / or fixed to a solid support, such as, but not limited to, slides, beads, micropores, multi-well plates, nitrocellulose, PDVF membranes, flow cells, or paper. In embodiments, fresh or preserved samples can be configured on a solid support for analysis and imaging according to the methods disclosed herein. In some embodiments, biological samples are configured in a closed microfluidic system for reagent delivery to the sample.

[0052] Exemplary non-limiting tissue samples include biopsy samples, surgical excisions, and surgical aspirates. Exemplary non-limiting liquid biological samples include blood, bile, bone marrow aspirates, breast milk, cerebrospinal fluid, plasma, saliva, semen, serum, sputum, feces, swabs (oral, nasal, and vaginal fluids), synovial fluid, and urine.

[0053] In embodiments, target molecules include molecules such as proteins, nucleic acids, carbohydrates, lipids, and combinations thereof. Such molecules may contain all natural components or one or more non-natural components (e.g., modified amino acids, nucleic acids, chemical moieties, etc.). Methods for detecting such molecules are well known in the art, and the design and use of appropriate detection probes (e.g., antibodies, fragments thereof, nanobodies, nucleic acids, aptamers, etc.) to specifically bind to such molecules is not the focus of this invention. Rather, this invention relates to the quenching or selective elimination of one or more signals generated by a signal generator associated with a detection probe (i.e., a reporter probe for detecting any of the aforementioned molecules).

[0054] For example, many protein detection methods involve using a protein-binding moiety (e.g., an antibody or fragment thereof) linked to or associated with a detectable label (e.g., by contacting the binding moiety with a secondary molecule containing the detectable label). Similarly, nucleic acid detection methods typically involve a nucleic acid-binding moiety (e.g., a probe that is wholly or partially complementary to a target nucleic acid) linked to or associated with a detectable label.

[0055] As used herein, the term "target probe" refers to a molecule capable of specifically binding to a target molecule. Exemplary non-limiting target probes include antibodies, nucleic acids, aptamers, etc. Target probes may themselves include detectable labels or may contact secondary or tertiary probes (e.g., "reporter probes" containing detectable labels).

[0056] As used herein, a "reporter probe" comprises a molecule that includes an optical signal generator (detectable label). By way of example, but not limitation, a reporter probe may comprise antibodies, nanobodies, nucleic acids, etc., chemically linked to a detectable label.

[0057] As used herein, the terms “reporter molecule,” “detectable marker,” and “optical signal generator” are used interchangeably and refer to molecules that provide a visual signal. By way of example, but not limitation, in some embodiments, the detectable marker is a dye or a tissue / cell staining agent.

[0058] In this implementation, the signal generator is a dye expressed by a cell or organism (e.g., genetically encoded). Exemplary non-limiting dyes include fluorescent proteins such as GFP, YFP, RFP, tdTomato, etc.

[0059] Tissue autofluorescence, which is not a dye itself, is a source of fluorescence "noise" in tissue imaging applications (see Figure 15 for example).

[0060] In some embodiments, the detectable marker contains a staining agent (e.g., a general tissue staining agent or a general cell staining agent) that allows visualization of cells or tissues in a biological sample. As is known in the art, staining agents highlight different features and / or components of cells or tissues. By way of example, but not limitation, two of the most common staining agents are hematoxylin and eosin. Hematoxylin is a basic dye that stains acidic molecules such as DNA / RNA blue, while eosin stains proteins pink. Sometimes these staining agents are used in combination and may be referred to as H&E staining. The use of two staining agents, alone or in combination, is useful in the context of this disclosure. Other non-limiting staining agents include: mucin staining agents for detecting and visualizing mucopolysaccharides (e.g., Alcian Blue, Mucin Carbomer Red, period acid-Schiff (PAS)); melanin staining agents for staining and visualizing melanin (e.g., Fontana-Masson); and trichrome staining agents for lipid visualization (e.g., Gomori trichrome staining, Mallory trichrome staining, Sudan staining agents such as Red Oil O). Table 1 below provides exemplary non-limiting dyes that can be used in combination with the photobleaching techniques of this disclosure. Although some of the dyes listed in Table 1 may not be suitable for selective photobleaching or may not be photobleached at all using the compounds and methods disclosed herein, such dyes may still be used in combination with other detectable markers that can be photobleached.

[0061] Table 1: Exemplary dyes

[0062] Detectable markers (a term used interchangeably with "optical signal generator" herein) are well known in the art. By way of example, but not limitation, detectable markers include dyes, such as fluorophores from the rhodamine, cyanine, and BODIPY dye families. Exemplary dyes include AF488, AF532, AF594, AF647, Atto532, AF546, AF555, AF568, Atto594, and Atto643.

