Enhancing fluorescence emission of near-infrared DYES in biological environments using biocompatible ionic liquids

Abstract

Disclosed herein is a method for enhancing molecular brightness of a near infrared (NIR) dye in aqueous solution, the method including at least the step of contacting the dye with a biocompatible ionic liquid (IL). In one aspect, the IL includes an organic cation such as, for example, choline, and an organic anion selected from decanoate, ethylenediamine tetraacetate, urocanate, nicotinate, pyridoxinate, orotate, citrate, hexadecanoate, biotinate, and deoxycholate, wherein the cation and anion are present in a ratio of from about 4:1 to about 1:1. Also disclosed are compositions containing the NIR dye and IL as well as methods of using the compositions for immunohistochemical staining, photothermal treatment, and medical imaging.

Description

ENHANCING FLUORESCENCE EMISSION OF NEAR-INFRARED DYES IN BIOLOGICAL ENVIRONMENTS USING BIOCOMPATIBLE IONIC LIQUIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 63 / 677,427, filed July 31 , 2024, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number 5R01 EB034086- 02 awarded by the National Institutes of Health and grant number 1757220 awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND

[0003] Over the last few decades, near-infrared (NIR) emitting materials have emerged as an extensive area of research due to their wide application in advanced electronics and medical research such as photodynamic therapy and bioimaging. NIR bioimaging materials utilize low- energy photons within the 700-1700 nm range (specifically NIR-I: 700-1000 nm and NIR-II: 1000- 1700 nm). This spectral region enables deeper tissue penetration, improving the visualization of internal anatomical structures by reducing light attenuation, scattering, and background interference caused by autofluorescence.

[0004] For practical applications, apart from inorganic semiconducting nanomaterials, molecularly tailorable organic dyes that can be functionalized and made biocompatible have recently gained more attention. In this direction, several synthetic strategies comprising an acceptor-donor based design with squarine, cyanine and BODIPY cores have been developed to match ideal NIR bioimaging agent criteria for the therapeutic window such as (a) aqueous solubility, (b) significant Stokes shift in the absorption / emission energy, and (c) effective absorption and emission profile along with high molar absorptivity, molecular brightness (M B) and fluorescence quantum yield (FQY). Clinical use of indocyanine green (ICG) as a contrast agent in medical procedures following FDA approval several decades ago served as another motivation for the dedicated research on squaraine and cyanine dyes. To enhance the NIR absorption, it is imperative to extend the TT-system around these dye motifs (squaraine and cyanine) via conjugated donors. However, in most cases the extension of rr-conjugation results in a decrease in the energy gap between the highest occupied molecular orbital (HOMO) and lowest occupiedmolecular orbital (LUMO), leading to more prevalent non-radiative decay events and low FQY of NIR dyes. Additionally, the extensive TT-conjugation leads to increased hydrophobicity and low aqueous solubility, posing additional requirements for the suitable functionalization for increasing aqueous solubility of NIR dyes.

[0005] To address these challenges, various strategies have been employed including the formulation of NIR dyes with proteins, encapsulation into nanoparticles, bioconjugation, and structural modifications to optimize molecular geometry. Each of these approaches, however, introduces its own set of challenges and complexities.

[0006] What is needed is a method for improving emissive properties of NIR dyes used in water. It would be desirable if the method were inexpensive and scalable and relied on materials that are biocompatible and efficient to produce. The present disclosure addresses these needs.SUMMARY

[0007] Disclosed herein is a method for enhancing molecular brightness of a near infrared (NIR) dye in aqueous solution, the method including at least the step of contacting the dye with a biocompatible ionic liquid (IL). In one aspect, the IL includes an organic cation such as, for example, choline, and an organic anion selected from decanoate, ethylenediamine tetraacetate, urocanate, nicotinate, pyridoxinate, orotate, citrate, hexadecanoate, biotinate, and deoxycholate, wherein the cation and anion are present in a ratio of from about 4:1 to about 1 :1. Also disclosed are compositions containing the NIR dye and IL as well as methods of using the compositions for immunohistochemical staining, photothermal treatment, and medical imaging.

[0008] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Many aspects of the present disclosure can be better understood with reference to thefollowing drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0010] FIGs. 1 A-1 B show molecular structure of (FIG. 1 A) choline-based ionic liquids possessing chemically diverse anions; (FIG. 1 B) different categories of NIR dyes used herein.

