Morphological marker staining

Abstract

Disclosed are systems and methods for labeling one or more morphological markers in a biological sample that are specific to one or more molecular features. [Solution] In particular, a system and method for labeling one or more morphological markers in a biological sample using covalently deposited narrowband detectable regions are described. Labeling of one or more morphological markers with narrowband detectable regions enables higher-order multiple assays by maintaining the available spectral bandwidth. Furthermore, compared to conventional counterstaining methods, the covalent deposition of one or more detectable regions provides flexibility and robustness regarding the order in which biomarkers and morphological markers are labeled in a given staining protocol.

Description

[Technical Field]

[0001] Cross-reference with related applications This disclosure asserts the benefit as of the filing date of U.S. Provisional Patent Application No. 63 / 176326, filed on 18 August 2021 (the full text of which is incorporated herein by reference).

[0002] Areas of disclosure This disclosure relates to labeling one or more morphological markers and / or one or more biomarkers with different detectable portions. [Background technology]

[0003]

[0001] Immunohistochemistry (IHC) refers to a method of detecting, localizing, and / or quantifying antigens, such as proteins, in a biological sample using antibodies specific to those antigens. In cellular samples, such as tissue samples, IHC offers the substantial advantage of providing information about the location of specific proteins within the biological sample. In situ hybridization (ISH) refers to a method of using nucleic acid probes to detect, localize, and / or quantify specific nucleic acid sequences in DNA and RNA that may be present in a sample. Both IHC and ISH can be performed on a variety of biological samples, such as tissue samples (e.g., fresh-frozen or formalin-fixed paraffin-embedded (FFPE)) and cytological samples, and can be used to detect a wide variety of specific antigen targets and sequence targets. Recognition of targets in a sample by antibodies and nucleic acid probes can be detected (e.g., visualized) using various labels (e.g., colorimetric labels, fluorescent labels, luminescent labels, radiometric labels). Amplification of the recognition event is desirable, as is the ability to reliably detect low-abundance cellular markers. For example, by depositing hundreds or thousands of labeled molecules at a marker site in response to a single antigen detection event, the ability to detect that recognition event is enhanced through amplification.

[0004]

[0002] Amplification is often accompanied by adverse events, such as nonspecific signals that appear as an increase in background signal. The increase in background signal interferes with clinical analysis by obscuring weak signals that may be associated with low but clinically important expressions. Therefore, while amplification of recognition events is desirable, amplification methods that minimize background signal are highly desirable.

[0005]

[0003] Even if nucleic acid and protein targets within a sample are precisely localized, further diagnostic information may be obtained from the position of these targets compared to specific morphological structures of cells and tissues. Conventional bright-field staining for visualizing morphological structures is generally performed using broadly absorbed dyes, so combining IHC and ISH detection with morphological staining in a single sample can be challenging, especially when there is a need or desire to multiplely detect multiple targets in their morphological contexts within a single sample. For example, the broad absorption of hematoxylin and eosin (H&E) staining contributes to strong absorption across the entire visible spectrum, obscuring other chromogenic compounds that should be visible under a microscope. H&E absorption complicates the quantification of target molecules by image analysis techniques that rely on separating the spectral contributions of visible dyes used for target molecule detection and morphological dyes. As a result, it is common practice to target-stain the first tissue section and morphologically stain the second tissue section in so-called "sequential slides." Alternatively, the tissue can be stained either targeted or morphologically first, the tissue section can be destained, and then the other targeted or morphological staining can be performed. Another possibility is to reduce the intensity of conventional morphological staining by diluting the stain or shortening the contact time of the sample with the stain, so as not to obscure the desired IHC and ISH signals. Each of these options has its own disadvantages.

[0006]

[0004] In serial slices taken from tissue with a microtome, a new set of cells is removed with each successive slice, so serial slices from a tissue block do not necessarily coincide morphologically with one another. Staining, destaining, and restaining can damage the structure and morphology of the tissue, especially when they must be repeated to achieve higher-order multiplexing. Taking advantage of the reduced staining intensity of conventional morphological stains can result in insufficient staining, making it impossible to distinguish fine morphological features. Furthermore, despite the low level of hematoxylin staining, most of the available detection spectra are not very useful for detecting biomarkers because the broad absorption of hematoxylin needs to be separated from the overlapping biomarker signals. Therefore, it is desirable to provide an improved method for establishing the morphological context of one or more biomarker signals while still enabling the detection of the biomarker signals themselves. [Overview of the project]

[0007]

[0005] In some embodiments, the Disclosure relates to methods for labeling one or more morphological markers in a biological sample to provide morphological context within the sample during manual or automated microscopic analysis. In some embodiments, the morphological marker labeling methods of the Disclosure are combined with biomarker detection methods to provide morphological context for the location of one or more biomarkers detected in a biological sample. For example, staining a combination of one or more morphological markers and one or more medically relevant biomarkers can be used to determine the location of one or more medically relevant biomarkers relative to morphological features (e.g., cell composition, nucleus, etc.) visualized by the staining of one or more morphological markers. In some embodiments, one or more morphological markers may be representative or specific to the same morphological feature. In other embodiments, one or more morphological markers may be representative or specific to the morphological features of different markers. The presence of biomarkers and the location of biomarkers relative to morphological features in a sample often indicate a particular medical condition and can be a determinant of a patient's eligibility for targeted therapy with a particular class of therapeutic agents. The presence and location of biomarkers also function as quality control for the staining method itself, allowing for the detection of abnormal staining patterns that indicate failures at various process stages, such as when reagents are not properly dispensed onto the sample. In some embodiments, one or more morphological markers and / or one or more biomarkers are stained with detectable regions, such as coumarin cores, phenoxazinone cores, 4-hydroxy-3-phenoxazinone cores, 7-amino-4-hydroxy-3-phenoxazinone cores, thioninium cores, phenoxazine cores, phenoxatiin-3-on cores, xanthene cores, heptamethicyanine cores, and croconate cores.

[0008]

[0006] In another embodiment, the morphological marker labeling method of the present disclosure frees up the spectral wavelength range for the detection of one or more biomarkers in a single sample, particularly for bright-field multiplexing where the biomarker signal would otherwise be masked by conventional chemical counterstaining methods. Thus, in some embodiments, a detectable portion having a narrow first absorption band is deposited over, over, or in close proximity to at least a portion of one or more morphological features in a cell or tissue sample, thereby maintaining a usable detection spectrum for detecting one or more biomarkers in the same sample, even if they are present in low abundance. In certain embodiments, a detectable portion having a narrow first absorption band is utilized in the UV portion (e.g., the UVA portion) of the electromagnetic spectrum. In other specific embodiments, a detectable portion having a narrow first absorption band is utilized in the near-IR (NIR) portion of the electromagnetic spectrum. In some embodiments, the detectable portion includes having a coumarin core, a phenoxazinone core, a 4-hydroxy-3-phenoxazinone core, a 7-amino-4-hydroxy-3-phenoxazinone core, a thioninium core, a phenoxazine core, a phenoxatiin-3-on core, a xanthene core, a heptamethyncyanine core, and a croconate core.

[0009]

[0007] By extending the available wavelength range to UV and NIR and by using detectable regions having a narrow first absorption band, it is possible to perform highly multiplexed assays. Thus, for example, in some embodiments, at least one morphological marker and five or more, e.g., seven or more, nine or more, ten or more, or eleven or more biomarkers may be detected in a single sample in spatial relation to its at least one morphological feature. In other embodiments, two or more morphological markers and at least four or more, e.g., six or more, eight or more, or ten or more biomarkers may be detected in a single sample in spatial relation to its two or more morphological markers. For example, Figure 13 shows a specific spectral palette of disclosed detectable regions that can be selected and deposited to detect morphological markers and biomarkers, and it is clear that very high-order multiplexing of multiple biomarker signals and morphological marker signals is possible.

[0010]

[0008] In some embodiments, the detectable portions are covalently deposited on the biological sample, producing a sample that can be processed flexibly with respect to the order in which specific parts of the biological sample (such as one or more morphological features and one or more biomarkers) are stained. For example, one or more morphological features can be detected first, followed by one or more biomarkers. Alternatively, one or more biomarkers can be detected first, followed by staining of one or more morphological features. Overall, biomarker staining and morphological staining can be performed in any order.

[0011]

[0009] In some embodiments, the covalent deposition of the chromophore or detectable portion is achieved using tyramide signal amplification (TSA) (also known as catalyzed reporter deposition (CARD)). U.S. Patent No. 5,583,001 discloses a method for detecting and / or quantifying an analyte using an analyte-dependent enzyme activation system that relies on catalytic reporter deposition to amplify a detectable labeled signal. The catalysis of the enzyme in the CARD or TSA method is enhanced by reacting a labeled phenol molecule with the enzyme. Modern methods utilizing TSA effectively increase the signal obtained from IHC and ISH assays without significant background signal amplification (see, for example, U.S. Patent Application Publication No. 2012 / 0171668, which is incorporated herein by reference in its entirety, for disclosure relating to tyramide amplification reagents). The reagents for these amplification techniques are being used on clinically important targets to provide robust diagnostic capabilities that were previously unattainable (VENTANA OptiView Amplification Kit, Ventana Medical Systems, Tucson, Arizona, catalog number 760-099).

[0012]

[0010] TSA utilizes a reaction catalyzed by horseradish oxidase (HRP) acting on tyramide. In the presence of H2O2, tyramide is converted into a highly reactive and short-lived radical intermediate that preferentially reacts with electron-rich amino acid residues on proteins. The covalently bound detectable portion can then be detected by various colorimetric visualization techniques and / or fluorescence microscopy. In IHC and ISH, where spatial and morphological context is extremely important, the short lifetime of the radical intermediate allows tyramide to covalently bind to tissue adjacent to the site of production, thereby producing a specific signal isolated to the location of protein and nucleic acid targets.

[0013]

[0011] In other embodiments, covalent deposition of the chromophore or detectable portion is carried out using quinone methide chemistry. U.S. Patent No. 10168336, granted January 1, 2019, entitled “Quinone Methide Analog Signal Amplification,” describes a technique similar to TSA ("QMSA") which can be used to increase signal amplification without significantly increasing background signal. In particular, U.S. Patent No. 10168336 describes a novel quinone methide analog precursor and a method for detecting one or more targets in a biological sample using the quinone methide analog precursor. In certain embodiments, the detection method comprises contacting the sample with a detection antibody or probe, and then contacting the sample with a labeled conjugate comprising an alkaline phosphatase (AP) enzyme and a binding portion, wherein the binding portion recognizes the antibody or probe (for example, by binding to a hapten or species-specific antibody epitope or a combination thereof). The alkaline phosphatase enzyme in this labeled conjugate interacts with a quinone methide analog precursor containing a detectable moiety, thereby forming a reactive quinone methide analog, which covalently binds to the biological sample proximal to or directly on the target. The detectable label is then detected visually or by imaging techniques, etc. U.S. Patent No. 10168336 is incorporated herein by reference in its entirety.

[0014]

[0012] Another technique for depositing detectable portions uses "click" chemistry to form covalent bonds between detectable portions in a sample and morphological or biomarkers. "Click" chemistry is a chemical concept independently defined by the Sharpless and Meldal groups, describing chemistry adapted to quickly and reliably produce substances by bonding small units together. "Click" chemistry has been applied to a collection of reliable and autonomous organic reactions (Kolb, HC; Finn, MG; Sharpless, KB Angew. Chem. Int. Ed. 2001, 40, 2004-2021). Click chemistry techniques are described in U.S. Patent Application Publication 2019 / 0204330 (materially incorporated herein) in the context of covalently depositing detectable labels onto biological samples. In this technique, a first reactive group capable of participating in a click chemistry reaction is covalently immobilized on a biological sample using either the tyramide deposition described above or the quinone methide deposition described above. Subsequently, a second component of a detection system having a corresponding second reactive group capable of participating in a click chemistry reaction is reacted with the first reactive group to covalently bond the second component to the biological sample. In certain embodiments, the technique described herein includes contacting the biological sample with a first detection probe specific to a first target. The first detection probe may be a primary antibody or a nucleic acid probe. The sample is then contacted with a first labeled conjugate containing a first enzyme. In some embodiments, the first labeled conjugate is a secondary antibody specific to either a primary antibody (such as the species from which the antibody was obtained) or a label (such as a hapten) conjugated to a nucleic acid probe. The biological sample is then contacted with a first element of a click conjugate pair. The first enzyme cleaves the first element of a click conjugate pair having a tyramide or quinone methide precursor, thereby converting the first element into a reaction intermediate that covalently bonds to the biological sample proximal to or directly on the first target. Next, the second element of the click conjugate pair is brought into contact with the biological sample.Here, the second element of the click conjugate pair comprises a first reporter portion (e.g., a chromophore) and a second reactive functional group, and the second reactive functional group of the second element of the first click conjugate pair can react with the first reactive functional group of the first element of the click conjugate pair. Finally, a signal from the first reporter portion is detected.

[0015]

[0013] In one embodiment, a method for detecting a biomarker in a morphological context within a biological sample is disclosed, which includes labeling at least a portion of a first morphological feature of the biological sample with a first detectable portion, wherein labeling the first morphological feature (e.g., the nucleus or a portion thereof) includes contacting a morphological marker specific to the first morphological feature (e.g., DNA or a histone marker) with a first detection probe that binds to the morphological marker. The method further includes covalently depositing the first detectable portion at or near the location where the first detection probe is bound to the morphological marker specific to the morphological feature. Labeling the first biomarker in the biological sample with a second detectable portion is also part of the method, wherein the second detectable portion differs from the first detectable portion, and labeling the first biomarker includes contacting the first biomarker with a second detection probe that binds to the first biomarker. The method further includes covalently depositing the second detectable portion at or near the location where a second antibody is bound to the first biomarker. In some embodiments, the first and second detectable markers include those having a coumarin core, a phenoxazinone core, a 4-hydroxy-3-phenoxazinone core, a 7-amino-4-hydroxy-3-phenoxazinone core, a thioninium core, a phenoxazine core, a phenoxatiin-3-on core, a xanthene core, a heptamethyncyanine core, and a croconate core.

[0016]

[0014] In more specific embodiments, the morphological feature is the nucleus or nuclear components within a cell, and the morphological marker is present in the nucleus or nuclear components within this cell. In even more specific embodiments, the first detection probe is an antibody against nuclear components (e.g., DNA, histone proteins, etc.) and one or more biomarkers in a biological sample. Examples of other suitable morphological features, as well as morphological markers and biomarkers, are described herein.

[0017]

[0015] In some embodiments, labeling of one or more morphological markers provides a positional context to one or more labeled biomarkers. In some embodiments, labeling of one or more morphological markers allows the morphology of cells and / or tissues to be detected and / or visualized simultaneously with one or more labeled biomarkers. In some embodiments, labeling of one or more morphological markers serves as a substitute for special staining. In other embodiments, labeling of one or more morphological markers serves as a substitute for counterstaining, such as hematoxylin. The above and other advantageous uses of staining samples according to the methods of this disclosure are described herein.

[0018]

[0016] Another aspect of the present disclosure is a method for detecting one or more targets in a biological sample, comprising: labeling a first morphological marker with a first detectable portion, wherein the first detectable portion has a first absorbance peak having an FWHM of less than about 200 nm and an absorption maximum wavelength (λ) between 330 nm ± 10 and 950 nm ± 10. max Labeling a first biomarker with a second detectable portion, wherein the second detectable portion is different from the first detectable portion, and the second detectable portion has a first absorbance peak with an FWHM of less than approximately 200 nm and a maximum absorption wavelength (λ) between 330 nm ± 10 and 950 nm ± 10. maxLabeling with . In some embodiments, the first detectable moiety and the second detectable moiety have a first absorbance peak with a FWHM of less than 160 nm. In some embodiments, the method further comprises labeling a second morphological marker with a third detectable moiety, the third detectable moiety being different from either the first or the second detectable moiety. In some embodiments, both the first morphological marker and the second morphological marker are specific to the same morphological feature.

[0019]

[0017] In some embodiments, the first absorbance peak having the FWHM of the first detectable moiety and / or the second detectable moiety is less than about 130 nm. In some embodiments, the first absorbance peak having the FWHM of the first detectable moiety and / or the second detectable moiety is less than about 100 nm. In some embodiments, the first absorbance peak having the FWHM of the first detectable moiety and / or the second detectable moiety is less than about 80 nm. In some embodiments, the first absorbance peak having the FWHM of the first detectable moiety and / or the second detectable moiety is less than about 60 nm.

[0020]

(0018

[0021]

[0019] In some embodiments, the first morphological marker includes DNA. In some embodiments, labeling of DNA with the first detectable portion includes: (a) contacting the biological sample with an anti-DNA primary antibody; (b) contacting the biological sample with an anti-species secondary antibody specific to the anti-DNA primary antibody, wherein the anti-species antibody is directly or indirectly conjugated to at least one enzyme; and (c) contacting the biological sample with a first detectable conjugate comprising (i) the first detectable portion and (ii) a tyramide portion, a quinone methide precursor portion, or a derivative or analog of the tyramide portion or the quinone methide precursor portion. In some embodiments, DNA labeling with a first detectable moiety includes: (a) contacting a biological sample with an anti-DNA primary antibody; (b) contacting a biological sample with an anti-species secondary antibody specific to the anti-DNA antibody, wherein the anti-species antibody is directly or indirectly conjugated to at least one enzyme; (c) contacting a biological sample with a first tissue-reactive conjugate comprising (i) a first element of a pair of reactive functional groups capable of participating in a click chemistry reaction and (ii) a tyramide moiety, a quinone methide precursor moiety, or a derivative or analog of a tyramide moiety or a quinone methide precursor moiety; and (d) contacting a biological sample with a detectable conjugate comprising (i) a first detectable moiety and (ii) a second element of a pair of reactive functional groups. In alternative embodiments, the primary antibody is labeled with a hapten, and the secondary antibody specifically binds to the hapten conjugated to the primary antibody.