[0063] As used herein, the terms "fluorophore," "fluorescent signal generator," and similar terms refer to compounds that emit light of different wavelengths when excited by exposure to light of a specific wavelength. Fluoresceins can be described based on their emission spectrum or "color."

[0064] As is known in the art, different detectable markers such as fluorophores are useful in multiplex assays, at least because different fluorophores can appear as different colors.

[0065] Table 2 summarizes (not exhaustive) the dyes commonly used in fluorescence optical imaging applications. It also lists the excitation wavelengths that can be generated using microscope optics or custom light sources. The excitation wavelength of each dye is used to promote the emission of another wavelength, and this second wavelength emission is the signal ultimately captured as an image. Within this capability, the dye molecules contain detectable tags.

[0066] Table 2: Summary of wavelengths used for photobleaching via standard emission filters in a microscope (column 2) or custom light sources (column 3). Excitation wavelengths are listed as ranges for each fluorescent dye.

[0067] Excitation wavelengths can also be used to decolorize dyes; this occurs if the dye is excited for an extended period of time, i.e. during photobleaching, a process that can be made more effective with SPS, PDS, and / or PMS.

[0068] The term "light range" or similar terms refers to a range of wavelengths of light. In some embodiments, the light range includes the wavelengths that excite the detectable marker and / or photobleach the detectable marker (e.g., fluorophores). In embodiments, the specified light range is the "excitation range" and / or the specified light range is the "photobleaching range". In some embodiments, the excitation range and photobleaching range are the same for a given detectable molecule. In some embodiments, the excitation range and photobleaching range overlap for a given detectable molecule. In some embodiments, the excitation range is within the photobleaching range for a given detectable molecule. In some embodiments, the photobleaching range is within the excitation range for a given detectable molecule. As used herein, the light range does not include white light. Exemplary light ranges are provided in Table 2.

[0069] White light can be effective for the photobleaching compositions and methods disclosed herein, and in some embodiments, white light is used, for example, for non-selective photobleaching. However, for selective photobleaching applications, a specific range of light or a set of light ranges is used.

[0070] In some embodiments, photobleaching of the detection molecules in accordance with this disclosure deactivates the detection molecules; as used in this context, deactivation refers to a chemical change in the optical signal generator that causes it to lose its ability to generate an optical signal.

[0071] As used in this article, the term "free radical initiator" refers to a substance with weak bonds having low dissociation energies and thus capable of generating free radicals under mild conditions.

[0072] As used herein, the term "sulfate radical initiator" refers to an agent that can generate sulfate radicals (SO4) upon dissociation under mild conditions.•- Sulfate radicals are substances that can promote free radical reactions. Sulfate radicals have a high oxidation potential and readily react with organic molecules (e.g., detector molecules). Examples of sulfate radical initiators are peroxymonosulfate and peroxydisulfate.

[0073] As used in this article, the term "photosensitizer" refers to a compound that absorbs light and transfers energy from incident light directly or through a chemical reaction to another nearby molecule.

[0074] The following provides an exemplary workflow for multiple imaging of proteins using an antibody detection method and for photobleaching the signal between target labeling and detection rounds using the compositions and methods of this disclosure.

[0075] Exemplary workflow (see) Figure 3 ) Sample preparation and antibody staining: The samples were fixed on glass slides, dewaxed, and treated for antigen retrieval. After washing several times in appropriate buffer, the samples were then fixed in the fluid chamber of the optical imaging system.

[0076] Autofluorescence signal detection: The background autofluorescence of the sample is imaged using the same channel settings as for fluorophore signal detection. More than one background signal can be collected for more than one channel.

[0077] Dilution of fluorophore-labeled antibodies: Use antibody dilution buffer to dilute one or more fluorophore-labeled antibodies to an appropriate concentration. If the antibody conjugates to fluorophores with different spectra, more than one protein can be stained simultaneously.

[0078] Antibody incubation: The working solution of the antibody is added manually to the sample and / or automatically dispensed by the imaging device. The sample is incubated at room temperature (RT) for 1 hour to allow the antibody to bind to its target.

[0079] Washing procedure: Wash the sample three times with phosphate-buffered saline (PBS). Each wash should last 5 minutes to remove unbound antibodies.

[0080] Signal detection: The antibody-fluorophore signal is imaged using a fluorescence microscope. Appropriate light sources and filter sets are used for optimal signal detection. Imaging can be performed on a single fluorophore or multiple different fluorophores.