[0011] FIGs. 2A-2F show [Ch][Doc] significantly enhances the fluorescence of the SO3SQ NIR dye. The formation of [Ch][Doc] aggregates in water plays a crucial role in the fluorescence enhancement of SO3SQ. (FIG. 2A) The fluorescence profile of 1 pmol L‘1SO3SQ at 720 nm as a function of increasing concentrations of [Ch][Doc], (FIG. 2B) Fluorescence intensity of SO3SQ in the presence of different ionic liquids, plotted against their concentrations in aqueous solution at 298.15 K. (FIG. 2C) The effect of varying water content in a water / methanol binary solution on the fluorescence of SO3SQ. (FIG. 2D) Fluorescence quantum yield of SO3SQ, (FIG. 2E) Zeta potential, and (FIG. 2F) Hydrodynamic diameter of SC>3SQ-[Ch][Doc] complexes. In plot 2B, [Ch][Cit]-1 , -2, and -3 represent molar ratios of [Ch][Cit], where 1 :1 is denoted as 1 , 2:1 as 2, and 3:1 as 3, and [Ch][EDTA] is 4:1.

[0012] FIGs. 3A-3F show [Ch][Doc] enhanced the molecular brightness (MB) of all the tested dyes, regardless of their chemical composition. Due to its compact molecular structure and compatible spatial geometry, SO3SQ exhibited the highest fluorescence enhancement in the presence of [Ch][Doc] among the dyes tested (FIG. 3A) The relative extent of MB of different dyes with and without [Ch][Doc] at their corresponding emission maxima; Comparison of (FIG. 3B) SQ, squaraine analogues; (FIG. 3C) C5, cyanine analogues; and (FIG. 3D) commercially available ICG and IR-1061 dyes in presence of [Ch][Doc] as a function of its concentration in aqueous solution; (FIG. 3E) IR-1061 in aqueous solution in the absence, and (FIG. 3F) in the presence of 200 mmol L’1[Ch][Doc], under irradiation with a 1064 nm laser at 30% power. * in FIG. 3A implies dyes in presence of [Ch][Doc],

[0013] FIGs. 4A-4F show ionic liquid resulted in the extended stabilization of the excited states lifetime and preservation of rotational diffusion of SO3SQ in aqueous solutions of [Ch][Doc] preventing non-radiative decay loss of energy. Transient spectra (FIGs. 4A, 4C), Normalized single-wavelength (FIG. 4D), and transient anisotropy (FIGs. 4E-4F) of 100 pmol L-1SO3SQ in aqueous solution and 500 mmol L'1of [Ch][Nic] and [Ch][Doc] in aqueous solution of SO3SQ respectively.

[0014] FIGs. 5A-5D show thermal and photostability of the SO3SQ in presence of [Ch][Doc] at physiological conditions as the function of time. (FIG. 5A) The photostability profile of 1 mol L’1SO3SQ in absence and presence of [Ch][Doc] (FIG. 5A) At power 10 and 100 mW / cm2, abbreviated as P-10 and P-100; (FIG. 5B) thermal stability of profile of SO3SQ in presence of [Ch][Doc] at temperature 25, 37 and 50 °C for 48 hours; (FIGs. 5C-5D) Representative plots of fluorescence emission intensity of SC>3SQ-[Ch][Doc] at 37 and 50 °C.

[0015] FIGs. 6A-5D show the morphology of [Ch][Doc] aggregates encapsulating the SO3SQ dye in its hydrophobic cavities. Supporting the hypothesis of SO3SQ molecule inclusion within cagelike aggregates of [Ch][Doc], (FIG. 6A) Scanning electron micrograph images of 30 mmol L’1of [Ch][Doc]-SC>3SQ complexes; (FIG. 6B) enlarged view of aggregates; (FIG. 6C) scanning electron micrograph images of 200 mmol L1of [Ch][Doc]-SC>3SQ complexes; (FIG. 6D) hydropdyanmic (Dh) diameter of [Ch][Doc]-SC>3SQ complexes in aqueous solution.