[0022]

[0020] In some embodiments, the first morphological marker includes a histone protein. In some embodiments, labeling of a histone protein with the first detectable moiety includes: (a) contacting the biological sample with an anti-histone primary antibody; (b) contacting the biological sample with an anti-species secondary antibody specific to the anti-histone primary antibody, wherein the anti-species antibody is directly or indirectly conjugated to at least one enzyme; and (c) contacting the biological sample with a first detectable conjugate comprising (i) the first detectable moiety and (ii) a tyramide moiety, a quinone methide precursor moiety, or a derivative or analog of the tyramide moiety or a quinone methide precursor moiety. In some embodiments, the labeling of histone proteins with a first detectable moiety includes: (a) contacting a biological sample with an anti-histone primary antibody; (b) contacting a biological sample with an anti-species secondary antibody specific to the anti-histone antibody, wherein the anti-species antibody is directly or indirectly conjugated to at least one enzyme; (c) contacting a biological sample with a first tissue-reactive conjugate comprising (i) a first element of a pair of i reactive functional groups capable of participating in a click chemistry reaction and (ii) a tyramide moiety, a quinone methide precursor moiety, or a derivative or analog of a tyramide moiety or a quinone methide precursor moiety; and (d) contacting a biological sample with a detectable conjugate comprising (i) a first detectable moiety and (ii) a second element of a pair of reactive functional groups. In alternative embodiments, the primary antibody is labeled with a hapten, and the secondary antibody specifically binds to the hapten conjugated to the primary antibody.

[0023]

[0021] In some embodiments, the first morphological marker is selected from the group consisting of cytosolic markers, nuclear markers, nuclear membrane markers, nucleolar markers, actin filament markers, centrosome markers, centriole satellite markers, intermediate filament markers, microtubule structure markers, mitochondrial markers, endoplasmic reticulum markers, Golgi apparatus markers, plasma membrane markers, and vesicle organelle markers. Suitable morphological markers, antibodies, and antibody sources are shown in Tables 4 to 10 below.

[0024]

[0022] In some embodiments, the first biomarker is a protein biomarker. In some embodiments, the first biomarker is selected from the group consisting of PD-L1, Ki-67, CD3, CD8, CD4, CD20, CD68, p40, p63, TTF-1, ERG, ERBB2 (HER2), α-methylacyl-CoA racemase (AMACR), and synaptophysin. In some embodiments, the first biomarker is a nucleic acid biomarker selected from the group consisting of ERBB2, EGFR, PTEN, p63, TOP2A, CCND1, RREB1, CKS1B, CDKN2C, MCL1, NTRK1, PBX1, ALK, N-MYC, BCL6, PIK3CA, RPN1, TERC, IGH, FGFR3, PDGFRA, EGR1, PDGFRB, and NSD1.

[0025]

[0023] In some embodiments, the first detectable portion includes a coumarin core. In some embodiments, the second detectable portion is in the visible spectrum or the infrared spectrum. In some embodiments, the second detectable portion is in the ultraviolet spectrum. In some embodiments, the first detectable portion and the second detectable portion are separated by a maximum absorption wavelength (λ) of at least 20 nm apart. max ) has.

[0026]

[0024] In some embodiments, the first detectable portion includes a phenoxazinone core, a 4-hydroxy-3-phenoxazinone core, a 7-amino-4-hydroxy-3-phenoxazinone core, a thionium core, a phenoxazine core, a phenoxatiin-3-one core, or a xanthene core. In some embodiments, the second detectable portion is in the ultraviolet spectrum or the infrared spectrum. In some embodiments, the second detectable portion is in the visible spectrum. In some embodiments, the first detectable portion and the second detectable portion are separated by a maximum absorption wavelength (λ) of at least 20 nm apart. max ) has.

[0027]

[0025] In some embodiments, the first detectable portion includes a heptamethyn cyanine core or a croconate core. In some embodiments, the second detectable portion is in the visible spectrum or the ultraviolet spectrum. In some embodiments, the second detectable portion is in the infrared spectrum. In some embodiments, the first detectable portion and the second detectable portion are separated by a maximum absorption wavelength (λ) of at least 20 nm apart. max ) has.

[0028]

[0026] Further aspects of the present disclosure include labeling the first morphological marker with a first detectable portion comprising a core selected from the group consisting of a coumarin core, a phenoxazinone core, a 4-hydroxy-3-phenoxazinone core, a 7-amino-4-hydroxy-3-phenoxazinone core, a thioninium core, a phenoxazine core, a phenoxatin-3-one core, a xanthene core, a heptamethyncyanine core, and a croconate core; labeling the first biomarker with A method for detecting one or more targets in a biological sample, comprising labeling with a second detectable region comprising a core selected from the group consisting of droxy-3-phenoxazinone core, 7-amino-4-hydroxy-3-phenoxazinone core, thioninium core, phenoxazine core, phenoxatiin-3-on core, xanthene core, heptamethynyanine core, and croconate core, wherein the first and second detectable regions are different and have at least 10 nm different maximum absorption wavelengths (λ) max ) has.

[0029]

[0027] In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion and the second detectable portion max ) differ by at least 20 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion and the second detectable portion is different. max ) differ by at least 30 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion and the second detectable portion differs by at least 30 nm. max ) differ by at least 40 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion and the second detectable portion differs by at least 40 nm. max ) differ by at least 50 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion and the second detectable portion is different. max ) differ by at least 60 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion and the second detectable portion differs by at least 60 nm. max ) differ by at least 70 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion and the second detectable portion is different. max) differ by at least 80 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion and the second detectable portion is different. max ) differ by at least 90 nm.

[0030]

[0028] In some embodiments, the first biomarker is a cancer biomarker. Suitable cancer biomarkers include Ki-67, PD-L1, ER, PR, ERBB2 (HER2), EGFR, AMACR, CD8, CD3, or ERG. In some embodiments, the first morphological marker includes DNA. In some embodiments, the first morphological marker includes histone proteins. In some embodiments, the first morphological marker is selected from the group consisting of cytosolic markers, nuclear markers, nuclear membrane markers, nucleolar markers, actin filament markers, centrosome markers, centriole satellite markers, intermediate filament markers, microtubule structure markers, mitochondrial markers, endoplasmic reticulum markers, Golgi apparatus markers, plasma membrane markers, and vesicle organelle markers.

[0031]

[0029] In some embodiments, the method of the present invention further includes labeling a second biomarker with a third detectable portion, wherein the third detectable portion is different from the first detectable portion and the second detectable portion, and the first detectable portion, the second detectable portion and the third detectable portion have maximum absorption wavelengths (λ) that are at least 10 nm different. max ) has. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion, the second detectable portion, and the third detectable portion. max ) differ by at least 20 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion, the second detectable portion, and the third detectable portion is different. max ) differ by at least 30 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion, the second detectable portion, and the third detectable portion is different. max) differ by at least 40 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion, the second detectable portion, and the third detectable portion is different. max ) differ by at least 30 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion, the second detectable portion, and the third detectable portion is different. max ) differ by at least 50 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion, the second detectable portion, and the third detectable portion is different. max ) differ by at least 60 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion, the second detectable portion, and the third detectable portion is different. max ) differ by at least 70 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion, the second detectable portion, and the third detectable portion is different. max ) differ by at least 80 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion, the second detectable portion, and the third detectable portion is different. max ) differ by at least 90 nm.

[0032]

[0030] In some embodiments, the first detectable portion and the second detectable portion are selected from the group consisting of: TIFF2026126215000002.tif182170 TIFF2026126215000003.tif202170 TIFF2026126215000004.tif200170 TIFF2026126215000005.tif223170 TIFF2026126215000006.tif234170 TIFF2026126215000007.tif178170 TIFF2026126215000008.tif235170 TIFF2026126215000009.tif208170 TIFF2026126215000010.tif203170 TIFF2026126215000011.tif215170 TIFF2026126215000012.tif217170 TIFF2026126215000013.tif125170

[0031] (Here, the symbol " "TIFF2026126215000014.tif8170" refers to a region where a detectable portion is conjugated to another portion of a detectable conjugate.

[0033]

[0032] Another aspect of the present disclosure is a biological sample comprising (a) a first morphological marker labeled with a first detectable portion and (b) a first biomarker labeled with a second detectable portion, wherein the first detectable portion and the second detectable portion each have a first absorbance peak having an FWHM of less than about 200 nm and a maximum absorption wavelength (λ) between 330 nm ± 10 and 950 nm ± 10. max ) has the maximum absorption wavelength (λ) of the first detectable portion. max ) and the maximum absorption wavelength of the second detectable portion (λ max ) are separated by at least 20 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The interval between the first detectable portion and the first detectable portion is at least 30 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The interval between the first detectable portion and the first detectable portion is at least 45 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The interval between this and the first detectable portion is at least 60 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The interval between the first detectable portion and the first detectable portion is at least 75 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ maxThe distance between them is at least 90 nm.

[0034]

[0033] In some embodiments, the first morphological marker is selected from the group consisting of cytosolic markers, nuclear markers, nuclear membrane markers, nucleolar markers, actin filament markers, centrosome markers, centriole satellite markers, intermediate filament markers, microtubule structure markers, mitochondrial markers, endoplasmic reticulum markers, Golgi apparatus markers, plasma membrane markers, and vesicle organelle markers. In some embodiments, the first morphological marker is DNA. In some embodiments, the first morphological marker is a histone protein.

[0035]

[0034] In some embodiments, the first detectable portion includes a coumarin core. In some embodiments, the second detectable portion is in the visible spectrum or the infrared spectrum. In some embodiments, the second detectable portion is in the ultraviolet spectrum. In some embodiments, the first detectable portion and the second detectable portion are separated by a maximum absorption wavelength (λ) that is at least 20 nm apart. max ) has.

[0036]

[0035] In some embodiments, the first detectable portion includes a phenoxazinone core, a 4-hydroxy-3-phenoxazinone core, a 7-amino-4-hydroxy-3-phenoxazinone core, a thionium core, a phenoxazine core, a phenoxatiin-3-one core, or a xanthene core. In some embodiments, the second detectable portion is in the ultraviolet spectrum or the infrared spectrum. In some embodiments, the second detectable portion is in the visible spectrum. In some embodiments, the first detectable portion and the second detectable portion are separated by a maximum absorption wavelength (λ) of at least 20 nm apart. max ) has.

[0037]

[0036] In some embodiments, the first detectable portion includes a heptamethyn cyanine core or a croconate core. In some embodiments, the second detectable portion is in the visible spectrum or the ultraviolet spectrum. In some embodiments, the second detectable portion is in the infrared spectrum. In some embodiments, the first detectable portion and the second detectable portion are separated by a maximum absorption wavelength (λ) of at least 20 nm apart. max ) has.

[0038]

[0037] Further aspects of the present disclosure include a biological sample comprising (a) a first biomarker labeled with a first detectable portion and (b) either DNA or a histone protein labeled with a second detectable portion, wherein the first detectable portion and the second detectable portion each have a first absorbance peak having an FWHM of less than about 200 nm and a maximum absorption wavelength (λ) between 330 nm ± 10 and 950 nm ± 10. max ) and; the maximum absorption wavelength (λ) of the first detectable portion max ) and the maximum absorption wavelength of the second detectable portion (λ max ) are separated by at least 20 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The interval between the first detectable portion and the first detectable portion is at least 30 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The interval between the first detectable portion and the first detectable portion is at least 45 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The distance between them is at least 60 nm.

[0039]

[0038] In some embodiments, the biological sample further comprises a second biomarker labeled with a third detectable portion, wherein the first detectable portion, the second detectable portion, and the third detectable portion have maximum absorption wavelengths (λ) that are at least 10 nm different. max) has. In some embodiments, the first detectable portion, the second detectable portion, and the third detectable portion have maximum absorption wavelengths (λ) that are at least 20 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, and the third detectable portion have maximum absorption wavelengths (λ) that are at least 30 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, and the third detectable portion have maximum absorption wavelengths (λ) that are at least 40 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, and the third detectable portion have maximum absorption wavelengths (λ) that are at least 50 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, and the third detectable portion have maximum absorption wavelengths (λ) that are at least 60 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, and the third detectable portion have maximum absorption wavelengths (λ) that are at least 200 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, and the third detectable portion have maximum absorption wavelengths (λ) that are at least 80 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, and the third detectable portion have maximum absorption wavelengths (λ) that are at least 90 nm different. max ) has.

[0040]

[0039] In some embodiments, the biological sample further comprises a third biomarker labeled with a fourth detectable portion, wherein the first detectable portion, the second detectable portion, the third detectable portion, and the fourth detectable portion have maximum absorption wavelengths (λ) that are at least 10 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, the third detectable portion, and the fourth detectable portion have maximum absorption wavelengths (λ) that are at least 20 nm different. max) has. In some embodiments, the first detectable portion, the second detectable portion, the third detectable portion, and the fourth detectable portion have maximum absorption wavelengths (λ) that are at least 30 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, the third detectable portion, and the fourth detectable portion have maximum absorption wavelengths (λ) that are at least 40 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, the third detectable portion, and the fourth detectable portion have maximum absorption wavelengths (λ) that are at least 50 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, the third detectable portion, and the fourth detectable portion have maximum absorption wavelengths (λ) that are at least 60 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, the third detectable portion, and the fourth detectable portion have maximum absorption wavelengths (λ) that are at least 70 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, the third detectable portion, and the fourth detectable portion have maximum absorption wavelengths (λ) that are at least 80 nm different. max ) has. In some embodiments, the first detectable portion, the second detectable portion, the third detectable portion, and the fourth detectable portion have maximum absorption wavelengths (λ) that are at least 90 nm different. max ) has.

[0041]

[0040] In some embodiments, the first detectable portion and the second detectable portion are selected from the group consisting of: TIFF2026126215000015.tif223170 TIFF2026126215000016.tif237170 TIFF2026126215000017.tif211170 TIFF2026126215000018.tif233170 TIFF2026126215000019.tif251170 TIFF2026126215000020.tif188170 TIFF2026126215000021.tif156170 TIFF2026126215000022.tif214170 TIFF2026126215000023.tif221170 TIFF2026126215000024.tif214170 TIFF2026126215000025.tif223170 TIFF2026126215000026.tif237170

[0041] (Here, the symbol " "TIFF2026126215000027.tif8170" refers to a region where a detectable portion is conjugated to another portion of a detectable conjugate.

[0042]

[0042] Further aspects of the present disclosure include (a) a first morphological marker labeled with a first detectable portion and (b) a first biomarker labeled with a second detectable portion, wherein the first detectable portion and the second detectable portion each have a first absorbance peak having an FWHM of less than about 200 nm and a maximum absorption wavelength (λ) between 330 nm ± 10 and 950 nm ± 10. max ) has the maximum absorption wavelength (λ) of the first detectable portion. max ) and the maximum absorption wavelength of the second detectable portion (λ maxA biological sample having at least 20 nm apart, prepared by contacting a first primary antibody specific to a first morphological marker; contacting a first secondary antibody specific to the first primary antibody and conjugated to an enzyme; contacting a first detectable conjugate comprising (a) a tyramide moiety, a quinone methide precursor moiety, or a derivative or analog of a tyramide moiety or a quinone methide precursor moiety and (b) a first detectable moiety; contacting a second primary antibody specific to a first biomarker; contacting a second secondary antibody specific to the second primary antibody and conjugated to an enzyme; and contacting a second detectable conjugate comprising (a) a tyramide moiety, a quinone methide precursor moiety, or a derivative or analog of a tyramide moiety or a quinone methide precursor moiety and (b) a second detectable moiety. In some embodiments, the biological sample does not contain hematoxylin. In some embodiments, the biological sample does not contain special stains.

[0043]

[0043] In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The interval between the first detectable portion and the first detectable portion is at least 30 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The interval between the first detectable portion and the first detectable portion is at least 45 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The interval between this and the first detectable portion is at least 60 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The interval between the first detectable portion and the first detectable portion is at least 75 nm. In some embodiments, the maximum absorption wavelength (λ) of the first detectable portion is max ) and the maximum absorption wavelength of the second detectable portion (λ max The distance between them is at least 90 nm.

[0044]

[0044] In some embodiments, the first morphological marker is selected from the group consisting of a cytosolic marker, a nuclear marker, a nuclear membrane marker, a nucleosome marker, an actin filament marker, a centrosome marker, a centriolar satellite marker, an intermediate filament marker, a microtubule structure marker, a mitochondrial marker, an endoplasmic reticulum marker, a Golgi apparatus marker, a plasma membrane marker, and a vesicular organelle marker. In some embodiments, the first morphological marker is DNA and / or a histone protein.

[0045]

[0045] In some embodiments, the first detectable moiety comprises a coumarin core. In some embodiments, the second detectable moiety is within the visible spectrum or within the infrared spectrum. In some embodiments, the second detectable moiety is within the ultraviolet spectrum. In some embodiments, the first detectable moiety and the second detectable moiety have maximum absorption wavelengths (λ max ) that are at least 20 nm apart.

[0046]

[0046] In some embodiments, the first detectable moiety comprises a phenoxazinone core, a 4-hydroxy-3-phenoxazinone core, a 7-amino-4-hydroxy-3-phenoxazinone core, a thionium core, a phenoxazine core, a phenothiazine-3-one core, or a xanthene core. In some embodiments, the second detectable moiety is within the ultraviolet spectrum or within the infrared spectrum. In some embodiments, the second detectable moiety is within the visible spectrum. In some embodiments, the first detectable moiety and the second detectable moiety have maximum absorption wavelengths (λ max ) that are at least 20 nm apart.

[0047]

[0047] In some embodiments, the first detectable portion includes a heptamethyn cyanine core or a croconate core. In some embodiments, the second detectable portion is in the visible spectrum or the ultraviolet spectrum. In some embodiments, the second detectable portion is in the infrared spectrum. In some embodiments, the first detectable portion and the second detectable portion are separated by a maximum absorption wavelength (λ) of at least 20 nm apart. max ) has.

[0048]

[0048] Another aspect of the present disclosure includes (a) a first morphological marker labeled with a first detectable portion and (b) a first biomarker labeled with a second detectable portion, wherein the first detectable portion and the second detectable portion each have a first absorbance peak having an FWHM of less than about 200 nm and a maximum absorption wavelength (λ) between 330 nm ± 10 and 950 nm ± 10. max ) has the maximum absorption wavelength (λ) of the first detectable portion. max ) and the maximum absorption wavelength of the second detectable portion (λ maxA biological sample that is at least 20 nm apart, contacting with a first primary antibody specific to a first morphological marker; contacting with a first secondary antibody conjugated to an enzyme and specific to the first primary antibody; contacting with a first tissue-reactive moiety comprising (a) a tyramide moiety, a quinonemethide precursor moiety or a derivative or analog of a tyramide moiety or a quinonemethide precursor moiety and (b) a first reactive functional group capable of participating in a click chemistry reaction; contacting with a first detectable conjugate comprising (a) a first detectable moiety and (b) a second reactive functional group; contacting with a second primary antibody specific to a first biomarker; contacting with a second secondary antibody conjugated to an enzyme and specific to the second primary antibody; contacting with a second tissue-reactive moiety comprising (a) a tyramide moiety, a quinonemethide precursor moiety or a derivative or analog of a tyramide moiety or a quinonemethide precursor moiety and (b) a first reactive functional group capable of participating in a click chemistry reaction; contacting with a second detectable conjugate comprising (a) a second detectable moiety and (b) a second reactive functional group. In some embodiments, the biological sample does not contain hematoxylin. In some embodiments, the biological sample does not contain special staining.