[0081] Fluorescence signal removal In one embodiment, sodium peroxydisulfate (and / or sodium peroxymonosulfate) is treated as follows: 5 mg / ml sodium peroxydisulfate (SPS) in PBS is manually added or automatically dispensed onto the sample. The sample is then irradiated in one of the following ways: under low to moderate intensity light (irradiance 10-200 mW / cm²). 2Irradiation for 3-10 minutes under light (e.g., white light, 420-750 nm) irradiates the entire sample chamber and excites and irradiates all selected fluorophores. Alternatively, high irradiance (600-1500 mW / cm²) can be used. 2 The sample is illuminated by a beam of light for 5-10 seconds. The light originates from the microscope optics in the imaging system and is used to capture an image of the sample within a given field of view; illuminating the entire sample requires repeated repositioning of the sample on the beam. In some embodiments, the pH of the sample can be adjusted (if desired) to a pH range of about 5 to about 9 before illuminating (see, for example...). Figure 8A In some embodiments, the pH may be adjusted to between approximately 6-9, 7-9, or approximately 7-8 prior to irradiation. Methods and buffer solutions for pH adjustment are well known in the art.

[0082] Washing: Wash the sample five times with PBS to completely remove SPS.

[0083] Background fluorescence imaging: Acquire images of background autofluorescence in the same channels previously used for illumination to ensure complete removal of the signal.

[0084] Repeat subsequent staining / imaging cycles: Repeat the entire process for each subsequent staining or imaging cycle.

[0085] Exemplary non-restrictive applications Applications of the disclosed technologies include, but are not limited to, IF, mIF, FISH, smFISH (e.g., RNAscope™, HCR™ FISH, Stellaris™), Western blotting, tyramine-based signal detection and amplification (e.g., Opal system, Universal TSA), and nanobody-based antibody detection (e.g., FlexAble, Nanotag). Furthermore, the disclosed technologies enable the combination of some of the above applications into a single sequential workflow, allowing for the detection of multiple biomarker classes within a single tissue sample (e.g., sequential detection of protein epitopes using IF and sequential detection of mRNA transcripts using smFISH methods).

[0086] Exemplary non-limiting advantages The disclosed technique offers several key advantages compared to other existing methods for removing fluorescence signals from samples. Due to its reaction mechanism, PDS / PMS-enhanced photobleaching is selective and allows targeting specific reporter molecules in the sample based on their fluorophore excitation wavelength.

[0087] The reaction rate is determined by the irradiance of light on the target molecules within their specific excitation wavelength range. Molecules that do not undergo fluorescence excitation do not activate PDS / PMS and remain unchanged. Therefore, this method is relatively mild and does not damage the probe or target tissue.

[0088] Furthermore, enhanced photobleaching can deactivate fluorophores / dyes specifically designed for high photostability, which are typically resistant to conventional photobleaching procedures. It can decolorize any organic dye with fluorescent properties.

[0089] The compositions and methods disclosed herein also result in a reduction of the inherent autofluorescence of biological samples (see, for example, Figure 15).

[0090] Unlike chemical signal removal methods based on H2O2 or LiBH4 (IBEX, CyCIF), this reaction does not produce bubbles, allowing it to be used in closed microfluidic systems for delivering reagents to samples.

[0091] These characteristics enable this technology to be used almost universally in a wide range of multiplex imaging applications to remove fluorescent reporter signals. It is the only method that can be easily integrated into automated cyclic workflows for optical, fluorescence-based biomarker imaging at the protein, transcriptome, and genomic levels. The gentle and selective process also makes it suitable for sensitive sample materials such as freshly frozen tissues and fixed cell suspensions. The technology is rapid and utilizes readily available, chemically stable reagents. Working solutions are harmless and can be stored at 4°C for over a year. This allows for the standardized, ready-to-use preparation, transportation, and long-term storage of working solutions, increasing ease of use and reproducibility of experiments.

[0092] Table 3: Comparison of SPS / PMS-enhanced photobleaching with other publicly disclosed chemical or photochemical signal removal methods.

[0093] Definitions and Terms The definitions and terms used herein are for the purpose of describing a particular implementation and are not intended to be restrictive.

[0094] As used herein and in the claims, the singular forms “a,” “an,” and “the” include the plural forms unless the context clearly indicates otherwise. For example, unless the context clearly indicates otherwise, the terms “a probe” or “a label” shall be interpreted as meaning “one or more probes” and “one or more labels,” respectively. As used herein, the term “more than one” means “two or more types.”