[0016] FIGs. 7A-7G show a molecular dynamics simulation showing the de-aggregation of SO3SQ and surface mapping of water around SO3SQ in aqueous solution containing [Ch][Doc], Screenshots from MD trajectory at 0, 25, 50, 75, and 100 ns of a simulation for SO3SQ dye (FIG. 7A) in absence and (FIG. 7B) in the presence of [Ch][Doc] in water; structure and interactions formed by the most populated MD trajectory cluster for SO3SQ (FIG. 7C) in absence and (FIG. 7D) in the presence of [Ch][Doc] in water; (FIG. 7E) hydrophobic (orange) / hydrophilic (light blue) surface map for SO3SQ in water; (FIG. 7F) plot illustrating a radius of gyration within 100 ns MD simulation of SO3SQ dyes in water (blue) and SO3SQ in water in the presence of an [Ch][Doc] (green); and (FIG. 7G) plot illustrating a solvent accessible surface area within 100 ns molecular dynamics simulation of SO3SQ dyes in water (blue) and SO3SQ in water in the presence of [Ch][Doc] (green).

[0017] FIGs. 8A-8D show cytotoxicity and hemolysis profile shows the high biocompatibility of [Cho][Doc]; The bright staining of HEK293 cells detect human a-tubulin protein using AlexaFluor 647 conjugated antibody dye with [Ch][Doc], (FIG. 8A) Cell Viability; (FIG. 8B) Hemolysis with human red blood cells as function of concentration of [Cho][Doc], (FIG. 8C) Staining of HEK293 cells using DAPI, AlexaFluor 647 and merge profile; (FIG. 8D) Quantitative plot of maximum emission intensity of Alexa Fluor647 against concentration of [Ch][Doc], Significant difference in brightness observed between treatment as determined by ANOVA and post-hoc Tukey’s HSD test.

[0018] FIGs. 9A-9B show successful encapsulation of SO3SQ dye in [Ch][Doc] aggregatesdemonstrating the protective action of [Ch][Doc] towards HEK293 cells during photothermal treatment. (FIG. 9A) Live / cell assay showing the population of live and dead HEK293 cell after photothermal treatment using SO3SQ dye with and without [Ch][Doc] (scale bar = 500 pm); (FIG. 9B) Quantitative plot of number of dead cells without (control) and with [Ch][Doc] after laser exposure.

[0019] FIGs. 10A-10F show fluorescence emission profile of 1 pmol L’1of SO3SQ in aqueous media in presence of different ionic liquids as the function of their concentration at 298.15K.

[0020] FIGs. 11A-11H show the fluorescence emission profile of different NIR dyes in aqueous medium at concentration 1 pmol L‘1as a function of concentration of [Ch][Doc],

[0021] FIG. 12 shows normalized single-wavelength kinetics of 500 pmol L’1SO3SQ aqueous solution in presence of 0.5 M [Ch][Nic] solutions fit with biexponential decay functions.

[0022] FIG. 13 shows normalized single-wavelength kinetics of 100 pmol L’1SO3SQ aqueous solution in presence of 0.5 M [Ch][Nic] solutions fit with biexponential decay functions.

[0023] FIG. 14 shows normalized single-wavelength kinetics of 100 pmol L’1SO3SQ aqueous solution in presence of 0.5 M [Ch][Doc] solutions fit with biexponential decay functions.

[0024] FIG. 15 shows transient anisotropy of 100 pmol L-1SO3SQ aqueous solution in presence of 0.5 M [Ch][Nic] solutions (right axis, purple markers) overlaid with TA spectrum at 0.0 ps time delay.

[0025] FIG. 16 shows TA traces of parallel and perpendicular pump / probe orientation at 0.0 ps time delay of 500 pmol L1SO3SQ aqueous solution.

[0026] FIG. 17 shows TA traces of parallel and perpendicular pump / probe orientation at 0.0 ps time delay of 100 pmol L1SO3SQ aqueous solution in presence of 0.5 M [Ch][Nic] solutions.

[0027] FIG. 18 shows TA traces of parallel and perpendicular pump / probe orientation at 0.0 ps time delay of 100 pmol L1SO3SQ aqueous solution in presence of 0.5 M [Ch][Doc] solutions.