[0049]

[0049] In some embodiments, the maximum absorption wavelength (λ max ) of the first detectable moiety and the maximum absorption wavelength (λ max ) of the second detectable moiety are at least 30 nm apart. In some embodiments, the maximum absorption wavelength (λ max ) of the first detectable moiety and the maximum absorption wavelength (λ max ) of the second detectable moiety are at least 45 nm apart. In some embodiments, the maximum absorption wavelength (λ max ) of the first detectable moiety and the maximum absorption wavelength (λ max ) of the second detectable moiety are at least 60 nm apart. In some embodiments, the maximum absorption wavelength (λ max ) of the first detectable moiety and the maximum absorption wavelength (λ maxThe interval between the first detectable portion and the second detectable portion is at least 75 nm. In some embodiments, the interval between the maximum absorption wavelength (λ) of the first detectable portion and the maximum absorption wavelength (λ) of the second detectable portion is at least 90 nm.

[0050]

[0050] In some embodiments, the first morphological marker is selected from the group consisting of cytosolic markers, nuclear markers, nuclear membrane markers, nucleolar markers, actin filament markers, centrosome markers, centriole satellite markers, intermediate filament markers, microtubule structure markers, mitochondrial markers, endoplasmic reticulum markers, Golgi apparatus markers, plasma membrane markers, and vesicle organelle markers. In some embodiments, the first morphological marker is selected from the group consisting of DNA and histone proteins.

[0051]

[0051] In some embodiments, the first detectable portion includes a coumarin core. In some embodiments, the second detectable portion is in the visible spectrum or the infrared spectrum. In some embodiments, the second detectable portion is in the ultraviolet spectrum. In some embodiments, the first detectable portion and the second detectable portion are separated by a maximum absorption wavelength (λ) of at least 20 nm apart. max ) has.

[0052]

[0052] In some embodiments, the first detectable portion includes a phenoxazinone core, a 4-hydroxy-3-phenoxazinone core, a 7-amino-4-hydroxy-3-phenoxazinone core, a thionium core, a phenoxazine core, a phenoxatiin-3-one core, or a xanthene core. In some embodiments, the second detectable portion is in the ultraviolet spectrum or the infrared spectrum. In some embodiments, the second detectable portion is in the visible spectrum. In some embodiments, the first detectable portion and the second detectable portion are separated by a maximum absorption wavelength (λ) of at least 20 nm apart. max ) has.

[0053]

[0053] In some embodiments, the first detectable portion includes a heptamethyn cyanine core or a croconate core. In some embodiments, the second detectable portion is in the visible spectrum or the ultraviolet spectrum. In some embodiments, the second detectable portion is in the infrared spectrum. In some embodiments, the first detectable portion and the second detectable portion are separated by a maximum absorption wavelength (λ) of at least 20 nm apart. max ) has.

[0054]

[0054] Another further aspect of the present disclosure includes (a) a first morphological marker labeled with a first detectable portion and (b) a first biomarker labeled with a second detectable portion, wherein the first detectable portion and the second detectable portion each have a first absorbance peak having an FWHM of less than about 200 nm and a maximum absorption wavelength (λ) between 330 nm ± 10 and 950 nm ± 10. max ) has the maximum absorption wavelength (λ) of the first detectable portion. max ) and the maximum absorption wavelength of the second detectable portion (λ maxA biological sample having at least 20 nm apart, comprising: contact with a first primary antibody specific to a first morphological marker; contact with a first secondary antibody conjugated to an enzyme, specific to the first primary antibody; contact with a first detectable conjugate comprising (a) a tyramide moiety, a quinone methide precursor moiety, or a derivative or analog of the tyramide moiety or quinone methide precursor moiety and (b) a first detectable moiety; and contact with a second primary antibody specific to the first biomarker; A biological sample is prepared by contacting a second secondary antibody that is specific to a second primary antibody and conjugated to an enzyme; contacting a first tissue-reactive moiety comprising (a) a tyramide moiety, a quinone methide precursor moiety, or a derivative or analog of the tyramide moiety or a quinone methide precursor moiety, and (b) a first reactive functional group capable of participating in a click chemistry reaction; and contacting a second detectable conjugate comprising (a) a second detectable moiety and (b) a second reactive functional group. In some embodiments, the biological sample does not contain hematoxylin. In some embodiments, the biological sample does not contain special stains.

[0055]

[0055] In some embodiments, the biological sample is further prepared by contacting it with a third primary antibody that is specific to a second biomarker. In some embodiments, the first detectable conjugate and the second detectable conjugate are selected from the group consisting of: TIFF2026126215000028.tif139170TIFF2026126215000029.tif207170TIFF2026126215000030.tif199170TIFF2026126215000031.tif183170TIFF2026126215000032.tif212170TIFF2026126215000033.tif208170TIFF2026126215000034.tif222170 and TIFF2026126215000035.tif83170.

[0056]

[0056] Another aspect of the present disclosure includes (a) a first morphological marker labeled with a first detectable portion and (b) a first biomarker labeled with a second detectable portion, wherein the first detectable portion and the second detectable portion each have a first absorbance peak having an FWHM of less than about 200 nm and an absorption maximum wavelength (λ) between 330 nm ± 10 and 950 nm ± 10. max ) has the maximum absorption wavelength (λ) of the first detectable portion. max ) and the maximum absorption wavelength of the second detectable portion (λ max A biological sample having at least 20 nm apart, comprising: contact with a first primary antibody specific to a first morphological marker; contact with a first secondary antibody specific to the first primary antibody and conjugated to an enzyme; contact with a first tissue-reactive moiety comprising (a) a tyramide moiety, a quinone methide precursor moiety, or a derivative or analog of a tyramide moiety or a quinone methide precursor moiety and (b) a first reactive functional group capable of participating in a click chemistry reaction; and (a) a first detectable moiety. A biological sample prepared by contacting (a) a first detectable conjugate containing a second reactive functional group; contacting a second primary antibody specific to the first biomarker; contacting a second secondary antibody conjugated to an enzyme and specific to the second primary antibody; and contacting (a) a tyramide moiety, a quinone methide precursor moiety, or a derivative or analog of the tyramide moiety or quinone methide precursor moiety with (b) a second detectable moiety. In some embodiments, the biological sample does not contain hematoxylin. In some embodiments, the biological sample does not contain special stains.

[0057]

[0057] In some embodiments, the method for preparing a biological sample further includes contacting the biological sample with a third primary antibody specific to a second biomarker.

[0058]

[0058] In some embodiments, the first detectable portion and the second detectable portion are selected from the group consisting of: TIFF2026126215000036.tif210170TIFF2026126215000037.tif232170TIFF2026126215000038.tif48170TIFF2026126215000039.tif74170, TIFF2026126215000040.tif56170, TIFF2026126215000041.tif38170, TIFF2026126215000042.tif50170 , TIFF2026126215000043.tif72170.

[0059]

[0059] Further aspects of the present disclosure include a kit comprising (a) a primary antibody specific to a first morphological marker; (b) a primary antibody specific to a first biomarker; and (c) at least two detection conjugates, each comprising a different detectable portion, each detectable portion comprising a first absorbance peak having an FWHM of less than about 200 nm and a maximum absorption wavelength (λ) between 330 nm ± 10 and 950 nm ± 10. max ) and; the maximum absorption wavelength (λ) of the first detectable portion max ) and the maximum absorption wavelength of the second detectable portion (λ max ) are at least 20nm apart.

[0060]

[0060] In some embodiments, at least two detection conjugates are selected from the group consisting of: TIFF2026126215000044.tif197170TIFF2026126215000045.tif250170TIFF20261262150 00046.tif244170TIFF2026126215000047.tif107170TIFF2026126215000048.tif214170, TIFF2026126215000049.tif82170.

[0061]

[0061] The patent or application documents include at least one drawing made in color. A copy of the patent or published patent application accompanied by the color drawing will be provided to the Japan Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0062] [Figure 1]

[0062] An embodiment of the present disclosure describes a method for detecting one or more morphological markers and signals corresponding to one or more biomarkers in a biological sample. [Figure 2A]

[0063] One embodiment of the present disclosure describes a method for labeling one or more morphological markers and / or one or more biomarkers with a detectable portion. [Figure 2B]

[0064] One embodiment of the present disclosure describes a method for labeling one or more morphological markers and / or one or more biomarkers with a detectable portion. [Figure 2C]

[0065] One embodiment of the present disclosure describes a method for labeling one or more morphological markers and / or one or more biomarkers with a detectable portion. [Figure 2D]

[0066] One embodiment of the present disclosure describes a method for labeling one or more morphological markers and / or one or more biomarkers with a detectable portion. [Figure 3]

[0067] One embodiment of the present disclosure describes a method for detecting signals corresponding to one or more morphological markers and one or more biomarkers in a biological sample, utilizing a detectable conjugate comprising (i) a detectable portion and (ii) a tyramide portion, a derivative of a tyramide portion, a quinone methide precursor portion, or a derivative of a quinone methide precursor portion. [Figure 4]

[0068] This shows the deposition of a conjugate containing a quinone methide precursor moiety according to one embodiment of the present disclosure. [Figure 5]

[0069] This shows the deposition of a conjugate containing a tyramide moiety according to one embodiment of the present disclosure. [Figure 6]

[0070] One embodiment of the present disclosure describes a method for detecting signals corresponding to one or more morphological markers and one or more biomarkers in a biological sample. [Figure 7]

[0071] One embodiment of the present disclosure describes a method for detecting signals corresponding to one or more morphological markers and one or more biomarkers in a biological sample, utilizing a detectable conjugate comprising (i) a detectable portion and (ii) a reactive functional group that can participate in a click chemistry reaction. [Figure 8]

[0072] This shows the deposition of a conjugate containing a quinone methide precursor moiety according to one embodiment of the present disclosure. [Figure 9]

[0073] This shows the deposition of a conjugate containing a tyramide moiety according to one embodiment of the present disclosure. [Figure 10]

[0074] The plots of several conventional chromogens and the conventional chemical dye hematoxylin are shown, all of which exhibit absorbance over a broad spectral range. [Figure 11]

[0075] The absorbance over a broad spectrum of the conventional dye hematoxylin is compared with several different detectable regions of the present disclosure, which have absorbances over a relatively narrow spectrum. [Figure 12]

[0076] The images show bright-field microscopy images of formalin-fixed, paraffin-embedded (FFPE) tonsil tissue specimens stained with both hematoxylin and anti-ds DNA IHC using Cy7, a covalently deposited chromophore (CDC). These images were taken at 20x magnification; the left image uses a monochrome CMOS camera with illumination from a 770nm light-emitting diode (LED), while the right image uses a color (RGB) CMOS camera with white light from a tungsten halogen lamp. Since Cy7 absorbs strongly at 770nm and hematoxylin absorbs very little, the left image represents staining by anti-ds DNA IHC. While Cy7 absorbs very little visible light, hematoxylin absorbs broadly in the visible range, so the right image reflects the absorbance of hematoxylin. Comparing these two images of the same microscopic field, we can see that anti-ds DNA IHC provides specific nuclear staining for all cells, similar to hematoxylin, and therefore anti-ds DNA can replace hematoxylin as an effective nuclear counterstain. [Figure 13]

[0077] The images show bright-field microscopy images of FFPE tonsil tissue specimens stained with both hematoxylin and antihistone IHC using Cy7 CDC. These images were taken at 20x magnification; the left image was taken with a monochrome CMOS camera illuminated from a 770nm LED, and the right image was taken with a color (RGB) CMOS camera illuminated from a tungsten halogen lamp. The left image represents staining by antihistone IHC, as Cy7 absorbs strongly at 770nm and hematoxylin absorbs very little. The right image reflects hematoxylin absorbance, as Cy7 absorbs very little visible light, while hematoxylin absorbs broadly in the visible range. Comparing these two images of the same microscopic field, it can be seen that antihistone IHC provides specific nuclear staining for all cells, similar to hematoxylin, and therefore antihistones can replace hematoxylin as an effective nuclear counterstain. [Figure 14]

[0078] As shown in Figure 12, bright-field microscopy images of the same FFPE tonsil tissue specimen stained with both hematoxylin and anti-ds DNA IHC using Cy7 CDC are presented. These images were taken with a monochrome CMOS camera at 20x magnification; the left image was illuminated with a 770 nm LED, and the right image was illuminated with a 595 nM LED. Hematoxylin absorbs strongly at 595 nm, while Cy7 has minimal absorbance; therefore, as shown in Figure 12, the right image reflects the absorbance of hematoxylin, while the right image reflects anti-ds DNA IHC staining using Cy7 CDC. Presenting both hematoxylin and anti-ds DNA IHC staining in monochrome allows for a better comparison of staining intensity across the entire microscopic field. The staining patterns of the antibody and HTX appear similar as shown in Figure 12, but the antibody staining of anti-ds IHC appears to provide a more uniform level of nuclear staining across the entire field. Since the purpose of counterstaining is often to identify all cell nuclei regardless of cell type, uniform staining is a desirable characteristic, offering an unexpected advantage of IHC-based counterstaining over conventional hematoxylin counterstaining. [Figure 15]

[0079] As shown in Figure 13, bright-field microscopy images of the same FFPE tonsil tissue specimen stained with both hematoxylin and antihistone IHC using Cy7 CDC are presented. These images were taken with a monochrome CMOS camera at 20x magnification; the left image was illuminated with a 770nm LED, and the right image was illuminated with a 595nM LED. Hematoxylin absorbs strongly at 595nm, while Cy7 has minimal absorbance; therefore, as shown in Figure 13, the right image reflects the absorbance of hematoxylin, while the right image reflects antihistone IHC staining using Cy7 CDC. Presenting both hematoxylin and antihistone IHC staining in monochrome allows for a better comparison of staining intensity across the entire microscopic field. The staining patterns of the antibody and hematoxylin appear similar as shown in Figure 13, but antibody staining for antihistone IHC appears to provide a more uniform level of nuclear staining across the entire field. Since the purpose of counterstaining is often to identify all cell nuclei regardless of cell type, uniform staining is a desirable characteristic, offering an unexpected advantage of IHC-based counterstaining over conventional hematoxylin counterstaining. [Figure 16]

[0080] This document compares the uniformity of staining using conventional hematoxylin staining and the counterstaining method described herein. [Figure 17]

[0081] This shows a comparison of the broad spectral absorbance of hematoxylin with several different detectable regions that have relatively narrow spectral absorbances. [Figure 18]

[0082] The image shows color images of Rhod614 (left panel) and Rhod634 (right panel) CDC used in anti-ds DNA-assisted IHC in FFPE tonsils. A comparison of the color images in Figure 12 and Figure 13 (right panel) shows color similarity between hematoxylin and these two CDCs, indicating that either of these two CDCs may provide a hematoxylin-versus-stain alternative with similar color development. While similar color development to hematoxylin provides a familiar observation experience for microscopy technicians, it is not essential. However, both CDCs, as shown in Figure 16, provide a narrower absorption band than hematoxylin, reducing spectral overlap and thereby improving visual color discrimination and spectral separation in multiplex IHC images. [Figure 19]

[0083] The image shows color images of Rhod614 (left panel) and Rhod634 (right panel) CDC used in antihistone-assisted IHC in FFPE tonsils. A comparison of the color images in Figure 12 and Figure 13 (right panel) shows color similarity between hematoxylin and these two CDCs, indicating that either of these two CDCs may provide a hematoxylin-versus-staining alternative with similar color development. While similar color development to hematoxylin provides a familiar observation experience for microscopy technicians, it is not essential. However, both CDCs, as shown in Figure 16, provide a narrower light absorption band than hematoxylin, reducing spectral overlap and thereby improving visual color discrimination and spectral separation in multiple IHC images. [Figure 20]

[0084] This disclosure shows a comparison of the absorbances of the spectra of several detectable parts. [Figure 21]

[0085] This figure compares the absorbance of the spectra of several detectable parts according to this disclosure. [Figure 22]

[0086] The four images were recorded with a monochrome camera (dual camera system), and the illumination channels were selected to align near the maximum absorption wavelengths of Dabsil, TAMRA, Rhod634, and Cy5.5, respectively. The fifth image is of the same microscope field, recorded with a color camera (dual camera system) using white light illumination. [Figure 23]

[0087] The images show a series of tissue sections stained with conventional hematoxylin counterstaining instead of anti-ds DNA counterstaining. The first four images, from left to right, are transmitted light images using LEDs filtered at 438, 549, 620, and 689 nm, respectively. These illumination channels were selected to align near the maximum absorption wavelengths of dabusil, TAMRA, hematoxylin, and Cy5.5, respectively. The fifth image is a white light illumination image of the same microscopic field, recorded with a color camera (dual camera system). [Figure 24]

[0088] The results shown by the absorption spectra of these two sections are obtained when a hematoxylin staining time (5 seconds) is selected to provide similar counterstaining absorbances for both hematoxylin and Rhod634 counterstaining according to this disclosure. [Figure 25]

[0089] From left to right, the images show three different microscope fields. The top image was recorded under 525nm LED illumination, where eosin absorbs light, while the corresponding bottom image was recorded under 770nm LED illumination, where Cy7 absorbs light, reflecting the presence of actin. [Figure 26]

[0090] The images show monochrome fluorescence recordings of FFPE tonsil tissue stained with anti-ds DNA IHC using Cy7 CDC, TAMRA CDC, and AMCA CDC at concentrations 1 / 10 of the typical chromophore concentration. [Figure 27]

[0091] The excitation and emission spectra of DAPI and AMCA are shown. [Modes for carrying out the invention]

[0063]

[0092] This specification discloses detectable portions and detectable conjugates comprising one or more detectable portions. In some embodiments, the detectable portions of this disclosure have narrow wavelengths and are suitable for multiplexing.

[0064]

[0093] definition

[0094] As used herein, the singular forms “a,” “an,” and “the” include multiple referents unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “includes” is defined inclusively, so that “includes A or B” means to include A, B, or A and B.

[0065]

[0095] Terms such as “comprising,” “including,” and “having” are used interchangeably and have the same meaning. Similarly, terms such as “comprises,” “includes,” and “has” are used interchangeably and have the same meaning. Specifically, each of these terms is defined to be consistent with the general definition of “comprising” in U.S. patent law, and is therefore understood to be a non-restrictive term meaning “at least,” and not to exclude additional features, limitations, aspects, etc. Thus, for example, “a device having components a, b, and c” means that the device includes at least components a, b, and c. Similarly, “a method involving steps a, b, and c” means that the method includes at least steps a, b, and c. Furthermore, while steps and methods may be outlined in a specific order in this specification, those skilled in the art will recognize that the order of steps and methods can be changed.

