Synthesis of Schiff reagents on glass slides

Abstract

The present disclosure relates to compositions, kits and methods for the synthesis of Schiff reagents in situ, i.e. Directly, on a substrate, such as within droplets disposed on a surface of a substrate. In some embodiments, the kit comprises at least a dye composition and a sulfite composition. In some embodiments, the dye composition includes a compound having a 4-dibenzylidene-2, 5-cyclohexadiene-1-imine core that is functionalized to include at least two substituted or unsubstituted amine groups, where the compound has a molecular weight in a range between about 300 g / mol and about 600 g / mol. In some embodiments, the dye composition optionally comprises an acid. In some embodiments, the sulfite composition comprises a sulfite or a source of SO2.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims the benefit of U.S. Provisional Patent Application No. 63 / 366,091, filed on June 9, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to compositions and methods for staining biological specimens. Background Art

[0004] The periodic acid Schiff assay ("PAS") is a technique originally described by McManus in 1946 that visualizes mucins, glycogen, basement membranes, and fungal organisms by combining periodic acid oxidation of polysaccharides and Schiff's reagent staining. PAS is a general carbohydrate stain that can be used to detect structures containing a high proportion of carbohydrate macromolecules, including polysaccharides. The main polysaccharide identified by histological staining of human tissue sections is glycogen. PAS staining allows microscopic visualization and assessment of glycogen levels in tissues. Disruption of the metabolic pathway that converts glucose to glycogen and vice versa may lead to glycogen accumulation. PAS staining is also used to examine basement membranes and is typically used to examine glomerular basement membranes in renal biopsies suspected of having glomerular disease. PAS stains can also be used to detect glycogen present in fungal cell walls to aid in the diagnosis of fungal infections. The glycogen content of the fungal cell walls of many fungi is similar; therefore, PAS staining is a reliable method to confirm or exclude the possibility of fungal infection.

[0005] The PAS assay is performed by reacting an analyte (e.g., a histological or cytological sample) with a predetermined amount of a Schiff's reagent (such as a commercially available Schiff's reagent). The Schiff's reagent can be derived from a single dye or a mixture of dyes, such as pararosaniline, rosaniline, carmine IP, and new carmine (see Figure 1 ).

[0006] Solutions containing Schiff's reagent will chemically combine with the aldehydes in the sample to form bright red, purple, or magenta products. Figure 2A As shown, the bisulfite molecule reacts with the pararosaniline dye to give a decolorized adduct with the central carbon sulfonated (see Figure 2B). The free and uncharged amine group belonging to the aromatic ring reacts with the aldehyde group to form an aldimine. The aldimine group is electrophilic and therefore reacts further with the bisulfite ion. Finally, a red, purple or magenta bisulfite adduct is formed. Histologically, the aldehyde is either attached to the tissue structure or produced by it. As a result, the tissue structure itself turns bright red. The mechanism is the same for all aldehydes in the tissue. The aldehyde condenses with the Schiff reagent to form a new compound that is attached to the tissue. In the process, the chromophore is reformed and the color is produced.

[0007] Schiff's reagent is an unstable reagent (see Figure 3 As described in 1960 [Kasten, FH (1960): The Chemistry of Schiff Reagent, Intl. Rev. Cytol. 10: 1-100.], the stability of Schiff's reagent ranges from "a few hours to six months or more, depending on the composition and storage method." Due to this instability, the storage and handling of Schiff's reagent are limited, and the staining quality of current PAS assays can change as the Schiff's reagent ages. In fact, it has been shown that once a vial of Schiff's reagent is opened, for example, one month after the vial was opened, the staining quality deteriorates over time. In some embodiments, vials of commercially available Schiff's reagent (opened and unopened) contain crystalline precipitates (see Figure 4 ), which in some cases may lead to false positives when used for staining, especially when detecting fungi in biological samples. It is desirable to use PAS Schiff reagent without crystalline precipitates to stain histological and / or cytological samples. Summary of the invention

[0008] Applicants have developed compositions, kits and methods for synthesizing Schiff's reagent in situ, i.e., directly on a substrate (e.g., a biological sample or a microscope slide), such as in a droplet disposed on the surface of a substrate. The phrase "Schiff's reagent synthesized in situ" and the like refers to a dye or dye mixture having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core and a sulfite or SO 2The in situ synthesis of Schiff's reagent can obtain a consistent dyeing comparable to the dyeing achieved using a commercially available Schiff's reagent while allowing for long-term stability of the reagent (e.g., stability of at least 1 year or at least 2 years). In addition, the in situ synthesis of Schiff's reagent prevents the formation of a precipitate, and thus avoids the precipitate being applied or dispensed to a substrate and / or a droplet disposed on the substrate. In addition, the in situ synthesis of Schiff's reagent allows "adjusting" the dyeing intensity by changing the components (or concentrations of components, such as dye concentration, acid concentration, and / or sulfite source concentration) used in the in situ synthesis and / or the reaction conditions (time, temperature) of the synthesis. These and other advantages are as described herein.

[0009] A first aspect of the present disclosure is a dye composition comprising a compound having any one of formula (I) to formula (XII):

[0010]

[0011]

[0012]

[0013] in

[0014] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl or cycloalkyl groups, -OH, -OC 1 -C 6 Alkyl or -N(R x )(R y ), where R x and R y is independently H or a branched or unbranched C optionally substituted with one or more halogen atoms 1 -C 6 An alkyl group, or one in which R 1 and R 2 Together, or R 3 and R 4 Together or R 5 or R 6 together to form optionally one or more C 1 -C 6 Alkyl groups or one or more -N(R x )(R y ) group substituted 5-membered or 6-membered ring or heterocyclic ring; and

[0015] A is a counterion; and

[0016] Wherein the concentration of the compound having any one of Formula (I) to Formula (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition.

[0017] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 6 an alkyl group or a cycloalkyl group; and wherein R x or R y At least one of is H. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 6 and R 6 At least two of them are -N(R x )(R y ), and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are C 1 -C 2 An alkyl group; and wherein R x or R y At least one of them is H.

[0018] In some embodiments, the compound having any one of Formula (I)-Formula (XII) is selected from the group consisting of:

[0019]

[0020] In certain embodiments, the compound with any one of formula (I) to formula (XII) has a molecular weight in the range between about 300g / mol to about 600g / mol. In certain embodiments, the compound with any one of formula (I) to formula (XII) has a molecular weight in the range between about 300g / mol to about 425g / mol. In certain embodiments, by the total volume of dye composition, concentration is in the range between about 0.15%w / v to about 2.5%w / v. In certain embodiments, by the total volume of dye composition, concentration is in the range between about 0.15%w / v to about 1.8%w / v.

[0021] In certain embodiments, the dye composition further comprises acid. In certain embodiments, acid is a strong acid. In certain embodiments, the strong acid is about 1M to about 1.3M hydrochloric acid. In certain embodiments, the strong acid is about 1M to about 1.3M nitric acid. In certain embodiments, the dye composition further comprises at least one of a solvent and / or an additive. In certain embodiments, the dye composition further comprises a solvent and at least one additive. In certain embodiments, the dye composition is substantially composed of a compound and a strong acid with any one of formula (I) to (XII).

[0022] A second aspect of the present disclosure is a sulfite composition comprising a sulfite or SO selected from the group consisting of: 2 Source: pyrosulfite, bisulfite, dithionite, thiosulfate, sulfite and sulfurous acid; of which sulfite or SO is based on the total weight of the sulfite composition. 2 The concentration of the source is in the range of about 0.1% w / v to about 18% w / v. In some embodiments, the concentration is in the range of about 1% w / v to about 9% w / v, based on the total weight of the sulfite composition. In some embodiments, the concentration is in the range of about 1% w / v to about 4% w / v, based on the total weight of the sulfite composition. In some embodiments, the sulfite or SO 2 The source is not thiosulfate. In some embodiments, the sulfite composition further comprises an acid.

[0023] A third aspect of the present disclosure is a kit comprising (i) a dye composition; and (ii) a sulfite composition; wherein the dye composition comprises a compound having any one of formula (I) to formula (XII):

[0024]

[0025]

[0026]

[0027] in

[0028] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl or cycloalkyl groups, -OH, -OC 1 -C 6 Alkyl or -N(R x )(R y ), where R x and R y is independently H or a branched or unbranched C optionally substituted with one or more halogen atoms 1 -C 6 An alkyl group, or one in which R 1 and R 2 Together, or R 3 and R 4 Together or R 5 or R 6 together to form optionally one or more C 1 -C 6 Alkyl groups or one or more -N(R x )(R y ) group substituted 5-membered or 6-membered ring or heterocyclic ring; and

[0029] A is a counterion; and

[0030] wherein the concentration of the compound of any one of formula (I) to formula (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition; and

[0031] wherein the sulfite composition comprises a sulfite or SO selected from the group consisting of 2 Source: pyrosulfite, bisulfite, dithionite, thiosulfate, sulfite and sulfurous acid; of which sulfite or SO is based on the total weight of the sulfite composition. 2 The concentration of the source ranges from about 0.1% w / v to about 18% w / v.

[0032] A fourth aspect of the present disclosure is a kit comprising (i) a dye composition that does not contain an acid; (ii) a sulfite composition; and (iii) an acid, wherein the dye composition comprises a compound having any one of formula (I) to formula (XII):

[0033]

[0034]

[0035]

[0036] in

[0037] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl or cycloalkyl groups, -OH, -OC 1 -C 6 Alkyl or -N(R x )(R y ), where R x and R y is independently H or a branched or unbranched C optionally substituted with one or more halogen atoms 1 -C 6 An alkyl group, or one in which R 1 and R 2 Together, or R 3 and R 4 Together or R 5 or R 6 together to form optionally one or more C 1 -C 6 Alkyl groups or one or more -N(R x )(R y ) group substituted 5-membered or 6-membered ring or heterocyclic ring; and

[0038] A is a counterion; and

[0039] wherein the concentration of the compound of any one of formula (I) to formula (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition; and

[0040] wherein the sulfite composition comprises a sulfite or SO selected from the group consisting of 2 Source: pyrosulfite, bisulfite, dithionite, thiosulfate, sulfite and sulfurous acid; of which sulfite or SO is based on the total weight of the sulfite composition. 2 The concentration of the source ranges from about 0.1% w / v to about 18% w / v.

[0041] In some embodiments, R1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 6 an alkyl group or a cycloalkyl group; and wherein R x or R y At least one of is H. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are C 1 -C 2 alkyl group; and wherein R x or R y At least one of them is H.

[0042] In some embodiments, the compound having any one of Formula (I)-Formula (XII) is selected from the group consisting of:

[0043]

[0044] In certain embodiments, the compound with any one of formula (I) to formula (XII) has a molecular weight in the range between about 300g / mol to about 600g / mol. In certain embodiments, the compound with any one of formula (I) to formula (XII) has a molecular weight in the range between about 300g / mol to about 425g / mol. In certain embodiments, by the total volume of dye composition, concentration is in the range between about 0.15%w / v to about 2.5%w / v. In certain embodiments, by the total volume of dye composition, concentration is in the range between about 0.15%w / v to about 1.8%w / v.

[0045] In some embodiments, the sulfite composition comprises a sulfite or SO selected from the group consisting of: 2 Source: pyrosulfite, bisulfite, dithionite, thiosulfate, sulfite and sulfurous acid; of which sulfite or SO is based on the total weight of the sulfite composition. 2 The concentration of the source is in the range of about 0.1% w / v to about 18% w / v. In some embodiments, the concentration is in the range of about 1% w / v to about 9% w / v based on the total weight of the sulfite composition. In some embodiments, the concentration is in the range of about 1% w / v to about 4% w / v based on the total weight of the sulfite composition.

[0046] In some embodiments, the acid is a strong acid. In some embodiments, the acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, hydrofluoric acid, trifluoroacetic acid, hydroiodic acid, sulfuric acid, nitric acid, chloric acid, and perchloric acid.