[0095] As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by those skilled in the art and will vary to some extent depending on the context in which they are used. Where there is use of a term that is unclear to those skilled in the art in the context in which the term is used, “about” and “approximately” will mean an addition or subtraction of up to 10% to the particular term, and “substantially” and “significantly” will mean more than an addition or subtraction of 10% to the particular term.

[0096] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising”. The terms “comprise” and “comprising” should be interpreted as “open-ended” transitional terms, which allow for the inclusion of additional components besides those recited in the claims. The terms “consist” and “consisting of” should be interpreted as “closed-ended” transitional terms, which do not allow for the inclusion of additional components besides those recited in the claims. The term “consisting essentially of” should be interpreted as partially closed and only allows for the inclusion of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0097] The phrase “such as” should be interpreted as “for example, including”. Furthermore, the use of any and all exemplary language, including but not limited to “such as”, is intended only to better illustrate the invention and does not impose a limitation on the scope of the invention unless otherwise claimed.

[0098] Furthermore, in cases where conventions such as "at least one of A, B, and C" are used, this syntactic structure is generally intended to be understood by those skilled in the art as to convey the meaning of the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having a single A, a single B, a single C, A and B together, A and C together, B and C together, and / or A, B, and C together). Those skilled in the art will further understand that, in practice, any disjunctive words and / or wording presenting two or more alternative terms, whether in the specification or in the drawings, should be understood to account for the possibility of including one, any, or both terms. For example, the phrase "A or B" should be understood to include the possibility of including "A" or "B" or "A and B".

[0099] All language expressions such as “up to,” “at least,” “greater than,” and “less than” include the stated number and refer to a range that can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group with 1-3 members refers to a group with 1, 2, or 3 members. Similarly, a group with 6 members refers to a group with 1, 2, 3, 4, 5, or 6 members, and so on.

[0100] The modal verb "may" refers to the preferred use or selection of one or more options or choices among the described implementation scheme or features included therein. Where no options or choices regarding a particular implementation scheme or its included features are disclosed, the modal verb "may" refers to an affirmative action regarding how to make or use the described implementation scheme or its included features, or a clear decision regarding the use of a specific technique related to the described implementation scheme or its included features. In this latter context, the modal verb "may" has the same meaning and connotation as the auxiliary verb "can".

[0101] Miscellaneous Unless otherwise indicated herein or clearly contradicted by the context, the steps of the methods described herein may be performed in any suitable order. Unless otherwise indicated herein or clearly contradicted by the context, these steps may be repeated or iterated any number of times to achieve the desired goal.

[0102] This document describes preferred aspects of the invention. Variations of those preferred aspects will become apparent to those skilled in the art upon reading the preceding description. The inventors expect those skilled in the art to appropriately adopt these variations, and the inventors intend to practice the invention in ways different from those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter set forth in the appended claims that are permitted under applicable law. Furthermore, unless otherwise indicated herein or otherwise clearly contradicted by the context, any combination of all possible variations of the elements described above is included within the scope of this invention.

[0103] Exemplary Implementation This document discloses methods, kits, systems, platforms, and compositions for visualizing targets in biological samples. Several non-limiting embodiments of the invention are provided below.

[0104] 1. A method for detecting more than one target in a biological sample, comprising: (a) Bind at least one target probe to one or more targets present in a biological sample containing more than one target; (b) Bind at least one reporter probe to at least one target probe bound in step (a); (c) Detect the signal from at least one reporter probe combined in step (b); (d) Contact the sample containing the bound target probe from step (a) and the reporter probe from step (b) with a sulfate radical initiator, which is capable of photoreacting with photoexciteable molecules (i.e., the reporter probe from step (b)). In this embodiment, the sulfate radical initiator, composed of peroxymonosulfate (PMS) or peroxydisulfate (PDS), is represented by the following structural formula: or

[0105] M+ is selected from the group composed of organic and inorganic cations; (e) Irradiation of the reporter probe in step (b) leads to the generation of sulfate radicals. (f) Repeat steps (a) to (e) above iteratively until all targets in the biological sample from step (a) have been recorded.

[0106] 2. The method according to embodiment 1, wherein the target probe in step (a) comprises any molecule for labeling epitopes or RNA transcripts within a biological sample. These include, but are not limited to, antibodies and RNA FISH probes.

[0107] 3. The method according to embodiment 1, wherein the reporting probe in step (b) includes a light signal generator, and the signal detected in step (c) is a light signal.

[0108] 4. The method according to embodiment 1, wherein the probe in step (b) includes a fluorescence signal generator, and the signal detected in step (c) is a fluorescence signal.

[0109] 5. The method according to embodiment 1, wherein the irradiation of the sample in step (e) is performed in the presence of a buffer solution with a pH of 5-9.