[0066]

[0096] As used herein, alkaline phosphatase (AP) is an enzyme that removes and transfers phosphate-grouped organic esters (by hydrolysis) by cleaving the phosphate-oxygen bond and temporarily forming an intermediate enzyme-substrate bond. For example, AP hydrolyzes naphthol phosphate ester (substrate) to phenol compounds and phosphates. The phenols bind to colorless diazonium salts (pigmentants) to produce insoluble, colored azo dyes. In another embodiment, AP hydrolyzes.

[0067]

[0097] As used herein, the term “antibody,” sometimes abbreviated as “Ab,” refers to immunoglobulins or immunoglobulin-like molecules (including, in non-limiting examples, IgA, IgD, IgE, IgG, and IgM, and combinations thereof) and similar molecules produced during an immune response in any vertebrate (e.g., mammals such as humans, goats, rabbits, and mice), as well as antibody fragments that specifically bind to a target molecule (or a population very similar to the target molecule) substantially excluding binding to other molecules. Furthermore, an antibody is a polypeptide ligand containing at least one light-chain immunoglobulin variable region or heavy-chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. Antibodies may consist of heavy and light chains, each having variable regions called a variable heavy (VH) region and a variable light (VL) region. Together, the VH and VL regions are responsible for binding to the antigen recognized by the antibody. The term antibody also includes intact immunoglobulins, as well as their variants and portions well known in the art.

[0068]

[0098] As used herein, the term “antigen” refers to a compound, composition, or substance that can be specifically bound by an antibody molecule or a product of specific humoral or cellular immunity, such as a T cell receptor. Antigens can be any type of molecule, including, for example, haptens, simple intermediate metabolites, sugars (e.g., oligosaccharides), lipids and hormones, as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids and proteins.

[0069]

[0099] As used herein, the term “biological sample” can be any solid or liquid sample excreted or secreted from any organism, including but not limited to single-celled or multicellular organisms such as bacteria, yeast, protozoa, and amoebas (e.g., plants or animals, including samples from healthy or appearing healthy human subjects or human patients suffering from a symptom or disease being diagnosed or examined, such as cancer). For example, a biological sample may be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous humor or vitreous fluid, or any bodily secretions, exudates, or effusions (e.g., bodily fluids obtained from an abscess or other site of infection or inflammation), or bodily fluids obtained from a joint (e.g., a normal joint or an affected joint). A biological sample may also be a sample obtained from any organ or tissue (including biopsy or autopsy specimens such as tumor biopsies), or it may include cells (whether primary or cultured) or culture media prepared by any cells, tissue or organ. In some examples, a biological sample is a nuclear extract. In certain examples, the sample is a quality control sample, for example, one of the cell pellet section samples of this disclosure. In other examples, the sample is a test sample. The sample can be prepared using any method known to those skilled in the art. The sample can be obtained from subjects for routine screening or from subjects suspected of having disorders such as genetic abnormalities, infections or neoplasms. Embodiments described in the methods of this disclosure can also be applied to samples that do not have genetic abnormalities, diseases, disorders, etc., referred to as "normal" samples. The sample may contain multiple targets to which one or more detection probes can specifically bind.

[0070]

[0100] As used herein, the term “conjugate” refers to two or more molecules or parts (including macromolecules or supramolecular parts) covalently bonded to a larger construct. In some embodiments, the conjugate includes one or more biomolecules (e.g., peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) covalently bonded to one or more other molecular parts.

[0071]

[0101] As used herein, the terms “couple” or “coupling” refer to the joining, bonding (e.g., covalent bonding) or linking of one molecule or atom to another molecule or atom.

[0072]

[0102] As used herein, the term “detectable portion” refers to a molecule or material that can produce a detectable signal (e.g., visually, electronically, or otherwise) indicating the presence (i.e., qualitative analysis) and / or concentration (i.e., quantitative analysis) of a label in a sample.

[0073]

[0103] As used herein, horseradish peroxidase (HRP) is an enzyme that can conjugate with labeled molecules. When incubated with a suitable substrate, HRP produces a colored, fluorescent, or luminescent derivative of the labeled molecule, enabling detection and quantification. HRP acts in the presence of an electron donor, first forming an enzyme-substrate complex and then oxidizing the electron donor. For example, HRP can act on 3,3'-diaminobenzidine tetrahydrochloride (DAB) to produce a detectable color. HRP can also act on labeled tyramide conjugates or tyramide-like reactive conjugates (i.e., ferrates, coumaric acid, caffeic acid, cinnamic acid, dopamine, etc.) to deposit a colored, fluorescent, or colorless reporter moiety for tyramide signal amplification (TSA).

[0074]

[0104] As used herein, the terms “multiplex,” “multiplexed,” or “multiplexing” refer to the simultaneous, substantially simultaneous, or sequential detection of multiple targets in a sample. Multiplexing may include the identification and / or quantification of multiple different nucleic acids (e.g., DNA, RNA, mRNA, miRNA) and polypeptides (e.g., proteins) individually and in any combination.

[0075]

[0105] As used herein, "quinone methide" refers to a quinone analog in which one of the carbonyl oxygens on the corresponding quinone is replaced by a methylene group (-CH2-) to form an alkene.

[0076]

[0106] As used herein, the term “specific binding entity” refers to a component of a specific binding pair. A specific binding pair is a pair of molecules that bind to each other in a manner that substantially excludes binding to other molecules (for example, a specific binding pair may have a binding constant that is at least 10⁻³M, 10⁻⁴M, or 10⁻⁵M greater than the binding constant of either of the two components of the binding pair to other molecules in a biological sample). Specific examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, avidins such as streptavidin, and protein A). A specific binding moiety may also include a molecule (or part thereof) that is specifically bound by such a specific binding protein.

[0077]

[0107] As used herein, the term “target” refers to any molecule whose presence, location, and / or concentration are determined or can be determined. Examples of target molecules include proteins, nucleic acid sequences, and haptens, such as haptens covalently bound to proteins. Target molecules are typically detected using one or more conjugates of a specifically binding molecule and a detectable label.

[0078]

[0108] When used in this specification, the symbol " "TIFF2026126215000050.tif8170" indicates the location where one part is joined to another part.

[0079]

[0109] As used herein, the terms “band,” “absorption band,” “peak,” “absorption peak,” “absorbance peak,” and “first absorption band” are used interchangeably. The terms "A" and "B" can be used interchangeably and all refer to the lowest energy absorption band of the chromophores in this disclosure. In this specification, all references to peak absorption wavelengths and FWHM refer to the spectral half-width of the first or lowest energy absorption band.

[0080]

[0110] overview

[0111] This disclosure relates to labeling one or more targets in a biological sample with one or more detectable parts, such as one or more different detectable parts. In some embodiments, one or more targets are one or more morphological markers and / or one or more biomarkers (each as described herein). In some embodiments, one or more targets include two or more morphological markers, e.g., three or more morphological markers, five or more morphological markers, seven or more morphological markers, etc. In some embodiments, one or more targets include two or more morphological markers and / or one or more biomarkers, e.g., two or more biomarkers, three or more biomarkers, etc. In some embodiments, one or more targets include one or more morphological markers and / or two or more biomarkers, e.g., three or more biomarkers, four or more biomarkers, etc.

[0081]

[0112] In some embodiments, labeling one or more morphological markers in a biological sample is considered to provide context for the detection and visualization of one or more biomarkers in the biological sample. In some embodiments, labeling one or more morphological markers provides positional context for one or more biomarkers. In some embodiments, labeling one or more morphological markers allows the morphology of cells and / or tissues to be detected and / or visualized simultaneously with one or more biomarkers. In some embodiments, labeling one or more morphological markers serves as a substitute for special stains, such as special stains that stain specific morphological structures or objects within cells. In other embodiments, labeling one or more morphological markers serves as a substitute for special stains (e.g., muticarmine) or counterstains, such as hematoxylin (see below).

[0082]

[0113] In some embodiments, the methods described herein facilitate the detection of one or more morphological markers and one or more biomarkers using bright-field microscopy. In some embodiments, the methods described herein facilitate the detection of one or more morphological markers and one or more biomarkers using one or more detectable conjugates. In some embodiments, the detectable conjugate comprises (i) a detectable moiety and (ii) a tyramide moiety, a quinone methide precursor moiety, a derivative or analog of a tyramide moiety, or a derivative or analog of a quinone methide precursor moiety. In other embodiments, the detectable conjugate comprises (i) a detectable moiety and (ii) a reactive functional group that can participate in click chemistry reactions. Suitable detectable conjugates and methods of using them are described herein.

[0083]

[0114] In some embodiments, each detectable portion coupled to a detectable conjugate has a predetermined full width at half maximum and a predetermined maximum absorption wavelength (as described herein). In some embodiments, the method described herein utilizes two or more detectable portions whose maximum absorption wavelength difference is at least 10 nm, at least 15 nm, at least 20 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 120 nm, at least 140 nm, at least 160 nm, at least 180 nm, at least 200 nm, and so on. In this way, one or more labeled morphological markers and one or more labeled biomarkers are distinguishable from each other with virtually no spectral overlap.

[0084]

[0115] In some embodiments, the present disclosure enables the labeling of one or more morphological markers and one or more biomarkers in a biological sample without the use of counterstaining such as hematoxylin. Thus, in some embodiments, the stained biological sample is substantially free of hematoxylin. Hematoxylin, a conventional bright-field nuclear counterstain, is an indicator of the morphology of cells and tissues in a specimen, but it has a broad spectral range of absorbance that is considered problematic for multiplexing with immunohistochemistry or in situ hybridization (see Figure 10). Because the broad range of absorbance has considerable overlap with spectrally adjacent chromogens, it is difficult to clearly distinguish individual stained biomarkers (see Figures 10 and 11). Although hematoxylin functions as an effective counterstain, its broad spectrum complicates the visual evaluation of labeled biomarkers, especially when evaluating two or more labeled biomarkers.

[0085]

[0116] Certain detectable regions (such as those described herein) have relatively narrow light absorption bands, making high-order brightfield multiplexing easy. For example, the absorption spectra of five detectable regions (Dabysl, R10, TAMRA, SR101, and Cy5) are plotted in Figure 11. The absorption spectrum of hematoxylin is also included in Figure 11, which helps to highlight the broad light absorption nature of hematoxylin and the resulting issue of spectral overlap between hematoxylin and the five detectable regions mentioned above (comparing hematoxylin with R110, TAMRA, SR101, and Cy5 in Figure 11). By reducing the spectral overlap between these detectable regions (see the descriptions of full width at half maximum and maximum absorption wavelengths herein), the visual distinction of biomarkers labeled with such detectable regions is improved. Nevertheless, counterstaining is still necessary to provide context to the labeled biomarkers.

[0086]

[0117] In IHC and ISH, hematoxylin staining is typically reduced to a level that does not interfere with the visualization or quantification of biomarker staining. In multiplex assays, hematoxylin is often reduced to the point where nuclear staining is almost invisible, thus representing a "trade-off" between the ability to identify / quantify one or more labeled biomarkers and the ability to distinguish nuclear staining (and thus contextual information such as cell and / or tissue morphology). Even with reduced hematoxylin staining levels, some spectral crosstalk still exists between hematoxylin and the chromogen or detectable portion.

[0087]

[0118] In some embodiments of this disclosure, morphological markers (as described herein) are labeled using any of the detectable portions described herein, thereby eliminating the need for counterstains such as hematoxylin. Thus, one or more labeled morphological markers and one or more labeled biomarkers can be detected, visualized, and / or quantified with minimal spectral crosstalk.

[0088]

[0119] Targets for labeling

[0120] The methods disclosed herein can label one or more targets in a biological sample, including “morphological markers” and “biomarkers” as described herein.

[0089]

[0121] Morphological markers

[0122] In some embodiments, one or more targets are protein markers, nucleic acid markers, or cellular components that enable the identification of different morphological features on or within different types of cells (or cellular components) in a biological sample, or on or within different types of tissues (hereinafter referred to as "morphological markers"). For example, the morphological feature may be a nucleus, and different morphological markers such as DNA and histone proteins can be used to facilitate the identification (e.g., visualization of the nucleus) or characterization of the nucleus. In some embodiments, two or more morphological markers specific to the same morphological feature (e.g., a nucleus) are stained at the detectable portion or in contact with it.

[0090]

[0123] Non-limiting examples of morphological markers (which can be used to identify various morphological features) include DNA, histone proteins, cytosolic markers, endoplasmic reticulum markers; nuclear membrane markers, nucleolus or substructures thereof; nucleus and substructures thereof; actin filaments, adhesion plaques or substructures thereof; centrosome and centriole satellite markers; intermediate filament or substructures thereof; microtubule structure or substructure markers; mitochondrial markers; markers for localizing endoplasmic reticulum proteins in different cell lines; Golgi apparatus markers; markers used to localize Golgi-associated proteins in different cell lines; plasma membrane markers; markers for monolocalized plasma membrane proteins highly expressed in different cell lines; and vesicle organelle markers.

[0091]

[0124] Specific, non-limiting examples of morphological markers are given below. In addition to the morphological markers listed herein, additional morphological markers and antibodies that specifically bind to those markers can be selected by referring to the Human Protein Atlas (https: / / www.proteinatlas.org / ). Methods for preparing antibodies for use in covalent detection schemes such as tyramide detection, quinone methide detection, or click detection are well known. Furthermore, antibodies available from Atlas Antibodies are typically available from Sigma-Aldrich.

[0092]

[0125] DNA; [Anti-ds DNA [DSD / 958] (ab215896) antibody obtained from Abcam (Cambridge, Massachusetts)]

[0093]

[0126] Histone protein; [Anti-histone H3 (ab1791) antibody obtained from Abcam (Cambridge, Massachusetts)]

[0094]

[0127] Cytosol markers (e.g., actin [anti-beta-actin antibody (ab8226) obtained from Abcam (Cambridge, Massachusetts)], adenylosuccinate lyase, ataxin 2, G3BP stress granule component 2, aminoacyl-tRNA synthetase complex interaction multifunctional protein 1, tyrosyl-tRNA synthetase, aspartyl-tRNA synthetase, SERPINE 1 mRNA-binding protein 1, coiled-coil domain-containing protein 43, glutamyl-prolyl-tRNA synthetase, histidyl-tRNA synthetase, ataxin 2-like protein, adenosine monophosphate deaminase 2, and RAB GTPase-activating protein 1);

[0095]

[0128] Table 1 - Cytosol Markers TIFF2026126215000051.tif159170

[0096]

[0129] For example, a cytosol can be characterized by labeling it with two or more cytosolic markers (such as any of the detectable portions disclosed herein). In some embodiments, labeling with two or more cytosolic markers can be combined with labeling with one or more biomarkers to characterize the morphological features of the cytosol and / or one or more biomarkers.

[0097]

[0130] Endoplasmic reticulum markers (e.g., heat shock protein 90 beta family component 1, calnexin [anti-calnexin antibody-ER marker (ab22595) obtained from Abcam], kinectin 1, protein disulfide isomerase family A component 3, reticulocarbine 1, ribosome-binding protein 1, Sec61 translocon beta subunit, cytochrome P450 family 51 subfamily A component 1);

[0098]

[0131] Table 2 - Endoplasmic reticulum markers TIFF2026126215000052.tif98170

[0099]

[0132] For example, the endoplasmic reticulum can be characterized by labeling it with two or more endoplasmic reticulum markers (such as any of the detectable regions disclosed herein). In some embodiments, labeling of two or more endoplasmic reticulum markers can be combined with labeling of one or more biomarkers to characterize the morphological features of the endoplasmic reticulum and / or one or more biomarkers.

[0100]

[0133] Nuclear membrane markers (e.g., Sad1 and UNC84 domain-containing protein 2, thymopoietin, Sad1 and UNC84 domain-containing protein 1, LEM domain-containing protein 2, lamin B1 [anti-lamin B1 antibody obtained from Abcam (EPR8985(B))][anti-lamin antibody obtained from Abcam (ab11575)], tosin 1A interacting protein 1, lamin B receptor, lamin B2);

[0101]

[0134] Table 3 - Nuclear membrane markers TIFF2026126215000053.tif70170

[0102]

[0135] For example, nuclear membranes can be characterized by labeling them with two or more nuclear membrane markers (such as any of the detectable regions disclosed herein). In some embodiments, labeling of two or more nuclear membrane markers can be combined with labeling of one or more biomarkers to characterize the morphological features of the nuclear membrane and / or one or more biomarkers.

[0103]

[0136] Markers of the nucleolus or its substructures (e.g., DEAD box helicase 47, ribosome-producing factor 1 homolog, UTP6, small subunit prosesome components, nucleolar protein 10, FtsJ RNA methyltransferase homolog 3, upstream-binding transcription factor RNA polymerase I);

[0104]

[0137] Table 4 - Nurculiolar Markers TIFF2026126215000054.tif70170

[0105]

[0138] For example, two or more nucleolar markers can be labeled (such as in any of the detectable portions disclosed herein) to characterize a nucleolus or a substructure thereof. In some embodiments, the labeling of two or more nucleolar markers can be combined with the labeling of one or more biomarkers to characterize the morphological features of a nucleolus and / or one or more biomarkers.

[0106]

[0139] Markers of the nucleus and its substructures (poly(ADP-ribose) polymerase 1, serine / arginine repeat matrix 2, RNA-binding motif protein 25, X-ray repair cross-complementation 6, heteronuclear ribonucleoprotein C (C1 / C2), TATA box-binding protein-related factor 15, actin-dependent regulatory factor subfamily a of DEAD / H box-containing SWI / SNF-related matrix-related chromatin, C-terminal binding protein 1, actin-dependent regulatory factor subfamily c component 2 of SWI / SNF-related matrix-related chromatin, and PDS5 aggregation-related factor A);

[0107]

[0140] Table 5 - Nuclear Markers TIFF2026126215000055.tif164170

[0108]

[0141] For example, a nucleus can be characterized by labeling it with two or more nuclear markers (such as any of the detectable portions disclosed herein). In some embodiments, labeling of two or more nuclear markers can be combined with labeling of one or more biomarkers to characterize the morphological features of the nucleus and / or one or more biomarkers.