[0047] A fifth aspect of the present disclosure is a method for staining a biological sample, the method comprising sequentially dispensing a dye composition and a sulfite composition to the biological sample; wherein the dye composition comprises a compound having any one of formula (I) to formula (XII):

[0048]

[0049]

[0050]

[0051] in

[0052] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl or cycloalkyl groups, -OH, -OC 1 -C 6 Alkyl or -N(R x )(R y ), where R x and R y is independently H or a branched or unbranched C optionally substituted with one or more halogen atoms 1 -C 6 An alkyl group, or one in which R 1 and R 2 Together, or R3 and R 4 Together or R 5 or R 6 together to form optionally one or more C 1 -C 6 Alkyl groups or one or more -N(R x )(R y ) group substituted 5-membered or 6-membered ring or heterocyclic ring; and

[0053] A is a counterion; and

[0054] wherein the concentration of the compound of any one of formula (I) to formula (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition; and

[0055] wherein the sulfite composition comprises a sulfite or SO selected from the group consisting of 2 Source: pyrosulfite, bisulfite, dithionite, thiosulfate, sulfite and sulfurous acid; of which sulfite or SO is based on the total weight of the sulfite composition. 2 The concentration of the source ranges from about 0.1% w / v to about 18% w / v.

[0056] In certain embodiments, the dye composition further comprises an acid. In certain embodiments, the acid is a strong acid. In certain embodiments, the dye composition further comprises hydrochloric acid. In certain embodiments, the dye composition further comprises nitric acid.

[0057] In some embodiments, the dye composition is dispensed before the sulfite composition. In other embodiments, the dye composition is dispensed after the sulfite composition is dispensed. In some embodiments, the temperature of the sample, the dye composition and / or the sulfite composition is in the range of 30°C to about 50°C. In some embodiments, the temperature of the sample, the dye composition and / or the sulfite composition is in the range of 30°C to about 40°C. In some embodiments, the sequentially dispensed dye composition and the sulfite composition are allowed to remain in contact with the sample for at least 4 minutes. In some embodiments, the sequentially dispensed dye composition and the sulfite composition are allowed to remain in contact with the sample for at least 8 minutes.

[0058] In some embodiments, the amount of the dye composition assigned to the sample is in the range of μ between about 50 μL and about 200 μL; and the amount of the sulfite composition assigned to the sample is in the range of about 50 μL to about 200 μL. In some embodiments, the amount of the dye composition assigned to the sample is in the range of about 100 μL to about 200 μL; and the amount of the sulfite composition assigned to the sample is in the range of about 100 μL to about 200 μL. In some embodiments, the amount of the dye composition assigned to the sample is in the range of about 100 μL to about 150 μL; and the amount of the sulfite composition assigned to the sample is in the range of about 100 μL to about 150 μL.

[0059] In certain embodiments, before distributing dye composition and / or sulfite composition, the sample is pretreated for a predetermined amount of time with a reagent that forms an aldehyde group. In certain embodiments, the sample is pretreated for a predetermined amount of time with periodic acid (e.g., 1 gram of periodic acid in 100 mL of water), e.g., between about 1 minute and about 10 minutes.

[0060] A sixth aspect of the present disclosure is a method for staining a biological sample, the method comprising simultaneously dispensing a dye composition and a sulfite composition to the biological sample; wherein the dye composition comprises a compound having any one of formulas (I) to (XII):

[0061]

[0062]

[0063]

[0064] in

[0065] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl or cycloalkyl groups, -OH, -OC 1 -C 6 Alkyl or -N(R x )(R y ), where R x and R y is independently H or a branched or unbranched C optionally substituted with one or more halogen atoms 1 -C 6 An alkyl group, or one in which R1 and R 2 Together, or R 3 and R 4 Together or R 5 or R 6 together to form optionally one or more C 1 -C 6 Alkyl groups or one or more -N(R x )(R y ) group substituted 5-membered or 6-membered ring or heterocyclic ring; and

[0066] A is a counterion; and

[0067] wherein the concentration of the compound of any one of formula (I) to formula (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition; and

[0068] wherein the sulfite composition comprises a sulfite or SO selected from the group consisting of 2 Source: pyrosulfite, bisulfite, dithionite, thiosulfate, sulfite and sulfurous acid; of which sulfite or SO is based on the total weight of the sulfite composition. 2 The concentration of the source ranges from about 0.1% w / v to about 18% w / v.

[0069] In some embodiments, the temperature of the sample, dye composition and / or sulfite composition is in the range of 30°C to about 50°C. In some embodiments, the temperature of the sample, dye composition and / or sulfite composition is in the range of 30°C to about 40°C. In some embodiments, the dye composition and sulfite composition dispensed simultaneously are allowed to remain in contact with the sample for at least 4 minutes. In some embodiments, the dye composition and sulfite composition dispensed simultaneously are allowed to remain in contact with the sample for at least 8 minutes.

[0070] In some embodiments, the amount of the dye composition assigned to the sample is in the range of about 50 μL to about 200 μL; and the amount of the sulfite composition assigned to the sample is in the range of about 50 μL to about 200 μL. In some embodiments, the amount of the dye composition assigned to the sample is in the range of about 100 μL to about 200 μL; and the amount of the sulfite composition assigned to the sample is in the range of about 100 μL to about 200 μL. In some embodiments, the amount of the dye composition assigned to the sample is in the range of about 100 μL to about 150 μL; and the amount of the sulfite composition assigned to the sample is in the range of about 100 μL to about 150 μL.

[0071] In certain embodiments, before distributing dye composition and / or sulfite composition, the sample is pretreated for a predetermined amount of time with a reagent that forms an aldehyde group. In certain embodiments, the sample is pretreated for a predetermined amount of time with periodic acid (e.g., 1 gram of periodic acid in 100 mL of water), e.g., between about 1 minute and about 10 minutes.

[0072] A seventh aspect of the present disclosure is a method for staining a biological sample, the method comprising sequentially dispensing a dye composition, a sulfite composition, and an acid to the biological sample; wherein the dye composition comprises a compound having any one of formula (I) to formula (XII):

[0073]

[0074]

[0075]

[0076] in

[0077] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl or cycloalkyl groups, -OH, -OC 1 -C 6 Alkyl or -N(R x )(R y ), where R x and R y is independently H or a branched or unbranched C optionally substituted with one or more halogen atoms 1 -C 6 An alkyl group, or one in which R 1 and R 2 Together, or R 3 and R 4 Together or R 5 or R 6 together to form optionally one or more C 1 -C 6 Alkyl groups or one or more -N(R x )(R y ) group substituted 5-membered or 6-membered ring or heterocyclic ring; and

[0078] A is a counterion; and

[0079] wherein the concentration of the compound of any one of formula (I) to formula (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition; and

[0080] wherein the sulfite composition comprises a sulfite or SO selected from the group consisting of 2 Source: pyrosulfite, bisulfite, dithionite, thiosulfate, sulfite and sulfurous acid; of which sulfite or SO is based on the total weight of the sulfite composition. 2 The concentration of the source ranges from about 0.1% w / v to about 18% w / v.

[0081] In some embodiments, the dye composition is distributed before distributing the sulfite composition and / or the acid. In some embodiments, the temperature of the sample, the dye composition, the acid and / or the sulfite composition is in the range of 30°C to about 50°C. In some embodiments, the temperature of the sample, the dye composition, the acid and / or the sulfite composition is in the range of 30°C to about 40°C. In some embodiments, the sequentially distributed dye composition, the acid and the sulfite composition are allowed to remain in contact with the sample for at least 4 minutes. In some embodiments, the sequentially distributed dye composition, the acid and the sulfite composition are allowed to remain in contact with the sample for at least 8 minutes.

[0082] In some embodiments, the amount of the dye composition assigned to the sample is in the range of about 50 μL to about 200 μL; wherein the amount of the acid assigned to the sample is in the range of about 50 μL to about 200 μL; and wherein the amount of the sulfite composition assigned to the sample is in the range of about 50 μL to about 200 μL. In some embodiments, the amount of the dye composition assigned to the sample is in the range of about 100 μL to about 200 μL; wherein the amount of the acid assigned to the sample is in the range of about 100 μL to about 200 μL; and wherein the amount of the sulfite composition assigned to the sample is in the range of about 100 μL to about 200 μL. In some embodiments, the amount of the dye composition assigned to the sample is in the range of about 100 μL to about 150 μL; wherein the amount of the acid assigned to the sample is in the range of about 100 μL to about 150 μL; and wherein the amount of the sulfite composition assigned to the sample is in the range of about 100 μL to about 150 μL.

[0083] In some circumstances, the acid is a strong acid. In some embodiments, the strong acid is hydrochloric acid. In some embodiments, the strong acid is nitric acid.

[0084] In certain embodiments, before distributing dye composition, acid and / or sulfite composition, the sample is pre-treated for a predetermined amount of time with a reagent that forms an aldehyde group. In certain embodiments, the sample is pre-treated for a predetermined amount of time with periodic acid (e.g., 1 gram of periodic acid in 100 mL of water), e.g., between about 1 minute and about 10 minutes.

[0085] An eighth aspect of the present disclosure is a method for staining a biological sample, the method comprising simultaneously dispensing a dye composition, a sulfite composition, and an acid to the biological sample; wherein the dye composition comprises a compound having any one of formula (I) to formula (XII):

[0086]

[0087]

[0088]

[0089] in

[0090] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl or cycloalkyl groups, -OH, -OC 1 -C 6 Alkyl or -N(R x )(R y ), where R x and R y is independently H or a branched or unbranched C optionally substituted with one or more halogen atoms 1 -C 6 An alkyl group, or one in which R 1 and R 2 Together, or R 3 and R 4 Together or R 5 or R 6 together to form optionally one or more C 1 -C 6 Alkyl groups or one or more -N(R x )(R y ) group substituted 5-membered or 6-membered ring or heterocyclic ring; and

[0091] A is a counterion; and

[0092] wherein the concentration of the compound of any one of formula (I) to formula (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition; and

[0093] wherein the sulfite composition comprises a sulfite or SO selected from the group consisting of 2 Source: pyrosulfite, bisulfite, dithionite, thiosulfate, sulfite and sulfurous acid; of which sulfite or SO is based on the total weight of the sulfite composition. 2 The concentration of the source ranges from about 0.1% w / v to about 18% w / v.

[0094] In some embodiments, the temperature of the sample, dye composition, acid and / or sulfite composition is in the range of 30°C to about 50°C. In some embodiments, the temperature of the sample, dye composition, acid and / or sulfite composition is in the range of 30°C to about 40°C. In some embodiments, the dye composition, acid and sulfite composition dispensed simultaneously are allowed to remain in contact with the sample for at least 4 minutes. In some embodiments, the dye composition, acid and sulfite composition dispensed simultaneously are allowed to remain in contact with the sample for at least 8 minutes.

[0095] In some embodiments, the amount of the dye composition assigned to the sample is in the range of about 50 μL to about 200 μL; wherein the amount of the acid assigned to the sample is in the range of about 50 μL to about 200 μL; and wherein the amount of the sulfite composition assigned to the sample is in the range of about 50 μL to about 200 μL. In some embodiments, the amount of the dye composition assigned to the sample is in the range of about 100 μL to about 200 μL; wherein the amount of the acid assigned to the sample is in the range of about 100 μL to about 200 μL; and wherein the amount of the sulfite composition assigned to the sample is in the range of about 100 μL to about 200 μL. In some embodiments, the amount of the dye composition assigned to the sample is in the range of about 100 μL to about 150 μL; wherein the amount of the acid assigned to the sample is in the range of about 100 μL to about 150 μL; and wherein the amount of the sulfite composition assigned to the sample is in the range of about 100 μL to about 150 μL.

[0096] In some circumstances, the acid is a strong acid. In some embodiments, the strong acid is hydrochloric acid. In some embodiments, the strong acid is nitric acid.

[0097] In certain embodiments, before distributing dye composition, acid and / or sulfite composition, the sample is pre-treated for a predetermined amount of time with a reagent that forms an aldehyde group. In certain embodiments, the sample is pre-treated for a predetermined amount of time with periodic acid (e.g., 1 gram of periodic acid in 100 mL of water), e.g., between about 1 minute and about 10 minutes.

[0098] A ninth aspect of the present disclosure is a stained biological sample prepared by: (i) dispensing a dye composition into the biological sample, the dye composition comprising (a) a compound having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core functionalized to include at least two substituted or unsubstituted amine groups, wherein the compound has a molecular weight ranging from about 300 g / mol to about 600 g / mol; and (b) an acid; and wherein the concentration of the compound ranges from about 0.15% w / v to about 2.5% w / v based on the total volume of the dye composition; and (ii) dispensing a dye composition comprising sulfite or SO 2 A sulfite composition derived from a source, wherein the sulfite or SO is present in an amount of 1% by weight based on the total weight of the sulfite composition. 2 The concentration of the source ranges from about 0.1% w / v to about 18% w / v. In some embodiments, the dye composition is first dispensed into the biological sample. In some embodiments, the dye composition and the sulfite composition are dispensed simultaneously. In some embodiments, the acid is a strong acid.