[0110] 6. The method according to embodiment 1, wherein irradiating the sample in step (e) is accomplished by exposing the sample to light that can excite the reporter probe, the light typically being light covering a wavelength range of 350 nm to 1.3 mm.

[0111] 7. The method according to embodiment 1, wherein the irradiation of the sample in step (e) is performed in the presence of a buffer containing a sulfate radical initiator (i.e., PMS and / or PDS).

[0112] 8. A method for detecting more than one target in a biological sample, comprising: (a) Binding more than one probe to more than one target present in a biological sample containing more than one target, wherein the more than one probe includes a first group of probes and a second group of probes; (b) Detect the first set of signals from the first set of probes combined in step (a); (c) Contact the sample containing the binding probe from step (ab) with a sulfate radical initiator, wherein the sulfate radical initiator is as described in Embodiment 1. Figure 3 Defined (d) Irradiation of the sample from step (a) (e) Binding more than one probe from the second group to more than one target present in a biological sample containing more than one target. (f) Generate a second set of signals from the second set of probes combined in step (e). (g) Detect the second set of signals.

[0113] 9. The method according to embodiment 1, wherein the probe comprises a binder and a signal generator, wherein the binder and the signal generator are implemented in a single entity.

[0114] 10. The method according to embodiment 1, wherein the probe comprises a first binder for binding a target and a second binder for binding the first binder, and carries a signal generator.

[0115] 11. The method according to embodiment 8, wherein the single entity is a small molecule probe.

[0116] 12. An automated method for photoactivated chemical bleaching of biological samples loaded and / or captured in a flow cell device, comprising the following automated steps: a) Bind at least one probe to one or more targets present in the biological sample; b) Detect signals from at least one reporting probe combined in or after step (a); c) The flow cell is filled with a sulfate radical initiator, wherein the sulfate radical initiator is as described in Embodiment 1. Figure 3 Defined; d) Inactivate the signal from the reporting probe by exposing the sample to light; e) Optionally, elute the sulfate radical initiator; and repeat steps 11a) and 11b) with at least one other probe for another round of imaging. Example

[0117] The following examples are illustrative and are not intended to limit the scope of the claimed subject matter.

[0118] Example 1 - Used to generate Figure 10 Methods for handling data and images in [the context of the text] Formalin-fixed paraffin-embedded (FFPE) human tonsil tissue was sectioned into 5 μm sections using a microtome. Sections were mounted on microscope slides, dried, and baked at 60°C for 1 hour. Baked sections were stored at 4°C. Each section was dewaxed twice by sequential immersion in RotiClear (Carl Roth GmbH, Germany) for 5 minutes each time, followed by rehydration in a series of fractionated ethanol solutions (2 x 100% ethanol, 1 x 90%, 1 x 70%, 1 x 50%, and finally 5 minutes each in PBS). After rehydration, the samples were subjected to an antigen retrieval program in CC1 buffer (Roche, Germany) at 95°C for 20 minutes, followed by cooling in PBS at room temperature for 5 minutes. The tissue sections were then sealed in a CellScape™ flow chamber, enabling multiple rounds of instrumental staining, imaging, and signal removal. Tissues were stained for 1 hour at room temperature with a primary antibody labeled with fluorophores targeting pancytokeratin (clone AE-1 / AE-3, AF488) and histone H3 (clone 1B1-B2, AF647) and propidium iodide (PI) DNA dye, and then imaged on the instrument; the resulting data showed... Figure 10 The top row is labeled "Stain Image". Then, it was observed through continuous exposure to a 405 LP Edge Basic long-pass filter (420-750 nm, 800 mW / cm²). 2 The white light retained in the PBS was photobleached for 20 seconds. The staining agent was imaged again, and the resulting data were displayed. Figure 10 In the middle row, it is labeled "20s Standard Photobleaching". Finally, the flow cell containing the sample was filled with PBS containing 5 mg / ml sodium persulfate, and photobleaching was repeated with white light (800 mW / cm²). 2 Another 10 seconds, then imaging. The resulting data is displayed... Figure 10 The bottom row is labeled “10s photobleaching SPS”, and they clearly demonstrate the efficacy of SPS-mediated photobleaching in removing reporter signals from stained tissues.