[0109]

[0142] Markers of actin filaments, adhesion plaques, or substructures thereof (septin 9, chondroitin sulfate N-acetylgalactosaminyltransferase 1, FYVE, RhoGEF and PH domain-containing 4, zyxin, N-acyl sphingosinamide hydrolase 2, vinculin);

[0110]

[0143] Table 6 - Actin filament markers TIFF2026126215000056.tif87170

[0111]

[0144] For example, actin filaments, adhesion plaques, or substructures thereof can be characterized by labeling them with two or more markers (such as any of the detectable regions disclosed herein). In some embodiments, labeling of actin filaments, adhesion plaques, or substructures thereof with two or more markers can be combined with labeling of one or more biomarkers to characterize the actin filaments or adhesion plaques as morphological features and / or one or more biomarkers.

[0112]

[0145] Markers for centrosome and centriole satellite (McKusick-Kaufmann syndrome, outer compact filament of sperm tail 2, centrosome protein 97, kinesin family component 5B, progesterone immunomodulatory binding factor 1);

[0113]

[0146] Table 7 - Centrosome Markers TIFF2026126215000057.tif59170

[0114]

[0147] Markers of intermediate filaments or their substructures (keratin 19, keratin 4, desmin, nestin, keratin 17, keratin 13), markers of intermediate filament proteins across different cell lines (vimentin, keratin 8, keratin 7, keratin 19, Praja RING finger ubiquitin ligase 2, keratin 17, keratin 14, nestin, keratin 80, keratin 13);

[0115]

[0148] Table 8 - Intermediate diameter filament markers TIFF2026126215000058.tif87170

[0116]

[0149] For example, intermediate filaments can be characterized by labeling them with two or more intermediate filament markers (such as any of the detectable portions disclosed herein). In some embodiments, labeling of two or more intermediate filament markers can be combined with labeling of one or more biomarkers to characterize the morphological features of the intermediate filament and / or one or more biomarkers.

[0117]

[0150] Markers of microtubule structure or substructure (e.g., tubulin alpha 1a, dystroblevin-binding protein 1, calmodulin regulatory spectrin-related protein family component 2);

[0118]

[0151] Table 9 - Microtubule Markers TIFF2026126215000059.tif48170

[0119]

[0152] For example, two or more microtubule markers can be labeled (such as in any of the detectable portions disclosed herein) to characterize a microtubule structure or substructure. In some embodiments, the labeling of two or more microtubule markers can be combined with the labeling of one or more biomarkers to characterize the morphological features and / or one or more biomarkers of a microtubule structure or substructure.

[0120]

[0153] Mitochondrial markers (containing citrate synthase, leucine-rich pentatricopeptide repeat, 25 components of solute transporter family 24, 44 translocases of the inner mitochondrial membrane, glutaryl-CoA dehydrogenase, and 1 TNF receptor-related protein);

[0121]

[0154] Table 10 - Mitochondrial Markers TIFF2026126215000060.tif70170

[0122]

[0155] For example, mitochondria can be characterized by labeling with two or more mitochondrial markers (such as any of the detectable portions disclosed herein). In some embodiments, labeling with two or more mitochondrial markers can be combined with labeling with one or more biomarkers to characterize the morphological features of mitochondria and / or one or more biomarkers.

[0123]

[0156] Markers for localizing endoplasmic reticulum proteins in different cell lines (ribosomal protein L41, calreticulin, heat shock protein 90 beta family member 1, prolyl 4-hydroxylase subunit beta, protein kinase C substrate 80K-H, ribophorin II, ribophorin I, Sec61 translocon beta subunit, dolityl diphosphooligosaccharide--protein glycosyltransferase noncatalytic subunit);

[0124]

[0157] Golgi apparatus markers (Golgin B1, Golgin A5, polypeptide N-acetylgalactosaminyltransferase 2, zinc finger protein-like protein 1, Golgi reconstitution stacking protein 2, Golgi membrane protein 1, Golgi membrane endogenous protein 4, B cell receptor-related protein 31);

[0125]

[0158] Markers used to localize Golgi apparatus-related proteins in different cell lines (e.g., retention at endoplasmic reticulum sorting receptor 1, stromal cell-derived factor 4, coatmer protein complex subunit epsilon, caveolin 1, transmembrane p24 transporter protein 10, cerglycine, transmembrane p24 transporter protein 3, ATPase secretion pathway Ca2+ transporter 1, ADP-ribosylation factor GTPase-activated protein 2, phosphatidylinositol 4-kinase beta);

[0126]

[0159] Plasma membrane markers (syntaxin 4, solute transporter family 16 component 1, ezrin, erythrocyte membrane protein band 4.1-like 3, component catenin beta 1, ankyrin 3, solute transporter family 413);

[0127]

[0160] Markers of monolocalized plasma membrane proteins highly expressed in different cell lines (adapter-related protein complex 2mu1 subunit, G protein subunit beta2, moesin, ATPase Na+ / K+ transport subunit beta3, phosphatidylethanolamine-binding protein 1, catenin beta1, CD81 molecule, solute transporter family 1 component 5, ezrin, S100 calcium-binding protein A4); and

[0128]

[0161] Markers for vesicle organelles (e.g., ankyrin repeat and FYVE domain-containing 1, RAB5C component RAS oncogene family, alkylglycerone phosphatase synthase, acyl-CoA binding domain-containing 5, RAB7A member RAS oncogene family, perilipin 3).

[0129]

[0162] In some embodiments, one or more morphological markers are histone proteins (e.g., targeted with anti-histone antibodies). In some embodiments, one or more morphological markers are DNA (e.g., targeted with anti-DNA antibodies). In some embodiments, the morphological markers are both histone proteins and DNA.

[0130]

[0163] In some embodiments, one or more morphological markers are cell membrane markers. Examples of cell membrane markers include sodium-potassium ATPase (which is responsible for the extracellular transport of sodium ions and intracellular transport of potassium ions and can be targeted by anti-sodium-potassium ATPase antibodies); plasma membrane calcium ATPase (PMCA regulates intracellular calcium concentration by removing Ca2+ from cells and can be targeted by anti-calcium pump pan-ATPase antibodies); cadherins (transmembrane proteins that mediate calcium-dependent cell-cell adhesion. The Ca2+ binding domain of cadherins is highly conserved, making it possible to create antibodies effective against all components of the cadherin superfamily and thus they can be targeted by anti-pan-cadherin antibodies); CD98 (a transmembrane glycoprotein found in vertebrates; it forms part of the heterodimer neutral amino acid transport system and can be targeted by anti-CD98 antibodies); and caveolae (complex plasma membrane structures characterized by their location between covering pits and lipid rafts and can be targeted by anti-caveolin-1 antibodies).

[0131]

[0164] In some embodiments, one or more morphological markers are cytoplasmic markers. Examples of cytoplasmic markers include microtubules (highly dynamic polymers composed of 13 protofilaments made up of α-tubulin and β-tubulin heterodimers, constantly expanding and contracting between interphase and mitosis, and targeted by anti-alpha-tubulin antibodies), vimentin (class III intermediate filaments found in various non-epithelial cells, especially mesenchymal cells; vimentin is attached to the nucleus, endoplasmic reticulum, and the lateral or terminal sides of mitochondria, and targeted by anti-vimentin antibodies); desmin (class III intermediate filaments found in muscle cells; in adult striated muscle, these form a fibrous network connecting myofibrils and the plasma membrane from the periphery of the Z-line structure, and can be targeted by anti-desmin antibodies); and cytokeratin (intermediate filaments present in all epithelial cells and some non-epithelial cells; these can modulate the activity of kinases such as PKC and SRC through binding to integrin beta-1 (ITB1) and activated protein kinase C receptors, and may be targeted by anti-cytokeratin 19 antibodies).

[0132]

[0165] In some embodiments, one or more morphological markers are nuclear markers. Examples of nuclear markers include the nucleus (anti-KDM1 / LSD1 antibody); nuclear pore (anti-NUP98 antibody); nuclear membrane (anti-lamin A+C antibody); nuclear speckle (anti-SC35 antibody); nucleolus (anti-fibrillarian antibody); heterochromatin (anti-HP1 alpha antibody); and centromere (anti-CENPA antibody).

[0133]

[0166] In some embodiments, one or more morphological markers are organelle markers. Examples of organelle markers include the endoplasmic reticulum (anti-calreticulin antibody); the Golgi apparatus (anti-GM130 antibody); mitochondria (anti-ATP5A antibody); ribosomes (anti-RPS3 antibody); lysosomes (anti-M6PR ANTIBODY); endosomes (anti-EEA1 antibody); peroxisomes (anti-catalase antibody); and autophagosomes (anti-SQSTM1 / p62 antibody).

[0134]

[0167] biomarkers

[0168] In some embodiments, one or more targets within a biological sample are biomarkers. As used herein, the term “biomarker” refers to an indicator that can be detected in a biological sample (e.g., a predictive indicator, a diagnostic indicator, and / or a prognostic indicator), e.g., PD-L1. A biomarker may function as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by specific molecular, pathological, histological, and / or clinical features. In some embodiments, the biomarker is a gene. Examples of biomarkers include, but are not limited to, molecular markers based on polynucleotides (e.g., DNA and / or RNA), polynucleotide copy number variations (e.g., DNA copy number), polypeptides, polypeptide modifications and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and / or glycolipids. Exemplary embodiments include antigens, epitopes, cellular proteins, transmembrane proteins, and DNA or RNA sequences. The Her-2 / neu gene and protein are both exemplary embodiments of biomarkers.

[0135]

[0169] As described above, biomarker targets can be nucleic acid sequences or proteins. Throughout this disclosure, when referring to a target biomarker protein, it is understood that nucleic acid sequences associated with that protein can also be used as biomarker targets. In some embodiments, the biomarker target is a protein or nucleic acid molecule derived from a pathogen such as a virus, bacterium, or intracellular parasite, such as from a viral genome. For example, a biomarker target protein may be generated from a target nucleic acid sequence that is associated with a disease (e.g., correlation, causation, etc.).

[0136]

[0170] Biomarker-targeted nucleic acid sequences can vary substantially in size. Nucleic acid sequences can have an unlimited number of nucleic acid residues. For example, a biomarker-targeted nucleic acid sequence may have at least about 10 nucleic acid residues or at least about 20, 30, 50, 100, 150, 500, or 1000 residues. Similarly, biomarker-targeted polypeptides can vary substantially in size. A biomarker-targeted polypeptide may contain at least one epitope that binds to a peptide-specific antibody or fragment thereof. In some embodiments, the polypeptide may contain at least two epitopes that bind to a peptide-specific antibody or fragment thereof.

[0137]

[0171] In certain non-limiting embodiments, biomarker target proteins are generated by target nucleic acid sequences (e.g., genomic target nucleic acid sequences) associated with neoplasms (e.g., cancer). Numerous chromosomal abnormalities (including translocations and other rearrangements, amplifications, or deletions) have been identified in neoplastic cells, particularly cancer cells such as B-cell and T-cell leukemia, lymphoma, breast cancer, colon cancer, and neurological cancers. Therefore, in some embodiments, at least a portion of the biomarker target molecules are generated by nucleic acid sequences (e.g., genomic target nucleic acid sequences) that are amplified or deleted in at least a subset of cells in the sample.

[0138]

[0172] Oncogenes are known to cause several human malignancies. For example, chromosomal rearrangements involving the SYT gene located in the breakpoint region of chromosome 18q11.2 are common in synovial sarcoma soft tissue tumors. The t(18q11.2) translocation can be identified, for example, using probes with different labels. The first probe contains an FPC nucleic acid molecule generated from a target nucleic acid sequence extending peripherally from the SYT gene, and the second probe contains an FPC nucleic acid generated from a target nucleic acid sequence extending 3' or proximal to the SYT gene. When probes corresponding to these target nucleic acid sequences (e.g., genomic target nucleic acid sequences) are used in an in situ hybridization procedure, normal cells lacking t(18q11.2) in the SYT gene region show two fusion signals (generated by two adjacent labels) reflecting two intact copies of SYT. Abnormal cells with t(18q11.2) show a single fusion signal.

[0139]

[0173] In other embodiments, biomarker target proteins produced from nucleic acid sequences (e.g., genome-targeted nucleic acid sequences) are selected from tumor suppressor genes that are deleted (missing) in malignant cells. For example, the p16 region located on chromosome 9p21 (including D9S1749, D9S1747, p16(INK4A), p14(ARF), D9S1748, p15(INK4B), and D9S1752) is deleted in certain bladder cancers. Chromosomal deletions including the terminal region of the short arm of chromosome 1 (e.g., including SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322) and chromosomal deletions including the pericentral region of chromosome 19 (e.g., including MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, and GLTSCR1) are characteristic molecular features of certain types of solid tumors of the central nervous system.

[0140]

[0174] The embodiments described above are provided for illustrative purposes only and are not intended to limit the scope of the invention. Numerous other cytogenetic abnormalities that correlate with neoplastic transformation and / or proliferation are known to those skilled in the art. Biomarker target proteins produced by nucleic acid sequences (e.g., genome target nucleic acid sequences) that correlate with neoplastic transformation and are useful in the methods of the present disclosure are also EGFR genes (7p12; e.g., GENBANK TM Accession number NC-000007, nucleotides 55054219-55242525), C-MYC gene (8q24.21; e.g., GENBANK) TM Accession number NC-000008, nucleotides 128817498-128822856), D5S271 (5p15.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK TM Accession number NC-000008, nucleotides 19841058-19869049), RB1(13q14; e.g., GENBANK) TM Accession number NC-000013, nucleotide 47775912-47954023), p53 (17p13.1; e.g., GENBANK) TM Accession number NC-000017, complement, nucleotide 7512464-7531642), N-MYC(2p24; e.g., GENBANK TM Accession number NC-000002, complement, nucleotides 151835231-151854620), CHOP(12q13; e.g., GENBANK TM Accession number NC-000012, complement, nucleotides 56196638-56200567), FUS (16p11.2; e.g., GENBANK) TM Accession number NC-000016, nucleotide 31098954-31110601), FKHR (13p14; e.g., GENBANK) TM Accession number NC-000013, complement, nucleotide 40027817-40138734), and other examples: ALK (2p23; e.g., GENBANK) TMAccession number NC-000002, complement, nucleotide 29269144-29997936), Ig heavy chain, CCND1(11q13; e.g., GENBANK TM Accession number NC-000011, nucleotide 69165054.69178423), BCL2(18q21.3; e.g., GENBANK) TM Accession number NC-000018, complement, nucleotide 58941559-59137593), BCL6 (3q27; e.g., GENBANK TM Accession number NC-000003, complement, nucleotides 188921859-188946169), MALF1, AP1 (1p32-p31; e.g., GENBANK TM Accession number NC-000001, complement, nucleotides 59019051-59022373), TOP2A (17q21-q22; e.g., GENBANK) TM Accession number NC-000017, complement, nucleotide 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK) TM Accession number NC-000021, complement, nucleotide 41758351-41801948), ERG(21q22.3; e.g., GENBANK) TM Accession number NC-000021, complement, nucleotides 38675671-38955488); ETV1(7p21.3; e.g., GENBANK TM Accession number NC-000007, complement, nucleotide 13897379-13995289), EWS(22q12.2; e.g., GENBANK) TM Accession number NC-000022, nucleotides 27994271-28026505); FLI1 (11q24.1-q24.3; e.g., GENBANK TM Accession number NC-000011, nucleotides 128069199-128187521), PAX3 (2q35-q37; e.g., GENBANK) TMAccession number NC-000002, complement, nucleotides 222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK) TM Accession number NC-000001, nucleotide 18830087-18935219), PTEN (10q23.3; e.g., GENBANK) TM Accession number NC-000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g., GENBANK) TM Accession number NC-000019, complement, nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK) TM Accession number NC-000001, complement, nucleotides 40133685-40140274), REL(2p13-p12; e.g., GENBANK) TM Accession number NC-000002, nucleotide 60962256-61003682), and CSF1R (5q33-q35; e.g., GENBANK) TM Includes accession number NC-000005, complement, and nucleotides 149413051-149473128.

[0141]

[0175] In other embodiments, the biomarker target protein is selected from viruses or other microorganisms associated with a disease or symptom. Detection of a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) of viral or microbial origin in a cell or biological sample indicates the presence of an organism. For example, the biomarker target peptide, polypeptide, or protein may be selected from the genomes of carcinogenic or pathogenic viruses, bacteria, or intracellular parasites (e.g., Plasmodium falciparum and other malaria parasite species, Leishmania species, Cryptosporidium parvum, Entamoeba histolytica, and Giardia lamblia, as well as species of Toxoplasma, Eimeria, Theileria, and Babesia).

[0142]

[0176] In some embodiments, biomarker target proteins are generated from nucleic acid sequences from the viral genome (e.g., genome target nucleic acid sequences). Exemplary viruses and corresponding genome sequences (GENBANK TMThe reference sequences (RefSeq) (accession numbers in parentheses) are: human adenovirus A (NC-001460), human adenovirus B (NC-004001), human adenovirus C (NC-001405), human adenovirus D (NC-002067), human adenovirus E (N-003266), human adenovirus F (NC-001454), human astrovirus (NC-001943), human BK polyomavirus (V01109; GI:60851), human bocavirus (NC-007455), human coronavirus 229E (NC-002645), human coronavirus HKU1 (NC-006577), human coronavirus NL63 ( NC-005831), Human Coronavirus OC43 (NC-005147), Human Enterovirus A (NC-001612), Human Enterovirus B (NC-001472), Human Enterovirus C (NC-001428), Human Enterovirus D (NC-001430), Human Erythrovirus V9 (NC-004295), Human Formyvirus (NC-001736), Human Herpesvirus 1 (Herpes Simplex Virus Type 1) (NC-001806), Human Herpesvirus 2 (Herpes Simplex Virus Type 2) (NC-001798), Human Herpesvirus 3 (Varicella-Zoster Virus) (NC-001348), Human Herpesvirus 4 Human herpesvirus 4 (Epstein-Barr virus type 1) (NC-007605), Human herpesvirus 4 (Epstein-Barr virus type 2) (NC-009334), Human herpesvirus 5 AD169 strain (NC-001347), Human herpesvirus 5 Merlin strain (NC-006273), Human herpesvirus A (NC-001664), Human herpesvirus 6B (NC-000898), Human herpesvirus 7 (NC-001716), Human herpesvirus 8 M type (NC-003409), Human herpesvirus 8Human papillomavirus type P (NC-009333), Human immunodeficiency virus 1 (NC-001802), Human immunodeficiency virus 2 (NC-001722), Human metapneumovirus (NC-004148), Human papillomavirus-1 (NC-001356), Human papillomavirus-18 (NC-001357), Human papillomavirus-2 (NC-001352), Human papillomavirus-54 (NC-001676), Human papillomavirus-61 (NC-001694), Human papillomavirus cand90 (NC-004104), Human papillomavirusRTRX7 (NC-004761), Human Papillomavirus 10 (NC-001576), Human Papillomavirus 101 (NC-008189), Human Papillomavirus 103 (NC-008188), Human Papillomavirus 107 (NC-009239), Human Papillomavirus 16 (NC-001526), ​​Human Papillomavirus 24 (NC-001683), Human Papillomavirus 26 (NC-001583), Human Papillomavirus 32 (NC-001586), Human Papillomavirus 3 Human papillomavirus type 4 (NC-001587), Human papillomavirus type 41 (NC-001354), Human papillomavirus type 48 (NC-001690), Human papillomavirus type 49 (NC-001591), Human papillomavirus type 5 (NC-001531), Human papillomavirus type 50 (NC-001691), Human papillomavirus type 53 (NC-001593), Human papillomavirus type 60 (NC-001693), Human papillomavirus type 63 (NC-001 458), Human papillomavirus 6b (NC-001355), Human papillomavirus 7 (NC-001595), Human papillomavirus 71 (NC-002644), Human papillomavirus 9 (NC-001596), Human papillomavirus 92 (NC-004500), Human papillomavirus 96 (NC-005134), Human parainfluenza virus 1 (NC-003461), Human parainfluenza virus 2 (NC-003443), Human parainfluenza virus 3 (NC-00179) 6) Includes human parechovirus (NC-001897), human parvovirus 4 (NC-007018), human parvovirus B19 (NC-000883), human respiratory syncytial virus (NC-001781), human rhinovirus A (NC-001617), human rhinovirus B (NC-001490), human spumaretrovirus (NC-001795), human T-lympotropic virus 1 (NC-001436), and human T-lympotropic virus 2 (NC-001488).