[0099] In certain embodiments, before distributing dye composition and / or sulfite composition, the sample is pretreated for a predetermined amount of time with a reagent that forms an aldehyde group. In certain embodiments, the sample is pretreated for a predetermined amount of time with periodic acid (e.g., 1 gram of periodic acid in 100 mL of water), e.g., between about 1 minute and about 10 minutes.

[0100] In some embodiments, the acid is hydrochloric acid or nitric acid. In some embodiments, the compound is selected from the group consisting of:

[0101] BRIEF DESCRIPTION OF THE DRAWINGS

[0102] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided to the Office upon request and payment of the necessary fee.

[0103] Figure 1 Depicts the possibility of reacting with sulfite or SO 2 Source various dyes that react to form Schiff's reagents.

[0104] Figure 2AThe reaction between bisulfite and pararosaniline to provide a decolorized adduct is demonstrated.

[0105] Figure 2B Decolorization of Schiff's reagent is shown.

[0106] Figure 3 The stability of Schiff's reagent derived from pararosaniline when exposed to oxidizing agents is demonstrated.

[0107] Figure 4 Depicted is a tissue sample stained with PAS Schiff's reagent, wherein a crystalline precipitate formed during storage of the PAS Schiff's reagent is deposited onto the tissue sample.

[0108] Figure 5 The overall method for the in situ preparation of Schiff reagent is depicted.

[0109] Fig. 6A and Figure 6B Staining of tissue samples using in situ synthesized Schiff's reagent prepared from pararosaniline dye stored at 2°C-8°C or 60°C for two weeks is demonstrated.

[0110] Figure 6C and Fig.6D Shown is the staining of a tissue sample using the commercial Schiff's reagent, which is new ( Figure 6C ) and has been open for two weeks ( Fig.6D ).

[0111] Fig. 7A and Figure 7B Shown are the staining intensity and contrast using the in situ synthesized Schiff's reagent as a function of reaction time.

[0112] Figure 7C Control samples stained with commercial Schiff's reagent are shown.

[0113] Fig. 8A and 8B The results show that the dye and sulfite / SO 2 Source concentration varies the staining intensity and contrast using in situ synthesized Schiff reagent.

[0114] Figure 8C Control samples stained with commercial Schiff's reagent are shown. DETAILED DESCRIPTION

[0115] The present disclosure provides compositions, kits and methods for staining biological samples, such as biological samples disposed on microscope slides. Specifically, the present disclosure provides in situ synthesized Schiff reagents that facilitate staining of biological samples in a manner equivalent to staining using commercially available Schiff reagents.

[0116] definition

[0117] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are presented unless indicated to the contrary.

[0118] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" is defined as inclusive, such as "comprising A or B" means comprising A, B, or A and B.

[0119] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including at least one element in a number of elements or a list of elements, but also including more than one element, and optionally including additional unlisted items. Only terms indicating the contrary, such as "only one of" or "exactly one of", or "consisting of..." used in the claims, will refer to the inclusion of exactly one element in a number of elements or a list of elements. In general, the term "or" used herein should be interpreted as indicating an exclusive alternative (i.e., "one or the other, but not both") only when it is preceded by an exclusive term such as "or", "one of", "only one", or "exactly one". "Substantially consisting of..." used in the claims should have the ordinary meaning used in the field of patent law.

[0120] As used herein, the terms "including", "comprising", "having", etc. are used interchangeably and have the same meaning. Similarly, "including", "comprising", "having", etc. are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of "including" in general U.S. patent law, so each term can be understood as an open term, which means "at least the following", and can also be interpreted as not excluding additional features, limitations, aspects, etc. Therefore, for example, "a device having components a, b and c" means that the device includes at least components a, b and c. Similarly, the phrase: "a method involving steps a, b and c" means that the method includes at least steps a, b and c. In addition, although the steps and processes may be outlined in a particular order herein, those skilled in the art will recognize that the order steps and processes may vary.

[0121] As used herein in the specification and claims, with respect to a list of one or more elements, the phrase "at least one" should be understood as at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, nor does it exclude any combination of elements in the list of elements. In addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers, the definition also allows other elements to be optionally present, whether or not they are related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") can refer to at least one, optionally including more than one A, without B (and optionally including elements other than B) in one embodiment; in another embodiment, at least one, optionally including more than one B, without A (and optionally including elements other than A); in yet another embodiment, at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0122] References throughout this specification to "one embodiment," "an embodiment," etc., mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in different places in this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0123] As used herein, unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to an unbranched (i.e., non-branched) or branched chain, or combinations thereof, which may be fully saturated, monounsaturated, or polyunsaturated, and may include groups having a specified number of carbon atoms (i.e., C 1 -C 10 "Alkyl" is not cyclized. Examples of saturated hydrocarbon groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, (cyclohexyl) methyl, such as homologues and isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups are alkyl groups with one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-butadienyl, 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl and higher homologues and isomers. Alkoxy is an alkyl group that is connected to the rest of the molecule by an oxygen atom (-O-).

[0124] As used herein, the term "analog" or "derivative" is used according to its ordinary meaning in the fields of chemistry and biology, and refers to a compound that is structurally similar to another compound (the so-called "reference" compound) but has a different composition (e.g., an atom is replaced by an atom of a different element, or a specific functional group is present, or a functional group is replaced by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound is different). Thus, an analog is a compound that is similar or comparable in function and appearance to a reference compound but has a different structure or origin.

[0125] As used herein, the term "antibody", sometimes abbreviated as "Ab", refers to immunoglobulins or immunoglobulin-like molecules, including (for example) but not limited to: IgA, IgD, IgE, IgG and IgM and combinations thereof; similar molecules produced during the immune response of any vertebrate (for example, in mammals such as humans, goats, rabbits and mice, etc.); and antibody fragments that specifically bind to a target molecule (or a group of highly similar target molecules) and substantially exclude binding to other molecules. "Antibody" further refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region, which specifically recognizes and binds to an epitope of an antigen. An antibody can be composed of a heavy chain and a light chain, each of which has a variable region, referred to as a variable heavy chain (VH) region and a variable light chain (VL) region. The VH region and the VL region are jointly responsible for binding to the antigen recognized by the antibody. The term "antibody" also includes complete immunoglobulins and variants and portions thereof well known in the art.

[0126] As used herein, the term "antigen" refers to a compound, composition or substance that can be specifically bound by a product of specific humoral or cellular immunity (such as an antibody molecule or a T cell receptor). Antigens can be any type of molecule, including, for example, haptens, simple intermediate metabolites, sugars (such as oligosaccharides), lipids and hormones, and macromolecules (such as complex carbohydrates (such as polysaccharides), phospholipids, nucleic acids and proteins).

[0127] As used herein, the term "biological sample" may be any solid or fluid sample obtained from, excreted or secreted by any living organism including, but not limited to, unicellular organisms (such as bacteria, yeast, protozoa and amoeba, etc.), multicellular organisms (such as plants or animals), including samples from healthy or apparently healthy human subjects or human patients affected by a condition or disease to be diagnosed or studied, 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 body, or any body secretion, transudate, exudate (e.g., fluid obtained from an abscess or any other infection or inflammation site) or fluid obtained from a joint (e.g., a normal joint or a joint affected by a disease). A biological sample may also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy), or may include cells (whether primary cells or cultured cells) or a culture medium conditioned by any cell, tissue or organ. In some instances, the biological sample is a nuclear extract. In some instances, the sample is a quality control sample, such as one of the disclosed cell pellet slice samples. In other instances, the sample is a test sample. The sample can be prepared using any method known to those of ordinary skill in the art. The sample can be obtained from a subject undergoing routine screening, or from a subject suspected of having a condition, such as a genetic abnormality, infection, or neoplasia. The embodiments of the methods disclosed in the present invention can also be applied to samples without genetic abnormalities, diseases, conditions, etc., which are referred to as "normal" samples. The sample may include a plurality of targets that can be specifically bound by one or more detection probes.

[0128] As used herein, C a To C b (where "a" and "b" are integers) refers to the number of carbon atoms in an alkyl, alkenyl, or alkynyl group, or the number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl, cycloalkynyl, or aryl group, or the total number of carbon atoms and heteroatoms in a heteroalkyl, heterocyclyl, heteroaryl, or heteroalicyclic group. That is, the ring of an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, or heteroalicyclic group may contain from "a" to "b" (inclusive) carbon atoms. Thus, for example, "C 1 To C 4 "Alkyl" groups refer to all alkyl groups having 1 to 4 carbons, i.e., CH 3 -、CH 3 CH 2 -、CH 3 CH 2 CH 2 -、(CH 3 ) 2 CH-、CH 3 CH 2 CH2 CH 2 , CH 3 CH 2 CH(CH 3 )- and (CH 3 ) 3 C-. If "a" and "b" are not specified for an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, or heteroalicyclic group, the broadest range described in these definitions is assumed.

[0129] As used herein, terms similar to "cycloalkyl" (e.g., cycloalkyl groups) refer to fully saturated (no double or triple bonds) monocyclic or polycyclic hydrocarbon ring systems. When composed of two or more rings, the rings can be connected together in a fused manner. The cycloalkyl group may contain 3 to 10 atoms in one or more rings, or 3 to 8 atoms in one or more rings. The cycloalkyl group may be unsubstituted or substituted. Typical cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[0130] As used herein, the term "halogen atom" or "halogen" means any of the radioactive stable atoms of the 7th main group of the periodic table of elements, such as fluorine, chlorine, bromine and iodine.

[0131] As used herein, the term "heteroatom" refers to boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and silicon (Si). In some embodiments, a "heterocycle" may contain one or more heteroatoms. In other embodiments, an aliphatic group may contain one or more heteroatoms or be substituted with one or more heteroatoms.

[0132] As used herein, the term "reactive group" or "reactive functional group" refers to a functional group that is capable of chemically associating, interacting, hybridizing, hydrogen bonding, or coupling with a functional group of a different moiety. In some embodiments, a "reaction" between two reactive groups or two reactive functional groups may mean the formation of a covalent bond between the two reactive groups or two reactive functional groups; or may mean that the two reactive groups or two reactive functional groups associate with each other, interact with each other, hybridize with each other, hydrogen bond with each other, etc. Thus, in some embodiments, a "reaction" includes a binding event, such as the binding of a hapten to an anti-hapten antibody or a guest molecule associated with a supramolecular host molecule.

[0133] As used herein, the term "specific binding entity" refers to a member of a specific binding pair. A specific binding pair is characterized by binding to each other to substantially exclude a pair of molecules that bind to other molecules (e.g., the binding constant of a specific binding pair can be at least 10-3M, 10-4M, or 10-5M greater than the binding constant of any one of the two members of the binding pair of other molecules in a biological sample). Specific examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, avidin (such as streptavidin) and protein A). Specific binding moieties may also include molecules (or portions thereof) that are specifically bound by such specific binding proteins.

[0134] As used herein, the term "stain", "staining" or similar terms generally refers to any treatment of a biological specimen that detects and / or distinguishes the presence, location and / or amount (such as concentration) of a specific molecule (such as a lipid, protein or nucleic acid) or a specific structure (such as a normal or malignant cell, cytoplasm, nucleus, Golgi apparatus or cytoskeleton) in the biological specimen. For example, staining can compare a specific molecule or a specific cell structure of a biological specimen with the surrounding part, and the intensity of the staining can determine the amount of the specific molecule in the specimen. Staining can be used not only with a bright field microscope, but also with other observation tools such as phase contrast microscopes, electron microscopes and fluorescence microscopes to assist in the observation of molecules, cell structures and organisms. Some staining performed by system 2 can make the outlines of cells clearly visible. Other staining performed by the system 2 can rely on specific cell components (such as molecules or structures) that are stained and do not stain other cell components or stain other cell components relatively little. Examples of various staining methods performed by the system 2 include, but are not limited to, histochemical methods, immunohistochemical methods, and other methods based on molecular reactions such as hybridization reactions between nucleic acid molecules (including non-covalent binding interactions). Specific staining methods include, but are not limited to, primary staining methods (such as H&E staining, cervical staining, etc.), enzyme-linked immunohistochemical methods, and in situ RNA and DNA hybridization methods, such as fluorescence in situ hybridization (FISH).