[0028] FIGs. 19A-19G show minimal impact of [Ch][Doc] on the photostability profile of SO3SQ in aqueous solution at P10. (FIGs. 19A-19B) Irradiation by the light source with and without [Ch][Doc] (FIGs. 19C-19D) In dark with and without [Ch][Doc]; (FIGs. 19E-19F) Increase in fluorescence emission upon cooling SO3SQ-[Ch][Doc] solution to room temperature after getting out of 37 and 50 °C; (FIG. 19G) fluorescence emission of SO3SQ-[Ch][Doc] solution at 25 °C at the function of time.

[0029] FIG. 20A shows scanning electron micrograph images of 30 mmol L’1of [Ch][Doc] complexes; FIG. 20B shows the same at 200 mmol L’1of [Ch][Doc] complexes.

[0030] FIGs. 21A-21B show minimal impact on critical aggregation concentration (cac) of [Ch][Doc] in presence of SO3SQ. (FIG. 21 A) Zeta potential and (FIG. 21 B) conductivity profile of [Ch][Doc] with and without SO3SQ.

[0031] FIGs. 22A-22C show quantification of brightness intensity of SO3SQ in HEK-293 live cells in (FIG. 22A) absence and (FIG. 22B) presence of [Ch][Doc]; (C) plot against intensity maximum of SO3SQ with and without [Ch][Doc], In each treatment group collected following confocal microscopy. Significant difference in brightness observed between treatment as determined by ANOVA and post-hoc Tukey HSD test.

[0032] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0033] Disclosed herein is a method to enhance the molecular brightness of near-infrared (NIR) dyes for bioimaging applications. In one aspect, the NIR-emitting forensic dye can be SO3SQ. In another aspect, ionic liquids (ILs) can be used to stabilize the dye’s excited states and increase fluorescent emission. In an aspect, ionic liquid choline deoxycholate (CDOC) was discovered to significantly enhance the molecular brightness and quantum yield of the NIR SO3SQ dye in water. In an aspect, the stabilization effect of CDOC on the NIR dye’s excited states represents an advancement in the development of more effective dyes for biomedical imaging, potentially leading to improved diagnostic capabilities in biological environments.

[0034] In a further aspect, this disclosure not only enhances the brightness of special dyes used for biological imaging but also significantly broadens their application in healthcare. In one aspect, by improving the visibility of these dyes with ionic liquids, medical professionals can achieve more detailed and accurate imaging, which is important for early detection and diagnosis of diseases. In another aspect, enhanced imaging can provide clearer insights into cellular behaviors andmolecular interactions within tissues, facilitating advancements in understanding complex diseases like cancer and neurological disorders.

[0035] Disclosed herein is the use of biocompatible ionic liquids (ILs) to enhance the optical properties of NIR dyes. A library of ILs paired with a variety of organic NIR dyes was systematically screened. In one aspect, formulations were identified that not only improved the brightness of these dyes but also maintained high biocompatibility, making them suitable for biological imaging applications.

[0036] Among the various combinations tested, the indolizine squaraine-based dye (SO3SQ) paired with choline deoxycholate ([Cho][Doc]) demonstrated the most promising results: SO3SQ showed a dramatic increase in molecular brightness from 365 to 34,900 in aqueous solutions with [Cho][Doc], representing a 96-fold increase compared to the control. This improvement is attributed to the stabilization of the dye’s excited states facilitated by IL aggregates that effectively "cage" the dye molecules, preventing aggregation quenching typically induced by water.

[0037] The stability and enhanced fluorescent properties of SO3SQ in the presence of [Cho][Doc] were corroborated through various experimental approaches, including transient absorption spectroscopy, excited state anisotropy measurements, dynamic light scattering, and steady-state fluorescence. Biocompatibility and Applications:[Cho][Doc] was found to be highly biocompatible with human cells, displaying low cytotoxicity levels and minimal hemolysis. This compatibility was demonstrated in ultra-bright staining applications of human a-tubulin protein in HEK293 cells, where [Cho][Doc] not only improved the resolution and brightness of cellular features but also exhibited photoprotective properties.