[0143]

[0177] In certain embodiments, the biomarker target protein is generated from a nucleic acid sequence (e.g., a genome target nucleic acid sequence) from an oncogenic virus such as Epstein-Barr virus (EBV) or human papillomavirus (HPV, e.g., HPV16, HPV18). In other embodiments, the target protein generated from a nucleic acid sequence (e.g., a genome target nucleic acid sequence) is derived from a pathogenic virus such as respiratory syncytial virus, hepatitis virus (e.g., hepatitis C virus), coronavirus (e.g., SARS virus), adenovirus, polyomavirus, cytomegalovirus (CMV), or herpes simplex virus (HSV).

[0144]

[0178] Detectable portion

[0179] The methods of this disclosure utilize one or more detectable moieties. In some embodiments, the detectable moieties are components of a detectable conjugate. In some embodiments, the detectable conjugate that can be used in the methods of this disclosure comprises a detectable moiety and one of the following: a tyramide moiety (or a derivative or analog thereof), a quinone methide precursor moiety (or a derivative or analog thereof), or a functional group that can participate in a "click chemistry" reaction (see also U.S. Patent No. 1,0041,950, and U.S. Patent Publications 2019 / 0204330, 2017 / 0089911, and 2019 / 0187130, the disclosures of which are incorporated herein by reference in their entirety). In other embodiments, the detectable conjugate that can be used in the methods of this disclosure comprises a detectable moiety and one of the following: a hapten, an enzyme, or an antibody.

[0145]

[0180] In some embodiments, a preferred detectable portion may be characterized according to the full width at half maximum of the first absorbance peak of the absorption band, which is referred to herein as FWHM ("full-width half-max"). FWHM represents the range of a function given by the difference between two extremes of an independent variable where the dependent variable is equal to half of its maximum value. In other words, it is the width of the spectral curve measured between points on the y-axis that are half of the maximum amplitude. This is given by the distance between points on the curve where the function reaches half of its maximum value. Essentially, FWHM is a parameter commonly used to represent the width of a "bump" on a curve or function. In some embodiments, the maximum absorption wavelength (λ) is used. max While ) can represent the wavelength of maximum absorption in the detectable portion, FWHM represents the width of the spectral absorbance.

[0146]

[0181] In some embodiments, the detectable portion has a narrow FWHM. In some embodiments, the detectable portion has a first absorbance peak with a full width at half maximum that is smaller than the FWHM of conventional dyes or pigments (e.g., those typically deposited by precipitation). For example, conventional pigments (e.g., DAB, Fast Red, Fast Blue, or nanoparticle silver stains used in SISH techniques) may have an FWHM of about 200 nm or more, whereas the detectable portion of the present disclosure may have an FWHM of less than about 200 nm, for example, less than about 150 nm, less than about 130 nm, less than about 100 nm, less than about 80 nm, or less than about 60 nm.

[0147]

[0182] In some embodiments, the FWHM of the detectable portion is 40% smaller than that of a conventional dye or pigment source (e.g., hematoxylin, eosin, or a special dye); 50% smaller than that of a conventional dye or pigment source; 55% smaller than that of a conventional dye or pigment source; 65% smaller than that of a conventional dye or pigment source; 70% smaller than that of a conventional dye or pigment source; 75% smaller than that of a conventional dye or pigment source; 80% smaller than that of a conventional dye or pigment source; 85% smaller than that of a conventional dye or pigment source; 90% smaller than that of a conventional dye or pigment source; or 95% smaller than that of a conventional dye or pigment source.

[0148]

[0183] In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 200 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 190 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 180 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 170 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 160 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 150 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 140 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 130 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 120 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 110 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 100 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 90 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 80 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 70 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 60 nm. In some embodiments, the detectable portion has a first absorbance peak with an FWHM of less than approximately 50 nm.

[0149]

[0184] Detectable portion of the ultraviolet spectrum

[0185] In some embodiments, the detectable moiety has a peak absorption wavelength within the ultraviolet spectrum. In some embodiments, the detectable moiety has a peak absorption wavelength of less than about 420 nm. In some embodiments, the detectable moiety has a peak absorption wavelength of less than about 415 nm. In some embodiments, the detectable moiety has a peak absorption wavelength of less than about 410 nm. In some embodiments, the detectable moiety has a peak absorption wavelength of less than about 400 nm. In some embodiments, the detectable moiety has a peak absorption wavelength of less than about 405 nm. In some embodiments, the detectable moiety of the compounds of the present disclosure has a peak absorption wavelength of less than about 395 nm. In some embodiments, the detectable moiety has a peak absorption wavelength of less than about 390 nm. In some embodiments, the detectable moiety has a peak absorption wavelength of less than about 385 nm. In some embodiments, the detectable moiety has a peak absorption wavelength of less than about 380 nm. In some embodiments, the detectable moiety has a peak absorption wavelength of less than about 375 nm. In some embodiments, the detectable moiety of the compounds of the present disclosure has a peak absorption wavelength of less than about 370 nm. In some embodiments, the detectable moiety has a peak absorption wavelength in the range of from about 100 nm to about 400 nm, from about 100 nm to about 390 nm, from about 100 nm to about 380 nm, or from about 100 nm to about 370 nm.

[0150]

[0186] In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 420 nm and an FWHM of less than 160 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 415 nm and an FWHM of less than 160 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 410 nm and an FWHM of less than 160 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 400 nm and an FWHM of less than 160 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 405 nm and an FWHM of less than 160 nm. In some embodiments, the detectable portion of the compound of this disclosure has a first absorbance peak having a peak absorption wavelength of less than approximately 395 nm and an FWHM of less than 160 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 390 nm and an FWHM of less than 160 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 385 nm and an FWHM of less than 160 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 380 nm and an FWHM of less than 160 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 375 nm and an FWHM of less than 160 nm. In some embodiments, the detectable portion of the compound of this disclosure has a first absorbance peak having a peak absorption wavelength of less than approximately 370 nm and an FWHM of less than 160 nm.

[0151]

[0187] In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 420 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 415 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 410 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 400 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 405 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion of the disclosed compound has a peak absorption wavelength of less than approximately 395 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 390 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 385 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 380 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 375 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion of the compound of this disclosure has a first absorbance peak having a peak absorption wavelength of less than approximately 370 nm and an FWHM of less than 130 nm.

[0152]

[0188] In some embodiments, the detectable moiety has a first absorbance peak with a peak absorption wavelength of less than about 420 nm and an FWHM of less than 100 nm. In some embodiments, the detectable moiety has a first absorbance peak with a peak absorption wavelength of less than about 415 nm and an FWHM of less than 100 nm. In some embodiments, the detectable moiety has a first absorbance peak with a peak absorption wavelength of less than about 410 nm and an FWHM of less than 100 nm. In some embodiments, the detectable moiety has a first absorbance peak with a peak absorption wavelength of less than about 400 nm and an FWHM of less than 100 nm. In some embodiments, the detectable moiety has a first absorbance peak with a peak absorption wavelength of less than about 405 nm and an FWHM of less than 100 nm. In some embodiments, the detectable moiety of the compounds of the present disclosure has a first absorbance peak with a peak absorption wavelength of less than about 395 nm and an FWHM of less than 100 nm. In some embodiments, the detectable moiety has a first absorbance peak with a peak absorption wavelength of less than about 390 nm and an FWHM of less than 100 nm. In some embodiments, the detectable moiety has a first absorbance peak with a peak absorption wavelength of less than about 385 nm and an FWHM of less than 100 nm. In some embodiments, the detectable moiety has a first absorbance peak with a peak absorption wavelength of less than about 380 nm and an FWHM of less than 100 nm. In some embodiments, the detectable moiety has a first absorbance peak with a peak absorption wavelength of less than about 375 nm and an FWHM of less than 100 nm. In some embodiments, the detectable moiety of the compounds of the present disclosure has a first absorbance peak with a peak absorption wavelength of less than about 370 nm and an FWHM of less than 100 nm.

[0153]

[0189] In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 420 nm and an FWHM of less than 80 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 415 nm and an FWHM of less than 80 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 410 nm and an FWHM of less than 80 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 400 nm and an FWHM of less than 80 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 405 nm and an FWHM of less than 80 nm. In some embodiments, the detectable portion of the compound of this disclosure has a first absorbance peak having a peak absorption wavelength of less than approximately 395 nm and an FWHM of less than 80 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 390 nm and an FWHM of less than 80 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 385 nm and an FWHM of less than 80 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 380 nm and an FWHM of less than 80 nm. In some embodiments, the detectable portion has a first absorbance peak having a peak absorption wavelength of less than approximately 375 nm and an FWHM of less than 80 nm. In some embodiments, the detectable portion of the compound of this disclosure has a first absorbance peak having a peak absorption wavelength of less than approximately 370 nm and an FWHM of less than 80 nm.

[0154]

[0190] In some embodiments, the detectable portion contains or is derived from coumarin (i.e., the detectable portion contains a coumarin core). An example of a suitable detectable portion having a coumarin core is described in U.S. Patent No. 1,004,1950 (the disclosure of which is incorporated herein by reference in its entirety). In some embodiments, the coumarin core is a coumarinamine core. In some embodiments, the coumarin core is a 7-coumarinamine core. In some embodiments, the coumarin core is a coumarinol core. In some embodiments, the coumarin core is a 7-coumarinol core.

[0155]

[0191] In some embodiments, the coumarin core contains (or is modified to contain) one or more electron-withdrawing groups (each electron-withdrawing group may be the same or different). In some embodiments, the coumarin core contains (or is modified to contain) one electron-withdrawing group. In some embodiments, the coumarin core contains (or is modified to contain) two electron-withdrawing groups. In some embodiments, the coumarin core contains (or is modified to contain) three electron-withdrawing groups. In some embodiments, the coumarin core contains (or is modified to contain) three different electron-withdrawing groups. In some embodiments, the coumarin core contains (or is modified to contain) four electron-withdrawing groups. In some embodiments, each electron-withdrawing group has an electronegativity range of about 1.5 to about 3.5.

[0156]

[0192] In some embodiments, the coumarin core contains (or is modified to contain) one or more electron-donating groups (each electron-donating group may be the same or different). In some embodiments, the coumarin core contains (or is modified to contain) one electron-donating group. In some embodiments, the coumarin core contains (or is modified to contain) two electron-donating groups. In some embodiments, the coumarin core contains (or is modified to contain) three electron-donating groups. In some embodiments, the coumarin core contains (or is modified to contain) three different electron-donating groups. In some embodiments, the coumarin core contains (or is modified to contain) four electron-donating groups. In some embodiments, each electron-donating group has an electronegativity range of about 1.5 to about 3.5. In some embodiments, one or more electron-withdrawing and / or electron-donating groups are incorporated to facilitate a shift to the “red” or “blue” spectrum.

[0157]

[0193] In some embodiments, the detectable portion having a coumarin core has wavelengths in the range of about 300 nm to about 460 nm. In some embodiments, the detectable portion having a coumarin core has wavelengths in the range of about 320 nm to about 440 nm. In some embodiments, the detectable portion having a coumarin core has wavelengths in the range of about 340 nm to about 430 nm. These ranges may change or shift as more or less electronegative material is introduced into the coumarin core.

[0158]

[0194] In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 460 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 455 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 450 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 445 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 440 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 435 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 430 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 425 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 420 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 415 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 410 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 405 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 400 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 395 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 390 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 385 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 380 nm + / - 10 nm.In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 375 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 370 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 365 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 360 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 355 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 350 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 345 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 340 nm + / - 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 335 nm ± 10 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 330 nm ± 10 nm.

[0159]

[0195] In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 460 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 455 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 450 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 445 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 440 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 435 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 430 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 425 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 420 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 415 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 410 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 405 nm ± 10 nm and a first absorbance peak having an FWHM of less than 160 nm.In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 400 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 395 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 390 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 385 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 380 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 375 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 370 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 365 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 3160 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 355 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 350 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm.In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 345 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 340 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 335 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 330 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm.

[0160]

[0196] In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 460 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 455 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 450 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 445 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 440 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 435 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 430 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 425 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 420 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 415 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 410 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 405 nm ± 10 nm and a first absorbance peak having an FWHM of less than 130 nm.In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 400 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 395 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 390 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 385 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 380 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 375 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 370 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 365 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 3130 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 355 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 350 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm.In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 345 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 340 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 335 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 330 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm.

[0161]

[0197] In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 460 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 455 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 450 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 445 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 440 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 435 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 430 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 425 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 420 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 415 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 410 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 405 nm ± 10 nm and a first absorbance peak having an FWHM of less than 100 nm.In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 400 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 395 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 390 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 385 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 380 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 375 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 370 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 365 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 360 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 355 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 350 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm.In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 345 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 340 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 335 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a coumarin core has a peak absorption wavelength of approximately 330 nm + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm.

[0162]

[0198] An example of a detectable part having a coumarin core is: TIFF2026126215000061.tif163170TIFF2026126215000062.tif94170

[0199] In the above formula, the symbol " "TIFF2026126215000063.tif8170" refers to the site where the detectable portion (in this case, the coumarin core) is bound (directly or indirectly) to another portion of the detectable conjugate (e.g., the tyramide portion, the quinone methide portion, a functional group that can participate in a "click chemistry" reaction, an antibody, an enzyme, a hapten, etc.).

[0163]

[0200] Other suitable detectable parts having a coumarin core are described in U.S. Patent No. 1,0041,950, the entire disclosure of which is incorporated herein by reference, provided that those coumarin-based compounds have a first absorbance peak with an FWHM of less than approximately 200 nm.

[0164]

[0201] Further examples are disclosed herein.

[0165]

[0202] Detectable portion of the visible spectrum

[0203] In some embodiments, the detectable portion has a peak absorption wavelength within the visible spectrum. In some embodiments, the detectable portion has a peak absorption wavelength from about 400 nm to about 760 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 440 nm to about 720 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 460 nm to about 680 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 500 nm to about 640 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 540 nm to about 600 nm.

[0166]

[0204] In some embodiments, the detectable portion has a peak absorption wavelength in the visible spectrum. In some embodiments, the detectable portion has a peak absorption wavelength from about 400 nm to about 760 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 440 nm to about 720 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 460 nm to about 680 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 500 nm to about 640 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a peak absorption wavelength of approximately 540 nm to approximately 600 nm and a first absorbance peak having an FWHM of less than 160 nm.

[0167]

[0205] In some embodiments, the detectable portion has a peak absorption wavelength from about 400 nm to about 760 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 440 nm to about 720 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 460 nm to about 680 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 500 nm to about 640 nm and a first absorbance peak having an FWHM of less than 130 nm.

[0168]

[0206] In some embodiments, the detectable portion has a peak absorption wavelength from about 400 nm to about 760 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 440 nm to about 720 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 460 nm to about 680 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 500 nm to about 640 nm and a first absorbance peak having an FWHM of less than 100 nm.

[0169]

[0207] In some embodiments, the detectable portion has a peak absorption wavelength from about 400 nm to about 760 nm and a first absorbance peak having an FWHM of less than 80 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 440 nm to about 720 nm and a first absorbance peak having an FWHM of less than 80 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 460 nm to about 680 nm and a first absorbance peak having an FWHM of less than 80 nm. In some embodiments, the detectable portion has a peak absorption wavelength from about 500 nm to about 640 nm and a first absorbance peak having an FWHM of less than 80 nm.

[0170]

[0208] In some embodiments, the detectable moiety comprises or is derived from phenoxazine or phenoxazinone (i.e., the detectable moiety comprises a phenoxazine or phenoxazinone core). In some embodiments, the detectable moiety derived from phenoxazine or phenoxazinone is 4-hydroxy-3-phenoxazinone or 7-amino-4-hydroxy-3-phenoxazinone.

[0171]

[0209] In some embodiments, the phenoxazine or phenoxazinone core comprises or is modified to comprise one or more electron withdrawing groups (each electron withdrawing group may be the same or different). In some embodiments, the phenoxazine or phenoxazinone core comprises or is modified to comprise one electron withdrawing group. In some embodiments, the phenoxazine or phenoxazinone core comprises or is modified to comprise two electron withdrawing groups. In some embodiments, the phenoxazine or phenoxazinone core comprises or is modified to comprise three electron withdrawing groups. In some embodiments, the phenoxazine or phenoxazinone core comprises or is modified to comprise three different electron withdrawing groups. In some embodiments, the phenoxazine or phenoxazinone core comprises or is modified to comprise four electron withdrawing groups.

[0172]

[0210] In some embodiments, the phenoxazine or phenoxazinone core contains (or is modified to contain) one or more electron-donating groups (each electron-withdrawing group may be the same or different). In some embodiments, the phenoxazine or phenoxazinone core contains (or is modified to contain) one electron-donating group. In some embodiments, the phenoxazine or phenoxazinone core contains (or is modified to contain) two electron-donating groups. In some embodiments, the phenoxazine or phenoxazinone core contains (or is modified to contain) three electron-donating groups. In some embodiments, the phenoxazine or phenoxazinone core contains (or is modified to contain) three different electron-donating groups. In some embodiments, the phenoxazine or phenoxazinone core contains (or is modified to contain) four electron-donating groups.