[0135] Whenever a group or moiety is described as "substituted" or "optionally substituted" (or "optionally having" or "optionally containing"), the group may be unsubstituted or substituted with one or more of the indicated substituents. Likewise, when a group is described as "substituted or unsubstituted", if substituted, the one or more substituents may be selected from one or more of the indicated substituents. If no substituents are indicated, it means that the indicated "optionally substituted" or "substituted" group may be substituted with one or more groups individually and independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclic, aralkyl, heteroaralkyl, (heteroalicyclic)alkyl, hydroxy, protected hydroxy, alkoxy, aryloxy, acyl, thiol, alkylthio, arylthio, cyano, cyanate, halogen, thiocarbonyl, O-carbamoyl The invention also includes but is not limited to alkyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxyl, protected C-carboxyl, O-carboxyl, isothiocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, amino, ether, amino (e.g., monosubstituted amino or disubstituted amino) and protected derivatives thereof. Any of the above groups may include one or more heteroatoms, including O, N or S. For example, when a portion is substituted by an alkyl group, the alkyl group may include a heteroatom selected from O, N or S (e.g., -(CH 2 -CH 2 -O-CH 2 -CH 2 )-).

[0136] As used herein, "% w / v" refers to weight / volume percentage concentration, expressed as grams of solute per 100 mL of solution.

[0137] As used herein, the term "about" or "substantially" means up to + / - 10% of the indicated value, for example + / - 10%, + / - 9%, + / - 8%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2% or + / - 1% of the indicated value.

[0138] Overview

[0139] Applicants have developed compositions, kits and methods for synthesizing Schiff's reagents in situ, i.e., directly on a substrate, such as in a droplet disposed on the surface of a substrate, such as on the surface of a biological sample. A general method for synthesizing Schiff's reagents in situ is described in Figure 5 Applicants have also developed methods for staining biological samples, such as those disposed on a substrate, using in situ synthesized Schiff reagents.

[0140] Composition

[0141] The present disclosure provides a dye composition and a sulfite or SO 2 Source composition. Each of these compositions can be simultaneously or sequentially distributed to a biological sample, such as a biological sample disposed on a slide. In addition, each of these compositions can be included in one or more kits used with an automated staining system.

[0142] Dye composition

[0143] In some embodiments, the present disclosure provides a composition (hereinafter referred to as "dye composition") comprising one or more dyes (hereinafter referred to as "dye") with 4-benzhydryl-2,5-cyclohexadiene-1-imine core. In some embodiments, the dye composition comprises optional acid, one or more optional solvents, and optional one or more additives. In some embodiments, the dye composition comprises one or more dyes, acid and optional one or more additives. In other embodiments, the dye composition comprises one or more dyes, one or more solvents, and optional one or more additives. In some further embodiments, the dye composition comprises one or more dyes, acid, one or more solvents, and optional one or more additives. In some embodiments, the dye composition is stable for at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, etc. In some embodiments, the dye composition does not contain precipitate for at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, etc. In some embodiments, the dye composition is substantially free of precipitate for at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, etc.

[0144] In some embodiments, one or more dyes having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core are functionalized to include at least one substituted or unsubstituted amine group. In other embodiments, one or more dyes having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core are functionalized to include at least one substituted or unsubstituted amine group; and further functionalized to include one or more C 1 -C 4In other embodiments, one or more dyes having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core are functionalized to include at least two substituted or unsubstituted amine groups. In still other embodiments, one or more dyes having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core are functionalized to include at least two substituted or unsubstituted amine groups; and further functionalized to include one or more C 1 -C 4 In further embodiments, one or more dyes having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core are functionalized to include at least two substituted or unsubstituted amine groups; and further functionalized to include two or more C 1 -C 4 In some embodiments, at least one substituted amine group is replaced by a C 1 -C 4 Alkyl group substitution.

[0145] In some embodiments, one or more dyes having a 4-di-benzylidene-2,5-cyclohexadiene-1-imine core are functionalized to include at least one unsubstituted amine group. In other embodiments, one or more dyes having a 4-di-benzylidene-2,5-cyclohexadiene-1-imine core are functionalized to include at least one unsubstituted amine group; and further functionalized to include one or more C 1 -C 4 In other embodiments, one or more dyes having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core are functionalized to include at least two unsubstituted amine groups. In still other embodiments, one or more dyes having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core are functionalized to include at least two unsubstituted amine groups; and further functionalized to include one or more C 1 -C 4 Alkyl group.

[0146] In further embodiments, one or more dyes having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core have a molecular weight ranging from about 300 g / mol to about 600 g / mol, about 300 g / mol to about 500 g / mol, about 300 g / mol to about 450 g / mol, about 300 g / mol to about 400 g / mol, about 300 g / mol to about 375 g / mol, about 300 g / mol to about 350 g / mol, about 310 g / mol to about 340 g / mol, or about 310 g / mol to about 330 g / mol.

[0147] In some embodiments, one or more dyes having a 4-benzhydrylene-2,5-cyclohexadiene-1-imine core are of any one of Formula (I) to Formula (XII):

[0148]

[0149]

[0150]

[0151] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl or cycloalkyl groups, -OH, -OC 1 -C 6 Alkyl or -N(R x )(R y ), where R x and R y is independently H or a branched or unbranched C optionally substituted with one or more halogen atoms 1 -C 6 An alkyl group, or one in which R 1 and R 2 Together, or R 3 and R 4 Together or R 5 or R 6 together to form optionally one or more C 1 -C 6 Alkyl groups or one or more -N(R x )(R y ) group-substituted 5- or 6-membered ring or heterocyclic ring; and wherein A is a counterion.

[0152] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl or cycloalkyl groups, -OH, -OC 1 -C 6 Alkyl or -N(R x )(R y ), where Rx and R y are independently H or branched or unbranched C 1 -C 6 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl group or -N(R x )(R y ), where R x and R y are independently H or C 1 -C 2 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl group or -N(R x )(R y ), where R x and R y are independently H or -CH 3 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, branched or unbranched substituted or unsubstituted C 1 -C 6 Alkyl group or -NH 2 .

[0153] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ). In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched substituted or unsubstituted C 1 -C 6 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched substituted or unsubstituted C 1 -C 4 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched substituted or unsubstituted C 1 -C 3 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R 3 , R 4, R 5 and R 6 At least two of them are C 1 -C 2 Alkyl group.

[0154] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 6 an alkyl group or a cycloalkyl group; and wherein R x or R y At least one of is H. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 4 an alkyl group or a cycloalkyl group; and wherein R x or R y At least one of is H. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R 3 , R 4 , R 5 and R6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 3 an alkyl group or a cycloalkyl group; and wherein R x or R y At least one of is H. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are C 1 -C 2 An alkyl group; and wherein R x or R y At least one of them is H.

[0155] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 6 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C4 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 3 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH2 and wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 2 Alkyl group.

[0156] In some embodiments, the molecular weight of the compound of formula (I) to formula (III) ranges from about 300 g / mol to about 600 g / mol; about 300 g / mol to about 550 g / mol; about 300 g / mol to about 525 g / mol; about 300 g / mol to about 500 g / mol; about 300 g / mol to about 475 g / mol; about 300 g / mol to about 450 g / mol; about 300 g / mol to about 425 g / mol; about 300 g / mol to about 400 g / mol; about 300 g / mol to about 5 ... mol to about 390 g / mol; about 300 g / mol to about 380 g / mol; about 300 g / mol to about 370 g / mol; about 300 g / mol to about 360 g / mol; about 300 g / mol to about 365 g / mol; about 300 g / mol to about 350 g / mol; about 300 g / mol to about 345 g / mol; about 300 g / mol to about 340 g / mol; about 300 g / mol to about 335 g / mol; and about 300 g / mol to about 330 g / mol.

[0157] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 6 an alkyl group or a cycloalkyl group; and wherein R x or R y At least one of R is H and has a molecular weight ranging from about 300 g / mol to about 450 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 4 an alkyl group or a cycloalkyl group; and wherein R x or R y At least one of R is H and has a molecular weight ranging from about 300 g / mol to about 450 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 3 an alkyl group or a cycloalkyl group; and wherein R x or R y At least one of R is H and has a molecular weight ranging from about 300 g / mol to about 450 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ) and where R 1 , R 2 , R 3 , R 4 , R 6 and R 6 At least two of them are C 1 -C 2 alkyl group; and wherein R x or R y At least one of is H, and has a molecular weight ranging from about 300 g / mol to about 450 g / mol.

[0158] In some embodiments, R 1 , R 2 , R 3 , R4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 6 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 6 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 6 An alkyl group or a cycloalkyl group, and has a molecular weight ranging between about 300 g / mol to about 350 g / mol.

[0159] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 4 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 4 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 4 An alkyl group or a cycloalkyl group, and has a molecular weight ranging between about 300 g / mol to about 350 g / mol.

[0160] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 3 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 3 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are branched or unbranched, substituted or unsubstituted C 1 -C 3 An alkyl group or a cycloalkyl group, and has a molecular weight ranging between about 300 g / mol to about 350 g / mol.

[0161] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2, R 3 , R 4 , R 5 and R 6 At least two of them are C 1 -C 2 An alkyl group having a molecular weight in the range of about 300 g / mol to about 450 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are C 1 -C 2 An alkyl group having a molecular weight in the range of about 300 g / mol to about 400 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH 2 And where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are C 1 -C 2 An alkyl group and having a molecular weight ranging from about 300 g / mol to about 350 g / mol.

[0162] In some embodiments, the dye is selected from:

[0163]

[0164] or

[0165] In some embodiments, the total concentration of one or more dyes having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core in the dye composition, including any dye having any of Formulas (I) to (XIP), is between about 0.05% w / v to about 8% w / v, between about 0.1% w / v to about 6% w / v, between about 0.1% w / v to about 5% w / v, between about 0.1% w / v to about 4% w / v, between about 0.1% w / v to about 3.5% w / v, between about 0.1% w / v to about 3% w / v, between about 0.1% w / v to about 2.5% w / v, between about 0.1% w / v to about 2% w / v, between about 0.1% w / v to about 1.5% w / v, between about 0.1% w / v to about 2.5 ...2.5% w / v, between about 0.1% w / v to about 2.5% w / v, .1% w / v to about 1% w / v, about 0.15% w / v to about 4% w / v, about 0.15% w / v to about 3.5% w / v, about 0.15% w / v to about 3% w / v, about 0.15% w / v to about 2.5% w / v, about 0.15% w / v to about 2% w / v, about 0.15% w / v to about 1.5% w / v, about 0.15% w / v to about 1% w / v, about 0.2% w / v to about 4%, about 0.2% w / v to about 3.5%, about 0.2% w / v to about 3%, about 0.2% w / v to about 2.5%, about 0.2% w / v to about 2% w / v, about 0.2% w / v to about 1.5 % w / v, about 0.2% w / v to about 1% w / v, about 0.25% w / v to about 4%, about 0.25% w / v to about 3.5%, about 0.25% w / v to about 3%, about 0.25% w / v to about 2.5%, about 0.25% w / v to about 2% w / v, about 0.25% w / v to about 1.5% w / v, about 0.25% w / v to about 1% w / v, about 0.3% w / v to about 4%, about 0.3% w / v to about 3.5%, about 0.3% w / v to about 3%, about 0.3% w / v to about 2.5%, about 0.25% w / v to about 2% w / v, about 0.25% w / v to about 1.5% w / v, about 0.25% w / v to about 1% w / v, about 0.3% w / v to about 4%, about 0.3% w / v to about 3.5%, about 0.3% w / v to about 3%, about 0.3% w / v to about 2.5%, about 0.3% w / v to about 2% w / v, about 0.3% w / v to about 1.5% w / v / v, between about 0.3% w / v and about 1% w / v, between about 0.4% w / v and about 4%, between about 0.4% w / v and about 3.5%, between about 0.4% w / v and about 3%, between about 0.4% w / v and about 2.5%, between about 0.4% w / v and about 2% w / v, between about 0.4% w / v and about 1.5% w / v, between about 0.4% w / v and about 1% w / v, between about 0.5% w / v and about 4%, between about 0.5% w / v and about 3.5%, between about 0.5% w / v and about 3%, between about 0.5% w / v and about 2.5%, between about 0.5% w / v and about 2% w / v, between about 0.5% w / v and about 1.5% w / v, between about 0.4% w / v and about 1% w / v, between about 0.5% w / v and about 4%, between about 0.5% w / v and about 3.5%, between about 0.5% w / v and about 3%, between about 0.5% w / v and about 2.5%, between about 0.5% w / v and about 2% w / v, between about 0.5% w / v and about 1.5% w / v,5% w / v to about 1% w / v, about 0.6% w / v to about 4%, about 0.6% w / v to about 3.5%, about 0.6% w / v to about 3%, about 0.6% w / v to about 2.5%, about 0.6% w / v to about 2% w / v, about 0.6% w / v to about 1.5% w / v, about 0.6% w / v to about 1% w / v, about 0.8% w / v to about 4%, about 0.8% w / v to about 3.5%, about 0.8% w / v to about 3%, about 0.8% w / v to about 2.5%, about 0.8% w / v to about 2%, about 0.8% w / v to about 2%, about 1% w / v to about 4%, In the range of about 1% w / v to about 3.5%, about 1% w / v to about 3%, about 1% w / v to about 2.5%, about 1% w / v to about 2%, about 1.5% w / v to about 4%, about 1.5% w / v to about 3.5%, about 1.5% w / v to about 3%, about 1.5% w / v to about 2.5%, about 1.5% w / v to about 2%, about 2% w / v to about 4%, about 2% w / v to about 3.5%, about 2% w / v to about 3%, about 2% w / v to about 2.5%, about 2.5% w / v to about 4%, about 2% w / v to about 3.5%, or about 2.5% w / v to about 3%. .