[0038] In one aspect, ionic liquids (ILs) are a class of low melting point (< 100 °C) organic salts comprised of asymmetric anion-cation combinations. In another aspect, ILs possess remarkable physicochemical properties such as high solvation capacity, thermal stability, high flash point and low volatility, leading to their use in a variety of applications. In a further aspect, the category of ILs is broadened to include organic salts with melting points ranging from 25 - 250 °C, collectively known as group of uniform materials based on organic salts (GUMBOS) which retain all the characteristics of traditional ILs while expanding the chemical space for more cation / anion combinations. In one aspect, the precise tunability of the chemical forces in IL / GUMs (ILs and GUMBOS) achieved by altering cation / anion combinations offers a huge advantage over conventional solvents in creating designer solvents for specific applications. In an additionalaspect, crafting IL / GUMs with high safety profiles, namely biocompatibility and biodegradability, is another essential criterion for their use in biological applications.

[0039] In an aspect, 12 distinct ionic liquids have been synthesized and characterized in order to investigate their potential in enhancing the fluorescence properties of NIR dyes. In a further aspect, an exceptionally intense fluorescence was observed from the SO3SQ NIR dye with 200 mmol L’1of choline deoxycholate ([Ch][Doc]) which is a 96-fold higher increase in MB compared to SO3SQ in an aqueous solution. In one aspect, these promising results inspired further exploration of [Ch][Doc] to seven other NIR dyes with varying molecular structures in an attempt to gain insights into the structure-function relationships governing their interactions. In a further aspect, the photophysical properties of the SC>3SQ-[Ch][Doc] were investigated system using techniques such as steady-state and transient absorption spectroscopy, along with time-resolved anisotropy measurements. In another aspect, additional insights were provided by dynamic light scattering studies, scanning electron microscopy (SEM) and computational simulations, which helped clarify how [Ch][Doc] aggregates facilitate SO3SQ encapsulation. Further in this aspect, this encapsulation protects SO3SQ from fluorescence quenching in water, significantly enhancing its brightness. Importantly, these investigations also confirmed the biocompatibility of [Ch][Doc], as it exhibited low cytotoxicity and did not induce hemolysis within the tested concentration range. In yet another aspect, the utilization of biocompatible ionic liquids ([Ch][Doc]) for in vitro high- resolution cell imaging has been demonstrated herein. The incorporation of [Ch][Doc] into the imaging process led to a notable improvement in both the resolution and brightness of cellular features. In a still further aspect, it revealed significant photoprotective properties. In one aspect, this investigation underscores the capacity of biocompatible ionic liquids to manipulate and enhance the emission properties of organic NIR dye molecules in aqueous settings, thereby paving the way for advanced applications in bioimaging, diagnostics, and therapeutic delivery in biological environments.Method for Enhancing Molecular Brightness

[0040] In one aspect, disclosed herein is a method for enhancing molecular brightness of a near infrared (NIR) dye in aqueous solution, the method including at least the step of contacting the dye with a biocompatible ionic liquid (IL). Further in this aspect, the NIR dye can be selected from SO3SQ, SQ, NMe3SQ, C5, SO3C5, NMe3C5, ICG, IR-1061, a derivative or variant thereof, or any combination thereof.

[0041] In another aspect, the IL includes an organic cation and an anion in a ratio of from about 4:1 to about 1 :1 , or of from about 2:1 to about 1:1 , or of about 4:1, 3.5:1 , 3:1 , 2.5:1 , 2:1, 1.5:1 , or about 1:1 , or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.

[0042] In one aspect, the anion can be selected from decanoate, ethylenediamine tetraacetate, urocanate, nicotinate, pyridoxinate, orotate, citrate, hexadecanoate, biotinate, deoxycholate, a derivative or variant thereof, or any combination thereof. In another aspect, organic cation can be choline.

[0043] In one non-limiting aspect, the NIR dye is SO3SQ and the ionic liquid is choline deoxycholate, wherein the choline and the deoxycholate are in a ratio of 1 :1.

[0044] In any of these aspects, using the disclosed method, molecular brightness is increased from about 350 times an original level to about 35,000 times an original level, or from about 350 times to about 10,000 times, about 350 times to about 5,000 times, or about 350, 500, 100, 5000, 10,000, 15,000, 20,000, 25,000, 30,000, or about 35,000 times, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.Compositions Containing NIR Dyes and Biocompatible ILs

[0045] In another aspect, disclosed herein is composition including an NIR dye and a biocompatible IL in water. In a further aspect, in the disclosed composition, the NIR dye can be selected from SO3SQ, SQ, NMe3SQ, C5, SO3C5, NMe3C5, ICG, IR-1061 , a derivative or variant thereof, or any combination thereof.