[0173]

[0211] In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength in the range of about 580 nm to about 700 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength in the range of about 600 nm to about 680 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength in the range of about 620 nm to about 660 nm.

[0174]

[0212] In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 700+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 695+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 690+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 685+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 680+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 675+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 670 + / - 10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 665 + / - 10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 660 + / - 10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 655 + / - 10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 650 + / - 10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 645 + / - 10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 640 ± 10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 635 ± 10 nm.In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 630+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 625+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 620+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 615+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 610+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 605+ / -10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 600 + / - 10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 595 + / - 10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 590 + / - 10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 585 + / - 10 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of approximately 580 + / - 10 nm.

[0175]

[0213] In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 700+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 695+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 690+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 685+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 680+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 675+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 670+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 665+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxazinone core has a peak absorption wavelength of about 660 ± 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxazinone core has a peak absorption wavelength of about 655 ± 10 nm and a first absorbance peak having an FWHM of less than 160 nm.In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 650+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 645+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 640+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 635+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 630+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 625+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 620+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 615+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxazinone core has a peak absorption wavelength of about 610+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxazinone core has a peak absorption wavelength of about 605+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm.In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 600+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 595+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 590+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 585+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 580+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm.

[0176]

[0214] In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 700+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 695+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 690+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 685+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 680+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 675+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 670+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 665+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxazinone core has a peak absorption wavelength of about 660 ± 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxazinone core has a peak absorption wavelength of about 655 ± 10 nm and a first absorbance peak having an FWHM of less than 130 nm.In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 650+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 645+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 640+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 635+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 630+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 625+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 620+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 615+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 610 ± 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 605 ± 10 nm and a first absorbance peak having an FWHM of less than 130 nm.In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 600+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 595+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 590+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 585+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 580+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm.

[0177]

[0215] In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 700+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 695+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 690+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 685+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 680+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 675+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 670+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 665+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxazinone core has a peak absorption wavelength of about 660 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxazinone core has a peak absorption wavelength of about 655 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm.In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 650+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 645+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 640+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 635+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 630+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 625+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 620+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 615+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 610 ± 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 605 ± 10 nm and a first absorbance peak having an FWHM of less than 100 nm.In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 600+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 595+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 590+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 585+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a phenoxazine or phenoxadinone core has a peak absorption wavelength of about 580+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm.

[0178]

[0216] In some embodiments, the detectable portion includes or is derived from a core of thionium, phenoxazine, or phenoxathiin-3-one (i.e., the detectable portion includes a core of thionium or phenoxathiin-3-one).

[0179]

[0217] In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) one or more electron-withdrawing groups (each electron-withdrawing group may be the same or different). In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) one electron-withdrawing group. In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) two electron-withdrawing groups. In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) three electron-withdrawing groups. In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) three different electron-withdrawing groups. In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) four electron-withdrawing groups.

[0180]

[0218] In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) one or more electron-donating groups (each electron-withdrawing group may be the same or different). In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) one electron-donating group. In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) two electron-donating groups. In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) three electron-donating groups. In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) three different electron-donating groups. In some embodiments, the core of thionium, phenoxazine, or phenoxathiin-3-one contains (or is modified to contain) four electron-donating groups.

[0181]

[0219] In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength in the range of about 580 nm to about 720 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength in the range of about 600 nm to about 720 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength in the range of about 630 nm to about 720 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength in the range of about 645 nm to about 700 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength in the range of about 665 nm to about 690 nm.

[0182]

[0220] In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 580 nm to about 720 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 600 nm to about 720 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 630 nm to about 720 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength range of about 645 nm to about 700 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength range of about 665 nm to about 690 nm and a first absorbance peak having an FWHM of less than 160 nm.

[0183]

[0221] In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 580 nm to about 720 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 600 nm to about 720 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 630 nm to about 720 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 645 nm to about 700 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 665 nm to about 690 nm and a first absorbance peak having an FWHM of less than 130 nm.

[0184]

[0222] In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 580 nm to about 720 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 600 nm to about 720 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 630 nm to about 720 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 645 nm to about 700 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a wavelength in the range of about 665 nm to about 690 nm and a first absorbance peak having an FWHM of less than 100 nm.

[0185]

[0223] In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 720+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 715+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 710+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 705+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 700+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 695+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 690+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 685+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 680+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 675+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 670+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 665+ / -10 nm.In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 660+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 655+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 650+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 645+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 640+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 635+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 630+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 625+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 620+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 615+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 610+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 605+ / -10 nm.In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 600+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 595+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 590+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 585+ / -10 nm. In some embodiments, the detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of approximately 580+ / -10 nm.

[0186]

[0224] In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 720+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 715+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 710+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 705+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 700+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 695+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 690+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 685+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 680+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm.In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 675+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 670+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 665+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 660+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 655+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 650+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 645+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 640+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 635+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm.In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 630+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 625+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 620+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 615+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 610+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 605+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 600+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 595+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 590+ / -10 nm and a first absorbance peak with an FWHM of less than 160 nm.In some embodiments, a detectable portion having a core of thionium, phenoxazine, or phenoxathiin-3-one has a peak absorption wavelength of about 585+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm.

[0187]

[0225] In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 720+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 715+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 710+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 705+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 700+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 695+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 690+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 685+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 680+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm.In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 675+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 670+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 665+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 660+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 655+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 650+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 645+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 640+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 635+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm.In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 630+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 625+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 620+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 615+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 610+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 605+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 600+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 595+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 590+ / -10 nm and a first absorbance peak with an FWHM of less than 130 nm.In some embodiments, a detectable portion having a core of thionium, phenoxazine, or phenoxathiin-3-one has a peak absorption wavelength of about 585+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm.

[0188]

[0226] In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 720+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 715+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 710+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 705+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 700+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 695+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a core of thionium, phenoxazine, or phenoxathiin-3-one has a peak absorption wavelength of about 690+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a core of thionium, phenoxazine, or phenoxathiin-3-one has a peak absorption wavelength of about 685+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a core of thionium, phenoxazine, or phenoxathiin-3-one has a peak absorption wavelength of about 680+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm.In some embodiments, a detectable portion having a core of thionium, phenoxazine, or phenoxathiin-3-one has a peak absorption wavelength of about 675+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, a detectable portion having a core of thionium, phenoxazine, or phenoxathiin-3-one has a peak absorption wavelength of about 670+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, a detectable portion having a core of thionium, phenoxazine, or phenoxathiin-3-one has a peak absorption wavelength of about 665+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 660+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 655+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 650+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 645+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 640+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 635+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm.In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 630+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 625+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 620+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 615+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 610+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 605+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 600+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 595+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, a detectable portion having a thionium, phenoxazine, or phenoxathiin-3-one core has a peak absorption wavelength of about 590+ / -10 nm and a first absorbance peak with an FWHM of less than 100 nm.In some embodiments, a detectable portion having a core of thionium, phenoxazine, or phenoxathiin-3-one has a peak absorption wavelength of about 585+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, a detectable portion having a core of thionium, phenoxazine, or phenoxathiin-3-one has a peak absorption wavelength of about 580+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm.

[0189]

[0227] In some embodiments, the detectable portion includes or is derived from a xanthene core (i.e., the detectable portion includes a xanthene core).

[0190]

[0228] In some embodiments, the xanthene core contains (or is modified to contain) one or more electron-withdrawing groups (each electron-withdrawing group may be the same or different). In some embodiments, the xanthene core contains (or is modified to contain) one electron-withdrawing group. In some embodiments, the xanthene core contains (or is modified to contain) two electron-withdrawing groups. In some embodiments, the xanthene core contains (or is modified to contain) three electron-withdrawing groups. In some embodiments, the xanthene core contains (or is modified to contain) three different electron-withdrawing groups. In some embodiments, the xanthene core contains (or is modified to contain) four electron-withdrawing groups.

[0191]

[0229] In some embodiments, the xanthene core contains (or is modified to contain) one or more electron-donating groups (each electron-donating group may be the same or different). In some embodiments, the xanthene core contains (or is modified to contain) one electron-donating group. In some embodiments, the xanthene core contains (or is modified to contain) two electron-donating groups. In some embodiments, the xanthene core contains (or is modified to contain) three electron-donating groups. In some embodiments, the xanthene core contains (or is modified to contain) three different electron-donating groups. In some embodiments, the xanthene core contains (or is modified to contain) four electron-donating groups.

[0192]

[0230] In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength in the range of approximately 580 nm to approximately 650 nm. In some embodiments, the detectable portion having a xanthene core has a wavelength in the range of approximately 590 nm to approximately 640 nm. In some embodiments, the detectable portion having a xanthene core has a wavelength in the range of approximately 600 nm to approximately 630 nm. In some embodiments, when a conjugate including a detectable portion containing a xanthene core is applied to an issue, the above-mentioned optical absorption can be shifted towards the red spectrum by approximately 5 to approximately 10 nm.

[0193]

[0231] In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength in the range of about 580 nm to about 650 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a wavelength in the range of about 590 nm to about 640 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a wavelength in the range of about 600 nm to about 630 nm and a first absorbance peak with an FWHM of less than 160 nm. In some embodiments, when a conjugate including a detectable portion containing a xanthene core is applied to a tissue, the above-mentioned light absorption can be shifted towards the red spectrum of about 5 to about 10 nm.

[0194]

[0232] In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength in the range of about 580 nm to about 650 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a wavelength in the range of about 590 nm to about 640 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a wavelength in the range of about 600 nm to about 630 nm and a first absorbance peak with an FWHM of less than 130 nm. In some embodiments, when a conjugate including a detectable portion containing a xanthene core is applied to a tissue, the above-mentioned light absorption can be shifted towards the red spectrum of about 5 to about 10 nm.

[0195]

[0233] In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength in the range of about 580 nm to about 650 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a wavelength in the range of about 590 nm to about 640 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a wavelength in the range of about 600 nm to about 630 nm and a first absorbance peak with an FWHM of less than 100 nm. In some embodiments, when a conjugate including a detectable portion containing a xanthene core is applied to a tissue, the above-mentioned light absorption can be shifted towards the red spectrum of about 5 to about 10 nm.

[0196]

[0234] In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 650 + / - 10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 645 + / - 10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 640 + / - 10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 635 + / - 10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 630 + / - 10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 625 + / - 10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 620 + / - 10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 615 + / - 10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 610+ / -10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 605+ / -10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 600+ / -10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 595+ / -10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 590+ / -10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 585+ / -10 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 580+ / -10 nm.

[0197]

[0235] In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 650+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 645+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 640+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 635+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 630+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 625+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 620+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 615+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 610+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 605+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of about 600 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of about 595 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm.In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 590 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 585 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 580 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm.

[0198]

[0236] In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 650+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 645+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 640+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 635+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 630+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 625+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 620+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 615+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 610+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 605+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of about 600 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of about 595 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm.In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 590 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 585 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 580 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm.

[0199]

[0237] In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 650+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 645+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 640+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 635+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 630+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 625+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 620+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 615+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 610+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 605+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of about 600 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of about 595 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm.In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 590 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 585 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a xanthene core has a peak absorption wavelength of approximately 580 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm.

[0200]

[0238] Non-limiting examples of compounds having phenoxazinone, 4-hydroxy-3-phenoxazinone, 7-amino-4-hydroxy-3-phenoxazinone, thioninium, phenoxazine, phenoxatiin-3-oncore, or xanthene are: TIFF2026126215000064.tif241170TIFF2026126215000065.tif212170TIFF2026126215000066.tif194170TIFF2026126215000067.tif243170TIFF2026126215000068.tif228170TIFF2026126215000069.tif196170TIFF2026126215000070.tif103170,

[0239] In the above formula, the symbol " "TIFF2026126215000071.tif8170" refers to a site where a detectable moiety (here phenoxazinone, 4-hydroxy-3-phenoxazinone, 7-amino-4-hydroxy-3-phenoxazinone, thionium, phenoxazine, phenoxatiin-3-oncore, or xanthene core) is bound (directly or indirectly) to another part of the detectable conjugate (e.g., a tyramide moiety, a quinone methide moiety, a functional group that can participate in a "click chemistry" reaction, an antibody, an enzyme, a hapten, etc.). Further examples are disclosed herein.

[0201]

[0240] Detectable portion of the infrared spectrum

[0241] In some embodiments, the detectable portion has wavelengths in the infrared spectrum. In some embodiments, the detectable portion has wavelengths greater than approximately 740 nm. In some embodiments, the detectable portion has wavelengths greater than approximately 750 nm. In some embodiments, the detectable portion has wavelengths greater than approximately 760 nm. In some embodiments, the detectable portion has wavelengths greater than approximately 765 nm. In some embodiments, the detectable portion has wavelengths greater than approximately 770 nm. In some embodiments, the detectable portion has wavelengths greater than approximately 775 nm. In some embodiments, the detectable portion has wavelengths greater than approximately 780 nm. In some embodiments, the detectable portion has wavelengths greater than approximately 785 nm. In some embodiments, the detectable portion has wavelengths greater than approximately 790 nm. In some embodiments, the detectable portion has wavelengths in the range of approximately 760 nm to approximately 1 mm, approximately 770 nm to approximately 1 mm, or approximately 780 nm to approximately 1 mm.

[0202]

[0242] In some embodiments, the detectable portion has a wavelength greater than approximately 740 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 750 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 760 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 765 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 770 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 775 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 780 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 785 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 790 nm and a first absorbance peak having an FWHM of less than 160 nm.

[0203]

[0243] In some embodiments, the detectable portion has a first absorbance peak with a wavelength greater than approximately 740 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak with a wavelength greater than approximately 750 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak with a wavelength greater than approximately 760 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak with a wavelength greater than approximately 765 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak with a wavelength greater than approximately 770 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a first absorbance peak with a wavelength greater than approximately 775 nm and an FWHM of less than 130 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 780 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 785 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 790 nm and a first absorbance peak having an FWHM of less than 130 nm.

[0204]

[0244] In some embodiments, the detectable portion has a wavelength greater than approximately 740 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 750 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 760 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 765 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 770 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 775 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 780 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 785 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion has a wavelength greater than approximately 790 nm and a first absorbance peak having an FWHM of less than 100 nm.

[0205]

[0245] In some embodiments, the detectable portion includes or is derived from a heptamethyn cyanine core (i.e., the detectable portion includes a heptamethyn cyanine core).

[0206]

[0246] In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) one or more electron-withdrawing groups (each electron-withdrawing group may be the same or different). In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) one electron-withdrawing group. In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) two electron-withdrawing groups. In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) three electron-withdrawing groups. In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) three different electron-withdrawing groups. In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) four electron-withdrawing groups.

[0207]

[0247] In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) one or more electron-donating groups (each electron-withdrawing group may be the same or different). In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) one electron-donating group. In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) two electron-donating groups. In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) three electron-donating groups. In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) three different electron-donating groups. In some embodiments, the heptamethyn cyanine core contains (or is modified to contain) four electron-donating groups.

[0208]

[0248] In some embodiments, the detectable portion having a heptamethyn cyanine core has wavelengths in the range of about 780 nm to about 950 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has wavelengths in the range of about 810 nm to about 920 nm. In some embodiments, the detectable portion having heptamethyn cyanine has wavelengths in the range of about 840 nm to about 880 nm.

[0209]

[0249] In some embodiments, the detectable portion having a heptamethyn cyanine core has a wavelength in the range of about 780 nm to about 950 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a wavelength in the range of about 810 nm to about 920 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a wavelength in the range of about 840 nm to about 880 nm and a first absorbance peak having an FWHM of less than 160 nm.

[0210]

[0250] In some embodiments, the detectable portion having a heptamethyn cyanine core has a wavelength in the range of about 780 nm to about 950 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a wavelength in the range of about 810 nm to about 920 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a wavelength in the range of about 840 nm to about 880 nm and a first absorbance peak having an FWHM of less than 130 nm.

[0211]

[0251] In some embodiments, the detectable portion having a heptamethyn cyanine core has a wavelength in the range of about 780 nm to about 950 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a wavelength in the range of about 810 nm to about 920 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a wavelength in the range of about 840 nm to about 880 nm and a first absorbance peak having an FWHM of less than 100 nm.

[0212]

[0252] In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 950 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 945 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 940 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 935 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 930 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 925 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 920 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 915 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 910 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 905 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 900 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 895 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 890 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 885 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 880 ± 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 870 ± 10 nm.In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 865 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 860 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 855 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 850 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 845 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 840 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 835 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 830 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 825 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 820 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 815 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 800 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 795 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 790 + / - 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 785 ± 10 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 780 ± 10 nm.

[0213]

[0253] In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 950 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 945 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 940 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 935 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 930+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 925+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 920+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 915+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 910+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 905+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 900+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm.In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 895+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 890+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 885+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 880+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 870 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 865 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 860 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 855 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 850 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 845 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 840 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm.In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 835 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 830 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 825 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 820 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 815+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 800+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 795+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 790+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 785 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 780 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm.

[0214]

[0254] In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 950 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 945 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 940 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 935 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 930+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 925+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 920+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 915+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 910+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 905+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 900+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm.In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 895 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 890 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 885 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 880 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 870 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 865 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 860 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 855 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 850 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 845 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 840 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm.In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 835 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 830 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 825 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 820 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 815+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 800+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 795+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 790+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 785 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 780 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm.

[0215]

[0255] In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 950 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 945 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 940 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 935 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 930 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 925 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 920 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 915 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of about 910+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of about 905+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of about 900+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm.In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 895 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 890 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 885 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 880 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 870 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 865 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 860 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 855 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 850 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 845 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 840 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm.In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 835 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 830 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 825 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 820 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 815 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 800 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 795 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 790 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 785 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a heptamethyn cyanine core has a peak absorption wavelength of approximately 780 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm.

[0216]

[0256] In some embodiments, the detectable portion includes or is derived from a croconate core (i.e., the detectable portion includes a croconate core).

[0217]

[0257] In some embodiments, the croconate core contains (or is modified to contain) one or more electron-withdrawing groups (each electron-withdrawing group may be the same or different). In some embodiments, the croconate core contains (or is modified to contain) one electron-withdrawing group. In some embodiments, the croconate core contains (or is modified to contain) two electron-withdrawing groups. In some embodiments, the croconate core contains (or is modified to contain) three electron-withdrawing groups. In some embodiments, the croconate core contains (or is modified to contain) three different electron-withdrawing groups. In some embodiments, the croconate core contains (or is modified to contain) four electron-withdrawing groups.