[0166] In some embodiments, the total concentration of one or more dyes is about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, about 0.5% w / v, about 0.6% w / v, about 0.7% w / v, about 0.8% w / v, about 0.9% w / v, about 1% w / v, about 1.2% w / v, about 1.4% w / v, about 1.6% w / v, about 1.8% w / v, about 2% w / v, about 2.2% w / v, about 2.4% w / v, about 2.6% w / v, about 2.8% w / v, about 3% w / v, about 3.2% w / v, about 3.4% w / v, about 3.6% w / v, about 3.8% w / v, or about 4% w / v.

[0167] In some embodiments, one or more dyes have a molecular weight in the range of about 300 g / mol to about 375 g / mol, and wherein the total concentration of the one or more dyes is in the range of about 0.15% w / v to about 1.8% w / v. In other embodiments, one or more dyes have a molecular weight in the range of about 300 g / mol to about 350 g / mol, and wherein the total concentration of the one or more dyes is in the range of about 0.3% w / v to about 1.2% w / v.

[0168] In some embodiments, the one or more dyes are selected from:

[0169]

[0170] or

[0171] And wherein the total concentration of the one or more dyes is in the range of between about 0.15% w / v to about 1.8% w / v, such as between about 0.3% w / v to about 1.2% w / v.

[0172] acid

[0173] In certain embodiments, dye composition comprises acid.In certain embodiments, acid is strong acid.In certain embodiments, strong acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, hydrofluoric acid, trifluoroacetic acid, hydroiodic acid, sulfuric acid, nitric acid, chloric acid and perchloric acid.In certain embodiments, strong acid has the concentration in the scope between about 0.8M to about 2M.In other embodiments, strong acid has the concentration in the scope between about 0.9M to about 1.8M.In other embodiments, strong acid has the concentration in the scope between about 1M to about 1.5M.In certain embodiments, the concentration of strong acid is about 1M or higher than about 1M, for example, about 0.9M, about 0.95M, about 1.0M, about 1.05M, about 1.1M, about 1.15M, about 1.2M, about 1.25M, about 1.3M, about 1.35M, about 1.4M, about 1.45M, about 1.5M, about 1.55M, about 1.6M, about 1.65M etc. In some embodiments, the strong acid is hydrochloric acid, such as 1.2 M hydrochloric acid. In some embodiments, the strong acid is nitric acid, such as 1.25 M nitric acid.

[0174] In some embodiments, the acid is a weak acid. In some embodiments, the weak acid is formic acid, acetic acid, benzoic acid, oxalic acid, hydrofluoric acid, nitrous acid, sulfurous acid, and phosphoric acid.

[0175] In some embodiments, the pH value of the dye composition is in the range of about -0.5 to about 6.5. In some embodiments, the pH value is in the range of about -0.5 to about 6. In some embodiments, the pH value is in the range of about -0.5 to about 5.5. In some embodiments, the pH value is in the range of about -0.5 to about 5. In some embodiments, the pH value is in the range of about -0.5 to about 4.5. In some embodiments, the pH value is in the range of about -0.5 to about 4. In some embodiments, the pH value is in the range of about -0.5 to about 3.5. In some embodiments, the pH value is in the range of about -0.5 to about 3. In some embodiments, the pH value is in the range of about -0.5 to about 2.5. In some embodiments, the pH value is in the range of about -0.5 to about 2.

[0176] Solvents

[0177] In some embodiments, the dye composition includes one or more solvents. In some embodiments, the solvent is an alcohol. In some embodiments, the solvent is a polyol.

[0178] Non-limiting examples of suitable solvents include, but are not limited to, methanol, ethanol, propanol, butanol, amyl alcohol, amyl alcohol, fusel oil, hexanol, heptanol, octanol, cyclohexanol, benzyl alcohol, furfuryl alcohol, tetrahydrofurfuryl alcohol, hexane, heptane, octane, decane, petroleum ether, petroleum benzene, light petroleum, gasoline, kerosene, cyclohexane, benzene, toluene, xylene, tetralin, decahydronaphthalene, terpene oils, chloroform, carbon tetrachloride, dichloroethane, 1,1-dichloroethylene, trichloroethane, tetrachloroethane, trichloroethylene, tetrachloroethylene, trichloropropane, isopropyl chloride, dichloropropane, butyl chloride, pentyl chloride, hexyl chloride, vinyl bromide, tetrabromoethane, chlorobenzene, dichlorobenzene, trichlorobenzene, bromobenzene, chlorotoluene, isopropyl ether, dibutyl ether, diisoamyl ether, hexyl ether, methyl phenyl ether, ethyl phenyl ether, butyl phenyl ether, ethyl benzyl ether, dioxane, 2-methylfuran, tetrahydrofuran, tetrahydropyran, eucalyptol, acetone, methyl ethyl ketone, methyl propyl ketone, methyl butyl ketone, methyl amyl ketone, methyl hexyl ketone, diethyl ketone, ethyl butyl ketone, dipropyl ketone, diisobutyl ketone, diacetone alcohol, phorone, isophorone, cyclohexanone, methylcyclohexanone, acetophenone, ethyl formate, propyl formate, butyl formate, amyl formate, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, amyl acetate, methyl isoamyl acetate, methoxybutyl acetate, hexyl acetate, cyclohexyl acetate, benzyl acetate, methyl propionate , ethyl propionate, butyl propionate, pentyl propionate, methyl butyrate, ethyl butyrate, butyl butyrate, pentyl butyrate, methyl acetoacetate, ethyl acetoacetate, isopentyl isovalerate, methyl lactate, ethyl lactate, butyl lactate, pentyl lactate, methyl benzoate, diethyl oxalate, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monomethyl ether acetate, ethylene glycol monoethyl ether, ethylene glycol diethyl ether, ethylene glycol monoethyl ether acetate, ethylene glycol isopropyl ether, ethylene glycol monobutyl ether, ethylene glycol dibutyl ether, ethylene glycol monobutyl ether acetate, ethylene glycol monophenyl ether, methoxymethoxyethanol, ethylene glycol monoacetate, ethylene glycol diacetate, diethylene glycol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol mono In some embodiments, the solvent is DMSO.

[0179] additive

[0180] In some embodiments, the dye composition comprises one or more additives, such as antioxidants, stabilizers, and / or biocides.

[0181] The term "antioxidant" as used herein includes any compound or combination of compounds that prevent or slow down oxidation of components caused by destructive reactive oxygen species (ROS). Any known antioxidant can be used, including but not limited to tocopherol, phospholipids (PL), phytosterols, phycocyanin, vitamin E, vitamin A and vitamin C, beta carotene, coenzyme Q10, fatty acids ω-3, ω-6 and w-9, plant antioxidants such as polyphenols, terpenes such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, lecithin, sesamin, sesamol, sesamolin, alpha-tocopherol, gamma-tocopherol, salicylic acid, ascorbic acid, ascorbyl palmitate, fumaric acid, malic acid, sodium ascorbate and sodium pyrosulfite, and chelating agents such as disodium EDTA. Pharmaceutically acceptable nutritional dietary supplements may also be used as antioxidants and include plants, algae, and lichens and may include one or more extracts of propolis, red clover, soy, capers, almonds, milk thistle, green tea, pomegranate, orange red, grape seed, bilberry, vertigin, ginseng, English ivy, red algae, brown algae, green algae, and lichens.

[0182] In some embodiments, the antioxidant is selected from the group consisting of ascorbic acid and its salts, tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, ascorbyl palmitate, and propyl gallate.

[0183] In other embodiments, the antioxidant is selected from the group consisting of ascorbic acid, thioglycerol, thiosorbitol, thiourea, sodium thiosulfate, thioacetic acid, cysteine, methionine, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), ascorbyl palmitate, hydroquinone, gallic acid esters, nordihydroguaiaretic acid, vitamin E (α-tocopherol) and lecithin. Preferred antioxidants are micronized propyl gallate, micronized BHA, micronized BHT, vitamin E, ascorbic acid, sodium thiosulfate and cysteine.

[0184] In yet other embodiments, the antioxidant is selected from the group consisting of hydroquinone; n-alkyl gallates (such as n-propyl gallate, n-octyl gallate, and n-dodecyl gallate); reducing sugars such as sorbitol and mannitol; benzoates and hydroxybenzoates; certain acids such as citric acid, tartaric acid, lactic acid, isoascorbic acid, uric acid, tannic acid, and salts of these acids (such as Mg2+, NH4+, Na+, K+, and Ca2+ salts); chelating agents such as EDTA, which can remove metals that act as oxidants.

[0185] In yet further embodiments, the antioxidant is an oil-based antioxidant selected from the group consisting of ascorbyl palmitate, butylated hydroxytoluene (BHT); butylated hydroxyanisole (BHA); propyl gallate; and alpha-tocopherol.

[0186] In a preferred embodiment, the antioxidant is antioxidant.

[0187] In some embodiments, the composition comprises a stabilizer. In other embodiments, the composition comprises a biocide or preservative. Preservatives, such as antimicrobial agents. Suitable preservatives include, for example, isothiazolinones, diols, azides, and combinations thereof. Exemplary preservatives include 300 (2.30% 5-chloro-2-methyl-4-isothiazolin-3-one, 0.70% 2-methyl-4-isothiazolin-3-one, 2%-3% alkyl formate (stabilizer), and 93%-95% modifying diol; available from Sigma-Aldrich, St. Louis, MO), 950 (9.5%-9.9% 2-methyl-4-isothiazolin-3-one, Sigma-Aldrich) and sodium azide.

[0188] Sulfite or SO 2 Source composition

[0189] The present disclosure also provides a method comprising sulfite or SO 2 The composition of the source (hereinafter referred to as "sulfite or SO 2 In some embodiments, sulfite or SO 2 The source composition includes sulfite or SO 2 In some embodiments, the sulfite or SO 2 The source is selected from the group consisting of pyrosulfite, bisulfite, dithionite, thiosulfate, sulfite, and sulfurous acid. In some embodiments, sulfite or SO 2 The source composition is stable for at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, etc.