[0119] Example 2 - Used to generate Figure 11 Methods for handling data and images in [the context of the text] For HCR™ Gold RNA-FISH, FFPE tonsil samples were prepared as described in Example 1 above. After antigen retrieval, the tissue samples were hybridized with HCR™ RNA-FISH probes targeting ubiquitin c (UBC), keratin 19 (KRT19), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts at 42°C for 2 hours. The tissues were then sealed in a CellScape™ flow chamber. Detection of the HCR™ Gold probes was achieved using a target-specific fluorescently labeled HCR™ Gold amplifier to generate a signal at 42°C for 2 hours; the samples were then washed and imaged to produce Figure 11 Data in the top row. Samples in PBS were filtered through a 405 LP Edge Basic long-pass filter (420-750 nm, 800 mW / cm²). 2 After a 20-second photobleaching step, the staining agent was imaged again; the resulting partially destained RNA detection signal was displayed. Figure 11 In the middle row. Finally, the flow chamber was filled with PBS containing 5 mg / ml sodium persulfate, and photobleaching (800 mW / cm) was repeated. 2 10 seconds, then imaging. The resulting completely destaining RNA detection signal is shown in... Figure 11 In the bottom row.

[0120] Example 3 - Used to generate Figures 12A-12B Methods for handling data and images in [the context of the text] FFPE tonsil samples were prepared as described in Example 1 above. After antigen retrieval, the tissue samples were stained with primary antibodies labeled with fluorophores targeting CD45 (clone HI30, AF488) and histone H3 (clone 1B1-B2, AF647) at room temperature for 1 hour, and then imaged to produce... Figure 12A The data are shown in the figure. Subsequently, the chambers were filled with 5 mg / ml sodium persulfate in PBS, and each even-numbered field of view was photobleached using an FS488 excitation filter (454–497 nm), and each odd-numbered field of view was photobleached using an FS647 excitation filter (626–644 nm). After bleaching, the persulfate was washed off, and all areas were imaged again to produce… Figure 12B The image shows a chessboard pattern.

[0121] Example 4 - Method for generating the data and images in Figures 13 and 14 As described in Example 1 above, serial multiarray sections of normal and cancerous tissue (5 μm thickness) were prepared up to the antibody staining step. Prior to antibody staining, the tissues were treated differently to assess the potential effects of photobleaching on antibody labeling and staining performance. One section (control) was not photobleached before staining and followed the normal procedure outlined in Example 1 above. A second section (50 bleaching cycles) was photobleached again with 5 mg / ml SPS in PBS (532–555 nm; 100 mW / cm²). 2 (10 seconds) 50 times. All samples were then imaged to obtain background fluorescence images in each fluorescence channel from similar regions of interest in each consecutive slice. Samples from 50x photobleaching cycles were stained with DNA before the first photobleaching (cycle 1) and after 50 photobleaching cycles (cycle 50). Antibody and DNA staining was then performed at room temperature in CellScape™ storage buffer at the following dilutions for 1 h: (1) anti-CD3-AF488 [SP162] 1:500; (2) anti-panCK-AF532 [AE1 / AE2] 1:500; (3) anti-FoxP3-AF594 [SP97] 1:100; (4) anti-CD20-AF647 [L26] 1:500; (5) Sytox Orange DNA dye 1:10 6 The sections were then washed with CellScape wash buffer at a constant flow rate (0.5 mL / min) for 10 minutes, and each section was imaged using the same exposure and acquisition settings. The effects of antibody imaging data and SPS-mediated photobleaching on reporter signal generation are shown in Figure 13. The effects of SPS-mediated photobleaching on tissue integrity are shown in Figure 14.

[0122] References

[0123] In the foregoing description, it will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention exemplified herein may be suitably practiced where none of one or more elements or limitations not specifically disclosed herein are present. The terms and expressions used are descriptive rather than limiting, and their use is not intended to exclude any equivalents or portions thereof of the shown and described features, but rather to recognize that various modifications are possible within the scope of the invention. Therefore, it should be understood that although the invention has been exemplified by specific embodiments and optional features, modifications and / or variations of the concepts disclosed herein are available to those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0124] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. Unless otherwise stated, the use of any and all embodiments provided herein is intended only to better illustrate the invention and not to limit the scope of the invention. The language in this specification should not be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0125] This document cites numerous patent and non-patent references. All cited references are incorporated herein by reference in their entirety. In the event of any discrepancy between the definition of a term in this specification and its definition in the cited references, the definition in this specification shall prevail.

Claims

1. A method for detecting one or more targets in a biological sample, the method comprising: a. Contact the biological sample with a detectable marker and connect the detectable marker to one or more targets; b. Detect the signal from the detectable marker; c. After the detection in step (b), contact the sample from (b) with PMS, PDS, their salts, or any combination thereof, wherein PMS and PDS are represented by the following formula: or , d. Irradiating the sample of (c), wherein irradiation includes exposing the sample to light, optionally, wherein the light is light within a selected wavelength range, wherein the selected wavelength range is within the wavelength range required to detect the detectable marker; e. Optionally, repeat steps (a)-(d).