[0218]

[0258] In some embodiments, the croconate core contains (or is modified to contain) one or more electron-donating groups (each electron-withdrawing group may be the same or different). In some embodiments, the croconate core contains (or is modified to contain) one electron-donating group. In some embodiments, the croconate core contains (or is modified to contain) two electron-donating groups. In some embodiments, the croconate core contains (or is modified to contain) three electron-donating groups. In some embodiments, the croconate core contains (or is modified to contain) three different electron-donating groups. In some embodiments, the croconate core contains (or is modified to contain) four electron-donating groups.

[0219]

[0259] In some embodiments, the detectable portion having a croconate core has wavelengths in the range of about 780 nm to about 900 nm. In some embodiments, the detectable portion having a croconate core has wavelengths in the range of about 800 nm to about 880 nm. In some embodiments, the detectable portion having a croconate core has wavelengths in the range of about 820 nm to about 860 nm.

[0220]

[0260] In some embodiments, the detectable portion having a croconate core has a wavelength in the range of about 780 nm to about 900 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a wavelength in the range of about 800 nm to about 880 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a wavelength in the range of about 820 nm to about 860 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a wavelength in the range of about 780 nm to about 900 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a wavelength in the range of about 800 nm to about 880 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a wavelength in the range of about 820 nm to about 860 nm and a first absorbance peak having an FWHM of less than 130 nm.

[0221]

[0261] In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 900+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 895+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 890+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 885+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 880+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 870+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 865+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 860+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 855+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 850+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 845+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 840+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 835+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 830+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 825+ / -10 nm. In some embodiments, the detectable portion having a Croconate core has a peak absorption wavelength of approximately 820+ / -10 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 815 + / - 10 nm.In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 800 + / - 10 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 795 + / - 10 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 790 + / - 10 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 785 + / - 10 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 780 + / - 10 nm.

[0222]

[0262] In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 900+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 895+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 890+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 885+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 880+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 870+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 865+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 860+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 855+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 850+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 845 + / - 10 nm and a first absorbance peak having an FWHM of less than 160 nm.In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 840+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 835+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 830+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 825+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 820+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 815+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 800+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 795+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 790+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 785+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 780+ / -10 nm and a first absorbance peak having an FWHM of less than 160 nm.

[0223]

[0263] In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 900+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 895+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 890+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 885+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 880+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 870+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 865+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 860+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 855+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 850+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 845 + / - 10 nm and a first absorbance peak having an FWHM of less than 130 nm.In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 840+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 835+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 830+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 825+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 820+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 815+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 800+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 795+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 790+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 785+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 780+ / -10 nm and a first absorbance peak having an FWHM of less than 130 nm.

[0224]

[0264] In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 900+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 895+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 890+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 885+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 880+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 870+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 865+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 860+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 855+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 850+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 845 + / - 10 nm and a first absorbance peak having an FWHM of less than 100 nm.In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 840+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 835+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 830+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 825+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 820+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 815+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 800+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 795+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 790+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 785+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm. In some embodiments, the detectable portion having a croconate core has a peak absorption wavelength of approximately 780+ / -10 nm and a first absorbance peak having an FWHM of less than 100 nm.

[0225]

[0265] Non-limiting examples of detectable parts include: Includes heptamethic cyanine core or croconate core, including TIFF2026126215000072.tif215170TIFF2026126215000073.tif231170TIFF2026126215000074.tif217170TIFF2026126215000075.tif235170TIFF2026126215000076.tif73170,

[0266] In the above formula, the symbol " "TIFF2026126215000077.tif8170" refers to a site where a detectable portion (here including a heptamethyn cyanine core or a croconate core) is bound (directly or indirectly) to another portion of the detectable conjugate (e.g., a tyramide portion, a quinone methide portion, a functional group that can participate in a "click chemistry" reaction, an antibody, an enzyme, a hapten, etc.). Further examples are disclosed herein.

[0226]

[0267] Other detectable portions suitable for use in the methods of this disclosure include those having a diazocore, such as those disclosed in U.S. Patent No. 1,004,1950 (the disclosure of which is incorporated herein by reference in its entirety). Examples of such compounds include tartrazine: TIFF2026126215000078.tif48170

[0268] This has a peak absorption wavelength of less than approximately 472 nm and a first absorbance peak with an FWHM of less than 70 nm.

[0227]

[0269] Other detectable portions suitable for use in the methods of this disclosure include those having a triarylmethane core, such as those disclosed in U.S. Patent No. 1,004,1950 (whose disclosure is incorporated herein by reference in its entirety). Other detectable portions suitable for use in the methods of this disclosure include tetramethylrhodamine and diarylrhodamine, such as those disclosed in U.S. Patent No. 1,004,1950 (whose disclosure is incorporated herein by reference in its entirety).

[0228]

[0270] (i) comprising a tyramide or quinone methide precursor moiety, and (ii) bound to a detectable moiety, are non-exclusive examples of detectable conjugates: TIFF2026126215000079.tif125170TIFF2026126215000080.tif216170TIFF2026126215000081.tif73170 (maximum wavelength = 604), TIFF2026126215000082.tif75170 (maximum wavelength = 614), TIFF2026126215000083.tif76170 (maximum wavelength = 614), TIFF2026126215000084.tif75170 (maximum wavelength = 598), TIFF2026126215000085.tif73170 (maximum wavelength = 588), TIFF2026126215000086.tif60170 (maximum wavelength = 634), TIFF2026126215000087.tif72170 (maximum wavelength = 634), TIFF2026126215000088.tif70170 (maximum wavelength = 631), TIFF2026126215000089.tif30170 (maximum wavelength = 649), TIFF2026126215000090.tif27170 (maximum wavelength = 665), TIFF2026126215000091.tif128170 (maximum wavelength = 694), and TIFF2026126215000092.tif182170TIFF2026126215000093.tif214170TIFF2026126215000094.tif202170TIFF2026126215000095.tif95170.

[0229]

[0271] (i) containing a functional group that can participate in click chemistry reactions, and (ii) being bound to a detectable moiety, are non-exclusive examples of detectable conjugates: TIFF2026126215000096.tif65170 Maximum wavelength = 410, TIFF2026126215000097.tif60170 (maximum wavelength = 380~390), TIFF2026126215000098.tif178170TIFF2026126215000099.tif218170TIFF202612 6215000100.tif237170TIFF2026126215000101.tif188170TIFF2026126215000102 .tif195170TIFF2026126215000103.tif207170TIFF2026126215000104.tif203170TIFF2026126215000105.tif202170TIFF2026126215000106.tif161170 Maximum wavelength = 804, TIFF2026126215000107.tif59170, TIFF2026126215000108.tif76170TIFF2026126215000109.tif93170.

[0230]

[0272] While each of the exemplified compounds contains an azide group (i.e., N3), it will be understood by those skilled in the art that other functional groups that can participate in the "click chemistry" reaction may be substituted with azide groups containing any of the click functional groups listed in Table 11 below.

[0231] Table 11: Reactive functional groups that can participate in click chemistry reactions TIFF2026126215000110.tif137170

[0232]

[0273] In some embodiments, the detectable portion is selected from the following: TIFF2026126215000111.tif211170TIFF2026126215000112.tif233170TIFF2026126215000113.tif90170TIFF2026126215000114.tif215170 TIFF2026126215000115.tif140170TIFF2026126215000116.tif216170TIFF2026126215000117.tif188170TIFF2026126215000118.tif93170

[0233]

[0274] method

[0275] This disclosure also provides methods for detecting one or more morphological markers and / or one or more biomarkers in a biological sample. In some embodiments, this disclosure provides methods for labeling one morphological marker and two or more biomarkers using different detectable parts. In some embodiments, this disclosure provides methods for labeling one morphological marker and three or more biomarkers using different detectable parts. In some embodiments, this disclosure provides methods for labeling one morphological marker and four or more biomarkers using different detectable parts. In some embodiments, this disclosure provides methods for labeling one morphological marker and four or more biomarkers using different detectable parts.

[0234]

[0276] In some embodiments, the Disclosure provides a method for labeling two or more morphological markers (e.g., two or more morphological markers specific to the same or different morphological features) and two or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling two or more morphological markers (e.g., two or more morphological markers specific to the same or different morphological features) and three or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling two or more morphological markers (e.g., two or more morphological markers specific to the same or different morphological features) and four or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling two or more morphological markers (e.g., two or more morphological markers specific to the same or different morphological features) and five or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling two or more morphological markers and seven or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling two or more morphological markers (e.g., two or more morphological markers specific to the same or different morphological features) and nine or more biomarkers using different detectable portions. In some embodiments, the Disclosure provides a method for labeling two or more morphological markers and ten or more biomarkers using different detectable portions.

[0235]

[0277] In some embodiments, the Disclosure provides a method for labeling two or more morphological markers (e.g., those specific to the same morphological feature) and one or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling three or more morphological markers (e.g., those specific to the same morphological feature) and one or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling four or more morphological markers (e.g., those specific to the same morphological feature) and one or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling five or more morphological markers (e.g., those specific to the same morphological feature) and one or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling six or more morphological markers (e.g., those specific to the same morphological feature) and one or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling seven or more morphological markers (e.g., those specific to the same morphological feature) and one or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling eight or more morphological markers (e.g., those specific to the same morphological feature) and one or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling nine or more morphological markers (e.g., those specific to the same morphological feature) and one or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling ten or more morphological markers (e.g., those specific to the same morphological feature) and one or more biomarkers using different detectable parts. In some embodiments, the Disclosure provides a method for labeling eleven or more morphological markers (e.g., those specific to the same morphological feature) and one or more biomarkers using different detectable parts.

[0236]

[0278] In some embodiments, the Disclosure provides a method for labeling two or more morphological markers (e.g., those specific to the same morphological feature). In some embodiments, the Disclosure provides a method for labeling three or more morphological markers (e.g., those specific to the same morphological feature). In some embodiments, the Disclosure provides a method for labeling four or more morphological markers (e.g., those specific to the same morphological feature). In some embodiments, the Disclosure provides a method for labeling five or more morphological markers (e.g., those specific to the same morphological feature). In some embodiments, the Disclosure provides a method for labeling six or more morphological markers (e.g., those specific to the same morphological feature). In some embodiments, the Disclosure provides a method for labeling seven or more morphological markers (e.g., those specific to the same morphological feature). In some embodiments, the Disclosure provides a method for labeling eight or more morphological markers (e.g., those specific to the same morphological feature). In some embodiments, the Disclosure provides a method for labeling nine or more morphological markers (e.g., those specific to the same morphological feature). In some embodiments, the disclosure provides a method for labeling 10 or more morphological markers (e.g., those specific to the same morphological feature). In some embodiments, the disclosure provides a method for labeling 11 or more morphological markers (e.g., those specific to the same morphological feature).

[0237]

[0279] Referring to Figure 1, in some embodiments, a first morphological marker is labeled with a first detectable portion (step 101). In some embodiments, step 101 is repeated multiple times (step 102) to label one or more morphological markers with one or more detectable portions (the one or more detectable portions may be the same or different).

[0238]

[0280] Next, the first biomarker is labeled with a second detectable portion (step 103), where at least the first and second detectable portions are different. In some embodiments, step 103 is repeated multiple times (step 104) to label one or more biomarkers with one or more detectable portions, where each of the one or more detectable portions is different from the others and also different from the detectable portions used to label one or more morphological markers. In some embodiments, steps 101, 102, 103, and 104 can also be repeated as needed (step 105). Subsequently, signals from at least the first and second detectable portions are detected (step 106).

[0239]

[0281] In some embodiments, step 103 or 104 is performed first, followed by steps 101 and 102. In other embodiments, steps 101 and 103 are performed sequentially, and then both steps 101 and 103 are repeated one or more additional times. In yet another embodiment, steps 101 and 103 are performed simultaneously.

[0240]

[0282] In some embodiments, the first and second detectable portions are selected such that they have different peak absorption wavelengths and do not substantially overlap (e.g., at least 20 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, at least 120 nm, at least 130 nm, at least 140 nm, at least 150 nm, at least 170 nm, at least 190 nm, at least 210 nm, at least 230 nm, at least 250 nm, at least 270 nm, at least 290 nm, at least 310 nm, etc.).

[0241]

[0283] In some embodiments, the first and second detectable portions have different peak absorption wavelengths, and the different peak absorption wavelengths of the first and second detectable portions are separated by at least 20 nm, and each of the first and second detectable portions has an FWHM of less than 200 nm, for example less than 160 nm, less than 130 nm, less than 100 nm, etc. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, and the different peak absorption wavelengths of the first and second detectable portions are separated by at least 30 nm, and each of the first and second detectable portions has an FWHM of less than 200 nm, for example less than 160 nm, less than 130 nm, less than 100 nm, etc. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, and the different peak absorption wavelengths of the first and second detectable portions are separated by at least 4 In some embodiments, the first and second detectable portions are separated by 0 nm, and each of the first and second detectable portions has an FWHM of less than 200 nm, for example, less than 160 nm, less than 130 nm, less than 100 nm, etc. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, and the different peak absorption wavelengths of the first and second detectable portions are separated by at least 50 nm, and each of the first and second detectable portions has an FWHM of less than 200 nm, for example, less than 160 nm, less than 130 nm, less than 100 nm, etc. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, and the different peak absorption wavelengths of the first and second detectable portions are separated by at least 70 nm, and each of the first and second detectable portions has an FWHM of less than 200 nm, for example, less than 160 nm, less than 130 nm, less than 100 nm, etc.

[0242]

[0284] In some embodiments, the first and second detectable portions have different peak absorption wavelengths, the different peak absorption wavelengths of the first and second detectable portions are separated by at least 20 nm, and each of the first and second detectable portions has an FWHM of less than 160 nm. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, the different peak absorption wavelengths of the first and second detectable portions are separated by at least 30 nm, and each of the first and second detectable portions has an FWHM of less than 160 nm. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, the different peak absorption wavelengths of the first and second detectable portions are separated by at least 40 nm, and each of the first and second detectable portions has an FWHM of less than 160 nm. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, the different peak absorption wavelengths of the first and second detectable portions are separated by at least 50 nm, and each of the first and second detectable portions has an FWHM of less than 160 nm. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, the different peak absorption wavelengths of the first and second detectable portions are separated by at least 70 nm, and each of the first and second detectable portions has an FWHM of less than 160 nm.

[0243]

[0285] In some embodiments, the first and second detectable portions have different peak absorption wavelengths, the different peak absorption wavelengths of the first and second detectable portions are separated by at least 20 nm, and each of the first and second detectable portions has an FWHM of less than 130 nm. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, the different peak absorption wavelengths of the first and second detectable portions are separated by at least 30 nm, and each of the first and second detectable portions has an FWHM of less than 130 nm. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, the different peak absorption wavelengths of the first and second detectable portions are separated by at least 40 nm, and each of the first and second detectable portions has an FWHM of less than 130 nm. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, the different peak absorption wavelengths of the first and second detectable portions are separated by at least 50 nm, and each of the first and second detectable portions has an FWHM of less than 130 nm. In some embodiments, the first and second detectable portions have different peak absorption wavelengths, the different peak absorption wavelengths of the first and second detectable portions are separated by at least 70 nm, and each of the first and second detectable portions has an FWHM of less than 130 nm.

[0244]

[0286] In some embodiments, the first and second detectable portions have different peak absorption wavelengths, the diff...

Claims

1. A method for detecting a biomarker in a biological sample in a morphological context, (a) Labeling at least a portion of a first ubiquitous morphological feature of a biological sample using a first detectable portion, wherein the absorption FWHM of the first detectable portion is less than 200 nm, and the labeling of the first ubiquitous morphological feature includes (i) contacting a first morphological marker specific to at least a portion of the first ubiquitous morphological feature with a first detection probe that binds to the first morphological marker, and (ii) covalently depositing the first detectable portion on or proximal to the first morphological marker; and (b) Labeling a first biomarker in a biological sample using a second detectable portion, wherein the absorption FWHM of the second detectable portion is less than 200 nm, the second detectable portion is different from the first detectable portion, and the labeling of the first biomarker comprises (i) contacting the first biomarker with a second detection probe that binds to the first biomarker, and (ii) covalently depositing the second detectable portion on or near the first biomarker. Methods that include...

2. The method according to claim 1, wherein the first morphological marker is selected from the group consisting of markers for cytosol, markers for nucleus, nuclear membrane markers, markers for nucleolus, markers for actin filaments, markers for centrosomes, markers for centriole satellites, markers for intermediate filaments, markers for microtubule structures, mitochondrial markers, markers for endoplasmic reticulum, Golgi apparatus markers, plasma membrane markers, and vesicle organelle markers.

3. The method according to claim 2, wherein the first morphological marker is a marker for the nucleus.

4. The method according to claim 3, wherein the marker for the nucleus comprises DNA.

5. The method of claim 4, wherein the DNA labeling using a first detectable portion comprises (a) contacting a biological sample with an anti-DNA primary antibody; (b) contacting a biological sample with an anti-species secondary antibody specific to the anti-DNA primary antibody, wherein the anti-species antibody is directly or indirectly conjugated to at least one enzyme; and (c) contacting the biological sample with a first detectable conjugate comprising (i) a first detectable portion and (ii) a tyramide portion, a quinone methide precursor portion, or a derivative or analog of the tyramide portion or the quinone methide precursor portion.

6. The method according to claim 3, wherein the marker for the nucleus comprises a histone protein.

7. The method of claim 6, wherein the histone protein labeling using a first detectable moiety comprises (a) contacting a biological sample with an anti-histone primary antibody; (b) contacting a biological sample with an anti-species secondary antibody specific to the anti-histone primary antibody, wherein the anti-species antibody is directly or indirectly conjugated to at least one enzyme; and (c) contacting the biological sample with a first detectable conjugate comprising (i) a first detectable moiety and (ii) a tyramide moiety, a quinone methide precursor moiety, or a derivative or analog of the tyramide moiety or a quinone methide precursor moiety.

8. The method according to claim 1, wherein the first and second detectable portions are each independently conjugated with a tyramide or a derivative thereof, a quinone methide precursor portion or a derivative thereof, or a reactive functional group capable of participating in a click chemistry reaction; and the covalent deposition of the first and second detectable portions independently comprises one of tyramide signal amplification, quinone methide chemistry, or click chemistry.

9. The method according to claim 1, wherein the maximum absorption wavelength (λ max) of the first detectable portion and the maximum absorption wavelength (λ max) of the second detectable portion are separated by at least about 20 nm.

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