[0190] In some embodiments, sulfite or SO 2 Source composition of sulfite or SO 2The total concentration of the source is between about 0.1% w / v to about 21.5% w / v, between about 0.1% w / v to about 18% w / v, between about 0.1% w / v to about 12% w / v, between about 0.1% w / v to about 10% w / v, between about 0.1% w / v to about 9% w / v, between about 0.1% w / v to about 8% w / v, between about 0.1% w / v to about 7% w / v, between about 0.1% w / v to about 6% w / v, between about 0.1% w / v to about 5% w / v, between about 0.1% w / v to about 4% w / v, between about 0.1% w / v to about 3% w / v, between about 0.1% w / v to about 2% w / v, between about 0.1% w / v to about 1% w / v , between about 0.1% w / v and about 0.5% w / v, between about 0.1% and about 0.3% w / v, between about 0.5% w / v and about 21.5% w / v, between about 0.5% w / v and about 18% w / v, between about 0.5% w / v and about 12% w / v, between about 0.5% w / v and about 10% w / v, between about 0.5% w / v and about 9% w / v, between about 0.5% w / v and about 8% w / v, between about 0.5% w / v and about 7% w / v, between about 0.5% w / v and about 6% w / v, between about 0.5% w / v and about 5% w / v, between about 0.5% w / v and about 4% w / v, between about 0.5% w / v and about 3% w / v, between about 0. 5% w / v to about 2% w / v, about 0.5% w / v to about 1% w / v, about 1% w / v to about 21.5% w / v, about 1% w / v to about 18% w / v, about 1% w / v to about 12% w / v, about 1% w / v to about 10% w / v, about 1% w / v to about 9% w / v, about 1% w / v to about 8% w / v, about 1% w / v to about 7% w / v, about 1% w / v to about 6% w / v, about 1% w / v to about 5% w / v, about 1% w / v to about 4% w / v, about 1% w / v to about 3% w / v, about 2.5% w / v to about 21.5% w / v, about 2.5% w / v to about In some embodiments, the present invention relates to a method for preparing the present invention that is suitable for use in the present invention to provide a method for preparing the present invention that is suitable for use in the present invention to provide a method for preparing the present invention that is suitable for use in the present invention to provide a method for preparing the present invention that is suitable for use in the present invention to provide a method for preparing the present invention that is suitable for use in the present invention to provide a method for preparing the present invention that is suitable for use in the present invention to provide a method for preparing the present invention that is suitable for use in the present invention to provide a method for preparing the present invention that is suitable for use in the present invention to provide a method for preparing the present invention that is suitable for use in the present invention to provide a method for preparing the present invention that is suitable for use in the present invention

[0191] In which sulfite or SO 2 In those embodiments where the source is not thiosulfate, sulfite or SO 2 The source composition may comprise an acid (including any of the acids listed herein) and / or have a pH of less than about 9, such as less than about 8.5, such as less than about 8, such as less than about 7.5, such as less than about 7. In some embodiments, sulfite or SO 2 The pH of the source composition is about 5 to 6.9. In some embodiments, the sulfite or SO 2 The pH of the source composition is about 5 to 6.5. In some embodiments, the sulfite or SO 2 The pH of the source composition is about 5.5 to 6.5.

[0192] In sulfite or SO 2 In those embodiments where the source is thiosulfate, sulfite or SO 2 The pH of the source composition is greater than 7, such as greater than 7.5, such as greater than 8, such as greater than 8.5, or such as greater than 9. In some embodiments, the sulfite or SO 2 The source is thiosulfate, sulfite or SO 2 The source composition has a pH of about 10.

[0193] In some embodiments, sulfite or SO 2 The source composition comprises an antioxidant, including any of those listed herein. In some embodiments, sulfite or SO 2 The source composition comprises a solvent, including any of the solvents listed herein. In some embodiments, the sulfite or SO 2 The source composition includes an alcohol or a diol.

[0194] Reagent test kit

[0195] In some embodiments, the present disclosure provides a kit comprising at least two reservoirs, containers and / or dispensers (collectively referred to herein as "containers"), wherein the first container comprises a dye composition, and wherein the second container comprises a sulfite or SO 2 In some embodiments, the kit includes two containers, namely, a dye composition, which includes an acid, such as any one of a weak acid, a strong acid, an inorganic acid, or an organic acid; and a sulfite or SO 2 Source composition.

[0196] In other embodiments, the kit includes three containers, namely, a dye composition without an acid, a sulfite or SO 2In some embodiments, the acid composition comprises any of a weak acid, a strong acid, an inorganic acid, or an organic acid, including any of those described herein.

[0197] Sample staining method

[0198] The present disclosure also provides methods for forming a Schiff reagent in situ, such as directly on a surface of a substrate (e.g., a glass slide), on a sample disposed on a substrate, or in a droplet disposed on a substrate. In general, the method comprises: 2 The source composition, and / or the acid composition are applied or dispensed to a substrate, and the various components of the composition are allowed to react for a predetermined amount of time at a predetermined temperature. Subsequently, the in situ formed Schiff's reagent can be removed from the substrate, such as by washing the substrate with a washing reagent or buffer. As described herein, the appellant unexpectedly discovered that staining a sample using an in situ generated Schiff's reagent provides a staining quality and / or intensity that is at least equivalent to that using a commercial Schiff's reagent. In addition, the appellant unexpectedly discovered that the staining intensity can be "adjusted" by changing the time that any reagent is in contact with the sample; changing the temperature of the sample and / or the reagent in contact with the same sample; and / or changing the concentration of any reagent or reagent component (e.g., the concentration of the dye, the concentration of the sulfite source, and / or the concentration of the acid).

[0199] As used herein, phrase "being distributed to substrate refers to " being distributed to the surface of substrate (for example microscope slide), being distributed to the sample being arranged on substrate or being distributed to the droplet being arranged on substrate and / or sample.In certain embodiments, fluid or composition can be directly distributed to the sample being arranged on substrate.Or, fluid or composition can be distributed near the sample being arranged on substrate, and the fluid or composition distributed can be moved to sample subsequently, for example, by tilting substrate and / or using gas jet to promote fluid or composition to arrive sample from the surface of substrate.In another embodiment, fluid or composition can be distributed to the fluid or reagent " droplet" being present on the surface of substrate or sample.It will be appreciated by those skilled in the art that the sample being distributed to same substrate and / or being arranged on substrate by different compositions in order can be the same or roughly the same position, so that any fluid or composition distributed in sequence contacts with existing fluid droplet so that fluid and / or composition can mix and / or react.

[0200] In some embodiments, the dye composition, sulfite or SO can be manually added. 2 In other embodiments, the dye composition, sulfite or SO described herein is applied to the sample. 2The source composition and / or the acid composition are applied or dispensed to the biological sample (such as within an automated staining apparatus). Non-limiting examples of suitable automated staining systems are described herein.

[0201] In some embodiments, and in dispensing any dye composition, sulfite or SO 2 The sample is treated with an aldehyde group-forming reagent for a predetermined amount of time prior to dispensing any dye composition, sulfite or SO 2 Source composition and / or optional acid composition before, with periodic acid (for example, 1 gram of periodic acid in 100mL water) process sample and continue about 1 minute to about 10 minutes (for example, 5 minutes) time period.In certain embodiments, periodic acid is distributed to sample and makes it and sample keep contact predetermined about time (for example, about 1 minute to about 10 minutes).Subsequently, for example, use buffer, distilled water or other washing reagents to rinse or wash periodic acid from sample.

[0202] In some embodiments, the dye composition, sulfite or SO 2 The source composition, and / or the optional acid composition are sequentially dispensed onto the substrate, allowing for in situ synthesis of the Schiff reagent. In other embodiments, the dye composition, sulfite or SO 2 The source composition, and / or the optional acid composition, are simultaneously dispensed to the substrate, allowing for in situ synthesis of the Schiff reagent.

[0203] In some embodiments, the dye composition, sulfite or SO 2 The order of the source composition and / or the optional acid composition depends on the components included in the composition. For example, in some embodiments, if the dye composition includes an acid, the dye composition including the acid can be dispensed to the substrate first, followed by the sulfite or SO 2 In other embodiments, the sulfite or SO 2 The source composition is dispensed to the substrate, and then the dye composition comprising the acid can be subsequently dispensed to the substrate. In still other embodiments, the dye composition comprising the acid and the sulfite or SO 2 The source composition and the dye composition are dispensed simultaneously onto the substrate.

[0204] In some embodiments, a dye composition that does not contain an acid (or has a pH greater than about 6.9) is first dispensed to a substrate, and then an acid composition is dispensed to the substrate (e.g., to a fluid droplet containing the dye composition that does not contain an acid), thereby reducing the pH of the dye composition (e.g., to provide an acidified dye composition). In some embodiments, after dispensing the acid composition, the pH of the droplet is reduced to less than 6.9, such as less than 6.5, such as less than 6, such as less than 5.5, such as less than 5, such as less than 4.5, such as less than 4, such as less than 3.5, such as less than 3, such as less than 2.5, such as less than 2, etc. Subsequently, a sulfite or SO 2 The source composition is partitioned into the resulting acid dye composition to provide an in situ synthesized Schiff reagent.

[0205] In other embodiments, the acid composition is dispensed first, and then the dye composition without acid (or having a pH greater than about 6.9) is dispensed to produce an acidified dye composition on the surface of the substrate (e.g., the acidified composition has a pH less than 6.9). Subsequently, the sulfite or SO 2 The source composition is partitioned into the resulting acidified dye composition to provide an in situ synthesized Schiff reagent.

[0206] In yet other embodiments, a dye composition that does not contain an acid (or has a pH greater than about 6.9) and an acid composition are simultaneously dispensed to a substrate such that when the dispensed dye composition and acid composition mix on the surface of the substrate, the pH of the dye composition is reduced to provide an acidified dye composition. Subsequently, a sulfite or SO 2 The source composition is partitioned into the resulting acidified dye composition to provide an in situ synthesized Schiff reagent.

[0207] In some embodiments, sulfite or SO 2 The source composition is dispensed to the substrate, and then a dye composition that does not contain an acid (or has a pH greater than about 6.9) is dispensed to the substrate. Subsequently, an acid composition is dispensed to the substrate, allowing the in-situ synthesis of the Schiff reagent. In other embodiments, a sulfite or SO 2 The source composition is dispensed to the substrate, and then the acid composition is dispensed to the substrate. Subsequently, a dye composition that does not contain an acid (or has a pH greater than about 6.9) is dispensed to the substrate, allowing the in-situ synthesis of the Schiff reagent. In yet other embodiments, a sulfite or SO 2 The source composition is dispensed onto a substrate, and then the acid-free dye composition and the acid composition are simultaneously applied to the substrate, resulting in the in-situ synthesis of the Schiff reagent.

[0208] In some embodiments, the dye composition, sulfite or SO dispensed to the substrate 2The amounts of the source composition and the optional acid composition are independently in the following ranges: about 25 μL to about 500 μL, about 25 μL to about 400 μL, about 25 μL to about 300 μL, about 50 μL to about 300 μL, about 50 μL to about 250 μL, about 50 μL to about 225 μL, about 50 μL to about 200 μL, about 50 μL to about 175 μL, about 50 μL to about 150 μL, or about 100 μL to about 150 μL. By way of example only, about 50 μL to about 200 μL of the dye composition and about 50 μL to about 200 μL of the sulfite or SO 2 The source compositions may be dispensed to the substrate simultaneously or sequentially.

[0209] In some embodiments, the in situ synthesized Schiff's reagent is allowed to remain in contact with the sample for a predetermined amount of time. In this way, the staining intensity of the Schiff's reagent can be "adjusted" to meet the preferences of a pathologist or medical professional; to account for differences in the concentrations of components used to synthesize the Schiff's reagent or any composition described herein (e.g., dye concentration, acid concentration, and / or sulfite source concentration); to account for differences in sample type or sample fixation; and / or to account for temperature differences in sample and / or composition components. In some embodiments, the in situ synthesized Schiff's reagent is allowed to remain in contact with the sample for at least 1 minute, at least 2 minutes, at least 4 minutes, at least 8 minutes, at least 12 minutes, at least 16 minutes, at least 20 minutes, at least 24 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 80 minutes, at least 100 minutes, or at least 120 minutes.

[0210] In some embodiments, the sample and / or composition for forming the in-situ synthesized Schiff reagent is maintained at a predetermined temperature. For example, the predetermined temperature is a temperature higher than 25°C, higher than 30°C, or higher than 35°C. In other embodiments, the temperature of the sample and / or composition for forming the in-situ synthesized Schiff reagent is in the range of about 30°C to about 90°C, about 30°C to about 50°C, about 30°C to about 40°C, or about 35°C to about 41°C. In a further embodiment, the temperature of the sample and / or composition for forming the in-situ synthesized Schiff reagent is about 30°C. In a further embodiment, the temperature of the sample and / or composition for forming the in-situ synthesized Schiff reagent is about 32°C. In a further embodiment, the temperature of the sample and / or composition for forming the in-situ synthesized Schiff reagent is about 35°C. In a further embodiment, the temperature of the sample and / or composition for forming the in-situ synthesized Schiff reagent is about 37°C. In a further embodiment, the temperature of the sample and / or composition for forming the in-situ synthesized Schiff reagent is about 40°C.