2. The method of claim 1, wherein the light is a selected wavelength within the wavelength range required for detecting the detectable mark.

3. The method according to claim 1 or 2, wherein the detectable marker comprises a dye.

4. The method according to any one of the preceding claims, wherein step (a) includes a target probe.

5. The method according to any one of the preceding claims, wherein the dye is connected to the target probe.

6. The method according to any one of the preceding claims, wherein step (a) further comprises a reporting probe, wherein the dye is connected to the reporting probe.

7. The method according to any one of the preceding claims, wherein the biological sample comprises cells expressing the dye.

8. The method according to any one of the preceding claims, wherein the target comprises RNA or DNA.

9. The method according to any one of the preceding claims, wherein the target comprises a protein or a modified form of a protein, such as a form modified by glycosylation, phosphorylation, or methylation.

10. The method according to any one of the preceding claims, wherein the target comprises RNA or DNA, and the target probe comprises nucleic acid.

11. The method according to any one of the preceding claims, wherein the target comprises a protein and the target probe comprises an antibody.

12. The method according to any one of the preceding claims, wherein the biological sample comprises a tissue sample, optionally a biopsy sample.

13. The method according to any one of the preceding claims, wherein the biological sample comprises cells or cell cultures.

14. The method according to any one of the preceding claims, wherein the biological sample comprises blood, bile, bone marrow aspirate, milk, cerebrospinal fluid, plasma, saliva, serum, sputum, feces, swabs (oral, nasal, or vaginal fluid), synovial fluid, or urine.

15. The method according to any one of the preceding claims, wherein the sample is preserved, fixed and / or frozen, and optionally, wherein the sample is formalin fixed and paraffin embedded.

16. The method according to any one of the preceding claims, wherein step (d) is performed at a pH in the range of about 5 to about 9.

17. The method according to any one of the preceding claims, wherein the sample is disposed on a solid support.

18. The method of claim 17, wherein the solid support comprises one or more of a glass slide, a porous plate, a flow cell, beads, paper, nitrocellulose (e.g., for side flow measurement), or a PDVF membrane.

19. The method according to any one of the preceding claims, wherein the method is performed in situ.

20. The method according to any one of the preceding claims, wherein step (c) comprises PMS or a salt thereof.

21. The method according to any one of the preceding claims, wherein step (c) comprises PDS or a salt thereof.

22. The method according to any one of the preceding claims, wherein the method does not include adding an acid or an antioxidant buffer after step (b).

23. The method according to any one of the preceding claims, wherein the detectable marker comprises a dye, and wherein the dye comprises a fluorophore.

24. The method according to any one of the preceding claims, wherein the detectable marker comprises a staining agent.

25. The method according to any one of the preceding claims, wherein the detectable marker comprises a staining agent, and wherein the staining agent comprises one or more of hematoxylin and eosin.

26. The method according to any one of the preceding claims, wherein step (a) comprises more than one different detectable marker, wherein each detectable marker is connected to a different target.

27. The method according to any one of the preceding claims, wherein white light is used for irradiation.

28. A method for sequentially detecting more than one target in a biological sample, the method comprising: a. Contact the first target probe with the sample, wherein the first target probe is specific to a first target; b. Hybridize or bind the first target probe to the first target in the sample; c. Detecting a first fluorescently detectable marker, wherein the first fluorescently detectable marker is connected to the first target probe; d. After the detection in step (c), contact the sample from (c) with PMS, PDS, their salts, or any combination thereof, wherein PMS and PDS are represented by the following formula: or , e. Irradiating (d) the sample, wherein irradiation includes exposing the sample to light, optionally wherein the light is light within a selected wavelength range, wherein the selected wavelength range is within the wavelength range required to detect the first fluorescent detectable label; f. Repeat steps (a)-(e), including an Nth target probe and an Nth fluorescently detectable tag that are different from the first probe and the first fluorescently detectable tag, optionally wherein the light of the selected wavelength range is within the wavelength range required to detect the Nth fluorescently detectable tag.

29. The method of claim 28, wherein the fluorescent detectable label is chemically linked to the target probe.

30. The method of any one of claims 28-29, wherein step (c) includes a reporter probe, wherein the fluorescent detectable tag is connected to the reporter probe, and wherein the reporter probe is configured to hybridize or bind to the target probe.

31. The method according to any one of claims 28-30, wherein the target comprises RNA or DNA, and the target probe comprises nucleic acid.