[0211] In some embodiments, if the sample is a sample embedded in paraffin, one or more appropriate deparaffinizing fluids may be used to dewax the sample. After the deparaffinizing fluid is removed by the waste remover, any number of substances may be applied to the sample in succession. These substances may be used for pretreatment (e.g., protein crosslinking, nucleic acid exposure, etc.), denaturation, hybridization, washing (e.g., stringent washing), detection (e.g., connecting a display or marker molecule to a probe), amplification (e.g., amplifying proteins, genes, etc.), counterstaining, coverslipping, etc.

[0212] After specimen processing is complete, the user can transport the specimen slide to an imaging device for analysis or other downstream processing. For example, the imaging device can be a brightfield imager slide scanner. One brightfield imager is the iScan Coreo sold by Ventana Medical Systems, Inc. TM Bright field scanner. In automated embodiments, the imaging device is a digital pathology device disclosed in International Patent Application No. PCT / US2010 / 002772 (Patent Publication No.: WO / 2011 / 049608) entitled "IMAGING SYSTEM AND TECHNIQUES" or in U.S. Patent Publication No. 61 / 533,114, filed on September 9, 2011, entitled "IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME". International Patent Application No. PCT / US2010 / 002772 and U.S. Patent Application Publication No. 2014 / 0178169 are incorporated herein by reference in their entirety. In other embodiments, the imaging device includes a digital camera coupled to a microscope.

[0213] The method disclosed herein can be adapted for use with existing automated processing systems. For example, Ventana Medical Systems, Inc. is the assignee of multiple U.S. patents that disclose systems and methods for performing automated analysis, including U.S. Patent Nos.: 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, and U.S. Publication Application Nos.: 20030211630 and 20040052685, each of which is incorporated herein by reference. These systems can be adapted to be compatible with the present invention. In short, the automated slide handling system described in the aforementioned references is a high-capacity slide handling system that shuttles a plurality of slides between workstations that perform various slide handling operations on the slides to maintain a tray in a substantially horizontal position (to minimize cross contamination). In the process, fresh reagent can be applied to each slide, and the cross contamination of slide and reagent can be basically eliminated, because slide is processed respectively in the tray in an interval mode.In one configuration, system comprises radiant heater, deparaffinizer / stainer / solvent exchanger combined workstation, convection oven and coverslip device.In certain embodiments, the tray of the slide of the tissue sample of paraffin embedding can be heated under the radiant heater of system, so that the paraffin in the sample is diffused for making it more easily removed, and also the sample is adhered to the slide.In certain embodiments, then the tray can be transported to the multifunctional deparaffinizer / stainer / solvent exchanger workstation, where slide can be deparaffinized, dyed and solvent exchanged.In certain embodiments, then the tray of the stained slide ready to cover the cover glass can be shuttled to the coverslip device of system, where cover glass is added to the slide.Once slide is covered with cover glass, then the tray can be transported to the convection oven, to solidify the cover glass on the stained slide. In some embodiments, the high-volume stainer just described is commercially available from Ventana Medical Systems, Inc, Tucson, Ariz.

[0214] Examples of other commercially available sample processing systems with which the solutions and preparations described herein can be applied include: the VENTANA SYMPHONY (single slide stainer) and VENTANA HE 600 (single slide stainer) series, and the Dako CoverStainer (batch stainer) from Agilent Technologies; the Leica ST4020 Mini Linear Stainer (batch stainer), the Leica ST5020 Multistage Stainer (batch stainer), and the Leica ST5010 Automated Stainer XL Series (batch stainer) H&E stainers from Leica Biosystems Nussloch GmbH.

[0215] In some embodiments, the automated dyeing system comprises one or more reservoirs, containers, and / or dispensers, each of which may include any of the compositions disclosed herein. In some embodiments, the dye composition, sulfite, or SO 2 The source composition and the optional acid composition are separately dispensed onto the biological sample. In these embodiments, the solution may be allowed to mix by diffusion on the sample, or the solution may be mechanically mixed, for example, by stirring (such as with an air mixer or with a pipette).

[0216] In some embodiments, the automated sample handling equipment may include a carousel for holding a plurality of substrates, such as microscope slides, wherein each substrate includes a sample to be stained. In some embodiments, the automated staining equipment may also include: a device for rotating the carousel at a predetermined speed; and a mechanism for guiding and controlling the application of reagents (including solutions and preparations described herein) to substrates and samples during the rotation of the carousel. In some embodiments, once the slide is loaded into the instrument, the test protocol will determine which compositions (from which reservoir, dispenser or container) are dispensed onto the substrate at a specific time. At the appropriate time, in some embodiments, the dispenser bracket will rotate to align the correct composition above the substrate, and the instrument will dispense a predetermined amount of one or more disclosed compositions onto the substrate.

[0217] In some embodiments, the system is an automated slide handling system comprising: a slide tray that holds a plurality of slides in a substantially horizontal position (e.g., in two rows, wherein the slides are held at an angle between about 0.2 degrees and about 1.2 degrees to the horizontal plane); and one or more workstations (e.g., arranged in a vertical stack) that receive the slide tray and perform one or more slide handling operations on the slides in the slide tray. In some embodiments, the workstation can perform a slide handling operation on one or more individual slides in the slide tray (e.g., at least two or four slides in the slide tray), or it can perform a slide handling operation on all slides in the slide tray simultaneously. In some embodiments, the one or more workstations dispense reagents to the slides in the slide tray, while a large amount of reagents that contact the first slide do not contact the second slide, thereby minimizing cross contamination between slides. Such workstations may include one or more directional nozzles that dispense the composition of the present disclosure onto the slides. For example, the one or more directional nozzles may include a pair of directional nozzles that direct the dye composition, sulfite or SO 2 The one or more directional nozzles may further include a directional nozzle that dispenses the composition toward the bottom surface of the slide. In other specific embodiments, the one or more workstations may simultaneously dispense the dye composition, the sulfite or SO 2 The source composition and / or the optional acid composition are dispensed to at least two slides held in a slide tray within a given workstation, or the one or more workstations may simultaneously dispense the composition to all slides held in a slide tray within a given workstation. Additional system components and tray configurations (and control systems) are described in U.S. Pat. Nos. 8,663,991, 7,468,161, and 9,528,918, the disclosures of which are hereby incorporated by reference in their entirety.

[0218] In some embodiments, the present disclosure provides an apparatus for automatically processing biological samples, the apparatus comprising: at least one slide tray, the at least one slide tray holding a plurality of slides in a substantially horizontal position, wherein the biological samples are located on the slides; one or more workstations, the one or more workstations receiving the slide tray and performing one or more slide processing operations on the plurality of slides held in the slide tray; a transporter, the transporter moving the slide tray into and out of the one or more workstations; a fluidics module, the fluidics module being in fluid communication with the one or more workstations, the fluidics module supplying a dye composition, a sulfite or SO to the one or more workstations.2 a source composition and / or an optional acid composition; a pneumatic module in fluid communication with the one or more workstations and the fluidics module; wherein the pneumatic module provides vacuum and / or pressurized gas to the one or more workstations and the fluidics module; and a control module in electrical communication with the transporter, the one or more workstations, the fluidics module, and the pneumatic module, wherein the control module coordinates the functions of the components of the apparatus during processing of the biological sample. The apparatus may be adapted to deliver, for example, a dye composition, a sulfite, or a SO4-containing compound as described herein, sequentially or simultaneously. 2 One or more of a source composition and / or an optional acid composition.

[0219] Examples

[0220] Example 1 - Stability of Synthetic Reagents on Slides

[0221] Applicants have discovered that in situ synthesized Schiff's reagent promotes staining of biological samples in a manner equivalent to the staining observed using commercially available Schiff's reagent, even after the dye used to synthesize the Schiff's reagent has been stored at elevated temperatures for extended periods of time (e.g., 60° C. for two weeks). For example, Fig. 6A and 6B Shown are staining of tissue samples using in situ synthesized Schiff's reagent prepared from pararosaniline dye stored for two weeks at either 2°C-8°C or 60°C. Notably, the in situ synthesized Schiff's reagent stained equivalently regardless of the storage conditions of the precursor dye. In contrast, Figure 6C and 6D Shown is staining of tissue samples using commercial Schiff's reagent, which is new ( Figure 6C ) and has been open for two weeks ( Fig.6D ). Notably, the staining intensity of commercially available Schiff reagents opened for two weeks decreased compared to newly opened commercially available Schiff reagents or those prepared in situ.

[0222] Example 2 - Reaction Time

[0223] Fig. 7A and Figure 7BThe staining intensity and contrast using the in situ synthesized Schiff's reagent as a function of reaction time are shown. The time and temperature during incubation of the in situ synthesized Schiff's reagent on the tissue sample were varied simultaneously and independently to demonstrate that the staining intensity can be adjusted according to preference. Shorter incubation times between about 4 minutes and about 16 minutes and / or lower temperatures, such as 37°C, result in lighter staining intensities. Higher incubation times and temperatures, such as about 16 to about 32 minutes and / or about 40°C to about 60°C, result in darker staining intensities. Notably, the staining intensity and / or contrast increases as the in situ synthesized Schiff's reagent remains in contact with the sample for a longer period of time. This suggests that the staining intensity and / or contrast can be "tuned" by changing the time (and / or temperature) that the in situ synthesized Schiff's reagent remains in contact with the biological sample. Compared to a control sample stained using a commercially available Schiff's reagent ( Figure 7C ) compared to samples stained with the in situ synthesized Schiff's reagent had at least equivalent staining performance.

[0224] Example 3 - Dye Concentration

[0225] Fig. 8A and 8B The results show that the dye and sulfite / SO 2 The concentration of the source varies the staining intensity and contrast of the Schiff reagent synthesized in situ. For thiosulfate, between about 0.02 M and about 0.8 M were tested. At 0.02 M, the conditions are approximately equimolar to 0.6% pararosaniline, so the staining is very weak. It is noteworthy that with increasing dye concentration (sulfite or SO 2 The staining intensity and / or contrast increased as the concentration of the source was held constant. For strong acid concentrations, we tested concentrations as low as 0.01 M and as high as 4 M. Lower acid concentrations resulted in darker staining. This suggests that staining intensity and / or contrast can be "tuned" by varying the concentration of the dye used in the in situ synthesis of the Schiff's reagent. Compared to control samples stained with commercially available Schiff's reagent ( Figure 8C ) compared to samples stained with the in situ synthesized Schiff's reagent had at least equivalent staining performance.

[0226] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. If necessary, various aspects of the embodiments may be modified to provide other further embodiments using concepts from various patents, applications, and publications.

[0227] Although the present disclosure has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. Therefore, it should be understood that many modifications may be made to the illustrative embodiments, and other arrangements may be designed without departing from the spirit and scope of the present disclosure as defined in the appended claims.

Claims

1. A dye composition comprising a compound having any one of formula (I) to formula (XII): in R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, branched or unbranched substituted or unsubstituted C1-C6 alkyl or cycloalkyl, -OH, -O-C1-C6 alkyl, or -N(R x )(R y ), where R x and R y is independently H or a branched or unbranched C1-C6 alkyl group optionally substituted with one or more halogen atoms, or wherein R 1 and R 2 Together, or R 3 and R 4 Together or R 5 or R 6 Together they form a C1-C6 alkyl group or one or more -N(R x )(R y ) group substituted 5-membered or 6-membered ring or heterocyclic ring; and A is a counterion; and Wherein the concentration of the compound of any one of formula (I) to formula (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition.

2. The dye composition according to claim 1, wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least the other two of them are branched or unbranched, substituted or unsubstituted C1-C6 alkyl groups or cycloalkyl groups; and wherein R x or R y At least one of them is H.

3. The dye composition according to claim 1, wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are C1-C2 alkyl groups; and wherein R x or R y At least one of them is H.

4. The dye composition according to claim 1, wherein the compound having any one of formula (I) to formula (XII) is selected from the group consisting of:

5. The dye composition according to any one of claims 1 to 4, wherein the compound having any one of formula (I) to formula (XII) has a molecular weight in the range of about 300 g / mol to about 600 g / mol.

6. The dye composition according to any one of claims 1 to 4, wherein the compound having any one of formula (I) to formula (XII) has a molecular weight in the range of about 300 g / mol to about 425 g / mol.

7. The dye composition according to any one of claims 1 to 6, wherein the concentration is in the range of between about 0.15% w / v to about 2.5% w / v based on the total volume of the dye composition.

8. The dye composition according to any one of claims 1 to 6, wherein the concentration is in the range of between about 0.15% w / v to about 1.8% w / v based on the total volume of the dye composition.

9. The dye composition according to any one of claims 1 to 8, further comprising an acid.

10. The dye composition according to claim 9, wherein the acid is a strong acid.

11. The dye composition according to claim 10, wherein the strong acid is 1M to 1.3M hydrochloric acid.

12. The dye composition according to claim 10, wherein the strong acid is 1M to 1.3M nitric acid.

13. The dye composition according to any one of claims 1 to 9, further comprising at least one of a solvent and / or an additive.

14. The dye composition according to any one of claims 1 to 9, further comprising a solvent and at least one additive.

15. The dye composition of claim 1, consisting essentially of the compound of any one of Formula (I) to Formula (XII) and a strong acid.

16. A sulfite composition comprising a sulfite or SO2 source selected from the group consisting of: pyrosulfites, bisulfites, dithionites, thiosulfates, sulfites and sulfurous acids; wherein the concentration of the sulfite or SO2 source is in the range of about 0.1% w / v to about 18% w / v based on the total weight of the sulfite composition.

17. A sulfite composition wherein the concentration is in the range of between about 1% w / v to about 9% w / v based on the total weight of the sulfite composition.

18. A sulfite composition wherein the concentration is in the range of between about 1% w / v to about 4% w / v based on the total weight of the sulfite composition.

19. The sulfite composition of claim 16, wherein the sulfite or SO2 source is not thiosulfate.

20. The sulfite composition of claim 19, wherein the sulfite composition further comprises an acid.

21. A kit comprising: (i) a dye composition according to any one of claims 1 to 15; and (ii) a sulfite composition according to any one of claims 16 to 20.

22. A kit comprising: (i) a dye composition that does not contain an acid; (ii) a sulfite composition; and (iii) an acid, wherein each of the dye composition, the sulfite composition, and the acid is present in a separate container.

23. A kit according to claim 22, wherein the dye composition comprises a compound having any one of formula (I) to formula (XII): in R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, branched or unbranched substituted or unsubstituted C1-C6 alkyl or cycloalkyl, -OH, -O-C1-C6 alkyl, or -N(R x )(R y ), where R x and R y is independently H or a branched or unbranched C1-C6 alkyl group optionally substituted with one or more halogen atoms, or wherein R 1 and R 2 Together, or R 3 and R 4 Together or R 5 or R 6 Together they form a C1-C6 alkyl group or one or more -N(R x )(R y ) group substituted 5-membered or 6-membered ring or heterocyclic ring; and A is a counterion; and Wherein the concentration of the compound of any one of formula (I) to formula (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition.

24. The kit according to claim 23, wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least the other two of them are branched or unbranched, substituted or unsubstituted C1-C6 alkyl groups or cycloalkyl groups; and wherein R x or R y At least one of them is H.

25. The kit according to claim 23, wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the other two are C1-C2 alkyl groups; and wherein R x or R y At least one of them is H.

26. The kit according to claim 23, wherein the compound of any one of Formula (I) to Formula (XII) is selected from the group consisting of:

27. The kit according to any one of claims 23 to 26, wherein the compound of any one of Formula (I) to Formula (XII) has a molecular weight in the range of between about 300 g / mol to about 600 g / mol.

28. The kit according to any one of claims 23 to 26, wherein the compound of any one of Formula (I) to Formula (XII) has a molecular weight in the range of between about 300 g / mol to about 425 g / mol.

29. The kit of any one of claims 23 to 28, wherein the dye concentration is in the range of between about 0.15% w / v to about 2.5% w / v based on the total volume of the dye composition.

30. The kit of any one of claims 23 to 28, wherein the dye concentration is in the range of between about 0.15% w / v to about 1.8% w / v based on the total volume of the dye composition.

31. A kit according to any one of claims 23 to 30, wherein the sulfite composition comprises a sulfite or SO2 source selected from the group consisting of: pyrosulfites, bisulfites, dithionites, thiosulfates, sulfites and sulfurous acid; wherein the concentration of the sulfite or SO2 source is in the range of between about 0.1% w / v to about 18% w / v based on the total weight of the sulfite composition.

32. The kit of claim 31, wherein the sulfite source concentration is in the range of between about 1% w / v to about 9% w / v based on the total weight of the sulfite composition.

33. The kit of claim 31, wherein the sulfite source concentration is in the range of between about 1% w / v to about 4% w / v based on the total weight of the sulfite composition.

34. The kit of any one of claims 23 to 31 , wherein the acid is a strong acid.

35. The kit of any one of claims 23 to 31, wherein the acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, hydrofluoric acid, trifluoroacetic acid, hydroiodic acid, sulfuric acid, nitric acid, chloric acid and perchloric acid.

36. A method of staining a biological sample, the method comprising sequentially dispensing the dye composition and the sulfite composition of the kit according to claim 21 to the biological sample.

37. The method of claim 36, wherein the dye composition is dispensed before the sulfite composition.

38. The method of any one of claims 36 to 37, wherein the temperature of the sample, the dye composition and / or the sulfite composition is in the range of between 30°C and about 50°C.

39. The method of any one of claims 36 to 37, wherein the temperature of the sample, the dye composition and / or the sulfite composition is in the range of between 30°C and about 40°C.

40. The method of any one of claims 36 to 39, wherein the sequentially dispensed dye composition and sulfite composition are allowed to remain in contact with the sample for at least 4 minutes.

41. The method of any one of claims 36 to 39, wherein the sequentially dispensed dye composition and sulfite composition are allowed to remain in contact with the sample for at least 8 minutes.

42. The method of any one of claims 36 to 37, wherein the amount of the dye composition dispensed to the sample is in a range between about 50 μL to about 200 μL; and wherein the amount of the sulfite composition dispensed to the sample is in a range between about 50 μL to about 200 μL.

43. The method of any one of claims 36 to 37, wherein the amount of the dye composition dispensed into the sample ranges from about 100 μL to about 200 μL; and The amount of the sulfite composition dispensed into the sample is in a range between about 100 μL to about 200 μL.

44. The method of any one of claims 36 to 37, wherein the amount of the dye composition dispensed to the sample is in a range between about 100 μL to about 150 μL; and wherein the amount of the sulfite composition dispensed to the sample is in a range between about 100 μL to about 150 μL.

45. A method of staining a biological sample, the method comprising simultaneously dispensing the dye composition and the sulfite composition of the kit according to claim 21 into the biological sample.

46. ​​The method of claim 45, wherein the temperature of the sample, the dye composition and / or the sulfite composition is in the range of between 30°C and about 50°C.

47. The method of claim 45, wherein the temperature of the sample, the dye composition and / or the sulfite composition is in the range of between 30°C and about 40°C.

48. The method of any one of claims 45 to 47, wherein the simultaneously dispensed dye composition and sulfite composition are allowed to remain in contact with the sample for at least 4 minutes.

49. The method of any one of claims 45 to 47, wherein the simultaneously dispensed dye composition and sulfite composition are allowed to remain in contact with the sample for at least 8 minutes.

50. The method of any one of claims 45 to 47, wherein the amount of the dye composition dispensed to the sample is in a range between about 50 μL to about 200 μL; and wherein the amount of the sulfite composition dispensed to the sample is in a range between about 50 μL to about 200 μL.

51. The method of any one of claims 45 to 47, wherein the amount of the dye composition dispensed to the sample is in a range between about 100 μL to about 200 μL; and wherein the amount of the sulfite composition dispensed to the sample is in a range between about 100 μL to about 200 μL.

52. The method of any one of claims 45 to 47, wherein the amount of the dye composition dispensed to the sample is in a range between about 100 μL to about 150 μL; and wherein the amount of the sulfite composition dispensed to the sample is in a range between about 100 μL to about 150 μL.

53. A method of staining a biological sample, the method comprising sequentially dispensing the dye composition, the sulfite composition and the acid of the kit according to any one of claims 23 to 35 to the biological sample.

54. A method according to claim 53, wherein the dye composition is dispensed before the sulfite composition and / or the acid.

55. The method of any one of claims 53 to 54, wherein the temperature of the sample, the dye composition, the acid and / or the sulfite composition is in the range of between 30°C and about 50°C.

56. The method of any one of claims 53 to 54, wherein the temperature of the sample, the dye composition, the acid and / or the sulfite composition is in the range of between 30°C and about 40°C.

57. The method of any one of claims 53 to 56, wherein the sequentially dispensed dye composition, acid, and sulfite composition are allowed to remain in contact with the sample for at least 4 minutes.

58. The method of any one of claims 53 to 56, wherein the sequentially dispensed dye composition, acid, and sulfite composition are allowed to remain in contact with the sample for at least 8 minutes.

59. The method of any one of claims 53 to 56, wherein the amount of the dye composition dispensed to the sample is in the range of about 50 μL to about 200 μL; wherein the amount of the acid dispensed to the sample is in the range of about 50 μL to about 200 μL; and wherein the amount of the sulfite composition dispensed to the sample is in the range of about 50 μL to about 200 μL.

60. The method of any one of claims 53 to 56, wherein the amount of the dye composition dispensed to the sample is in a range between about 100 μL to about 200 μL; wherein the amount of the acid dispensed to the sample is in a range between about 100 μL to about 200 μL; and wherein the amount of the sulfite composition dispensed to the sample is in a range between about 100 μL to about 200 μL.

61. The method of any one of claims 53 to 56, wherein the amount of the dye composition dispensed to the sample is in a range between about 100 μL to about 150 μL; wherein the amount of the acid dispensed to the sample is in a range between about 100 μL to about 150 μL; and wherein the amount of the sulfite composition dispensed to the sample is in a range between about 100 μL to about 150 μL.

62. A method of staining a biological sample, the method comprising simultaneously dispensing the dye composition, the sulfite composition and the acid of the kit according to any one of claims 23 to 35 into the biological sample.

63. The method of claim 62, wherein the temperature of the sample, the dye composition, the acid and / or the sulfite composition is in the range of between 30°C and about 50°C.

64. The method of any one of claims 62 to 63, wherein the temperature of the sample, the dye composition, the acid and / or the sulfite composition is in the range of between 30°C and about 40°C.

65. The method of any one of claims 62 to 64, wherein the simultaneously dispensed dye composition, acid, and sulfite composition are allowed to remain in contact with the sample for at least 4 minutes.

66. The method of any one of claims 62 to 64, wherein the simultaneously dispensed dye composition, acid, and sulfite composition are allowed to remain in contact with the sample for at least 8 minutes.

67. A method according to any one of claims 62 to 64, wherein the amount of the dye composition dispensed to the sample is in a range between about 50 μL and about 200 μL; wherein the amount of the acid dispensed to the sample is in a range between about 50 μL and about 200 μL; and wherein the amount of the sulfite composition dispensed to the sample is in a range between about 50 μL and about 200 μL.

68. The method of any one of claims 62 to 64, wherein the amount of the dye composition dispensed to the sample is in a range between about 100 μL to about 200 μL; wherein the amount of the acid dispensed to the sample is in a range between about 100 μL to about 200 μL; and wherein the amount of the sulfite composition dispensed to the sample is in a range between about 100 μL to about 200 μL.

69. The method of any one of claims 62 to 64, wherein the amount of the dye composition dispensed to the sample is in a range between about 100 μL to about 150 μL; wherein the amount of the acid dispensed to the sample is in a range between about 100 μL to about 150 μL; and wherein the amount of the sulfite composition dispensed to the sample is in a range between about 100 μL to about 150 μL.

70. A stained biological sample, the stained biological sample being prepared by: (i) dispensing a dye composition into the biological sample, the dye composition comprising (a) a compound having a 4-benzhydryl-2,5-cyclohexadiene-1-imine core, which is functionalized to include at least two substituted or unsubstituted amine groups, wherein the compound has a molecular weight in the range of about 300 g / mol to about 600 g / mol; and (b) an acid; and wherein the concentration of the compound is in the range of about 0.15% w / v to about 2.5% w / v based on the total volume of the dye composition; and (ii) dispensing a sulfite composition comprising a sulfite or SO2 source, wherein the concentration of the sulfite or SO2 source is in the range of about 0.1% w / v to about 18% w / v based on the total weight of the sulfite composition.

71. The stained biological sample of claim 70, wherein the dye composition is first dispensed into the biological sample.

72. The stained biological sample of claim 70, wherein the dye composition and the sulfite composition are dispensed simultaneously.

73. The stained biological sample of claim 70, wherein the acid is a strong acid.

74. according to the biological sample through dyeing described in claim 70, wherein said compound is selected from the group consisting of following 75. The stained biological sample of claim 74, wherein the acid is hydrochloric acid or nitric acid.

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