32. The method according to any one of claims 28-31, wherein the target comprises a protein and the target probe comprises an antibody.

33. The method according to any one of claims 28-32, wherein the biological sample comprises a tissue sample, optionally a biopsy sample.

34. The method according to any one of claims 28-33, wherein the biological sample comprises cells or cell cultures.

35. The method according to any one of claims 28-34, wherein the biological sample comprises blood, bile, bone marrow aspirate, milk, cerebrospinal fluid, plasma, saliva, serum, sputum, feces, swabs (oral, nasal, or vaginal fluid), synovial fluid, or urine.

36. The method according to any one of claims 28-35, wherein the sample is preserved, fixed and / or frozen, and optionally, wherein the sample is formalin fixed and paraffin embedded.

37. The method according to any one of claims 28-36, wherein step (e) is carried out at a pH in the range of about 5 to about 9.

38. The method according to any one of claims 28-36, wherein in step (e) light of a selected wavelength range is used, wherein the selected wavelength range is within the wavelength range required to detect the first fluorescent detectable tag; and wherein in step (f) light of a selected wavelength range is used, wherein the selected wavelength range is within the wavelength range required to detect the Nth fluorescent detectable tag.

39. The method according to any one of claims 28-38, wherein the irradiation in steps (e), (f), or both is performed with white light.

40. A method for detecting more than one target in a biological sample, the method comprising: a. Contacting a sample with more than one type of target probe, which contains N subsets of target probes, wherein each probe in the subset of target probes is specific to a different target; b. Hybridize or bind the target probe to more than one target in the sample; c. Detecting a first fluorescently detectable marker, wherein the first fluorescently detectable marker is connected to a first subset of the more than one target probe; d. After the detection in step (c), contact the sample from (c) with PMS, PDS, their salts, or any combination thereof, wherein PMS and PDS are represented by the following formula: or , e. Irradiating (d) the sample, wherein irradiation includes exposing the sample to light, optionally wherein the light is light within a selected wavelength range, wherein the selected wavelength range is within the wavelength range required to detect the first fluorescent detectable label; f. Repeat steps (c)-(e), including an Nth fluorescently detectable tag connected to an Nth probe, wherein the Nth fluorescently detectable tag and the Nth subset of target probes are different from the first subset of target probes and the first fluorescently detectable tag, optionally wherein the light of the selected wavelength range is within the wavelength range required to detect the Nth fluorescently detectable tag.

41. The method of claim 40, wherein the fluorescent detectable label is chemically linked to the target probe.

42. The method according to any one of claims 40-41, wherein the fluorescently detectable label is a component in the detection molecule that is different from the target probe, and is configured to hybridize or bind to the target probe.

43. The method according to any one of claims 40-42, wherein the target comprises RNA or DNA, and the target probe comprises nucleic acid.

44. The method according to any one of claims 40-43, wherein the target comprises a protein and the target probe comprises an antibody.

45. The method according to any one of claims 40-44, wherein the biological sample comprises a tissue sample, optionally a biopsy sample.

46. ​​The method according to any one of claims 40-45, wherein the biological sample comprises cells or cell cultures.

47. The method according to any one of claims 40-46, wherein the biological sample comprises blood, bile, bone marrow aspirate, milk, cerebrospinal fluid, plasma, saliva, serum, sputum, feces, swabs (oral, nasal, vaginal fluid), synovial fluid, or urine.

48. The method according to any one of claims 40-47, wherein the sample is preserved, fixed and / or frozen, and optionally, wherein the sample is formalin fixed and paraffin embedded.

49. The method according to any one of claims 40-48, wherein the sample is disposed on a solid support.

50. The method according to any one of claims 40-49, wherein the solid support comprises one or more of a glass slide, a porous plate, a flow cell, beads, paper, and nitrocellulose (sideflow assay).

51. The method according to any one of claims 40-50, wherein the method is performed in situ.

52. The method according to any one of claims 40-51, wherein step (d) comprises PMS or a salt thereof.

53. The method according to any one of claims 40-52, wherein step (d) comprises PDS or a salt thereof.

54. The method according to any one of claims 40-53, wherein the method does not include adding an acid or an antioxidant buffer after step (c).

55. The method according to any one of claims 40-54, wherein in step (e) light of a selected wavelength range is used, wherein the selected wavelength range is within the wavelength range required to detect the first fluorescent detectable tag; and wherein in step (f) light of a selected wavelength range is used, wherein the selected wavelength range is within the wavelength range required to detect the Nth fluorescent detectable tag.

56. The method according to any one of claims 40-55, wherein the irradiation in steps (e), (f), or both is performed with white light.

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