[0046] In another aspect, the IL includes an organic cation and an anion in a ratio of from about 4:1 to about 1 :1 , or of from about 2:1 to about 1:1 , or of about 4:1, 3.5:1 , 3:1 , 2.5:1 , 2:1, 1.5:1 , or about 1:1 , or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.

[0047] In one aspect, the anion can be selected from decanoate, ethylenediamine tetraacetate, urocanate, nicotinate, pyridoxinate, orotate, citrate, hexadecanoate, biotinate, deoxycholate, a derivative or variant thereof, or any combination thereof. In another aspect, organic cation can be choline.

[0048] In one non-limiting aspect, the NIR dye is SO3SQ and the ionic liquid is choline deoxycholate, wherein the choline and the deoxycholate are in a ratio of 1 :1.

[0049] In any of these aspects, the composition is not cytotoxic. In another aspect, the composition causes substantially no hemolytic activity in human red blood cells.Methods of Using the CompositionsMedical Imaging

[0050] In one aspect, disclosed herein is a method for performing medical imaging in a subject, the method including at least the step of administering a disclosed composition to the subject and visualizing a signal from the NIR dye. In a further aspect, the signal comprises a fluorescence signal. In a still further aspect, the subject is a human.Photothermal Treatment

[0051] In one aspect, disclosed herein is a method for performing photothermal treatment for a disease or disorder in a subject, the method including at least the step of administering a disclosed composition to a treatment location in the subject and irradiating the treatment location. In a further aspect, irradiation is performed with NIR light.

[0052] In another aspect, the disease or disorder can be cancer and the treatment location can be a tumor. In any of these aspects, the subject can be a human or another mammal.Immunohistochemical Staining

[0053] In yet another aspect, disclosed herein is a method for enhancing immunohistochemical staining of a cell or tissue, the method comprising applying an NIR dye conjugated to an antibody and an IL to the cell or tissue. In a further aspect, in the disclosed composition, the NIR dye can be selected from SO3SQ, SQ, NMe3SQ, C5, SO3C5, NMe3C5, ICG, IR-1061 , Alexa Fluor 637, a derivative or variant thereof, or any combination thereof.

[0054] In another aspect, the IL includes an organic cation and an anion in a ratio of from about 4:1 to about 1 :1 , or of from about 2:1 to about 1:1 , or of about 4:1, 3.5:1 , 3:1 , 2.5:1 , 2:1, 1.5:1 , or about 1:1 , or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.

[0055] In one aspect, the anion can be selected from decanoate, ethylenediamine tetraacetate, urocanate, nicotinate, pyridoxinate, orotate, citrate, hexadecanoate, biotinate, deoxycholate, a derivative or variant thereof, or any combination thereof. In another aspect, organic cation can be choline.

[0056] In one non-limiting aspect, the NIR dye is Alexa Fluor 647 and the ionic liquid is choline deoxycholate, wherein the choline and the deoxycholate are in a ratio of 1 :1. In any of these aspects, the method can further include using fluorescence microscopy to visualize the cell or tissue.

[0057] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0058] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0059] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0060] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0061] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates ofpublication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0062] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0063] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0064] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.Definitions

[0065] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0066] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a dye,” “a wavelength,” or “an ionic liquid,” include, but are not limited to, mixtures, combinations, or ranges of two or more such dyes, wavelengths, or ionic liquids, and the like.

[0067] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0068] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. 'about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0069] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1 % to 5%” should be interpreted to include not only the explicitly recited values of about 0.1 % to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0070] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts,sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0071] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0072] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).

[0073] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.ASPECTS

[0074] The present disclosure can be described in accordance with the following numbered aspects, which should not be confused with the claims.

[0075] Aspect 1. A method for enhancing molecular brightness of a near infrared (NIR) dye in aqueous solution, the method comprising contacting the dye with a biocompatible ionic liquid (IL).

[0076] Aspect 2. The method of aspect 1 , wherein the NIR dye comprises SO3SQ, SQ, NMe3SQ, C5, SO3C5, NMe3C5, ICG, IR-1061 , a derivative or variant thereof, or any combination thereof.

[0077] Aspect 3. The method of aspect 1 or 2, wherein the IL comprises an organic cation and an anion in a ratio of from about 4: 1 to about 1 :1.

[0078] Aspect 4. The method of aspect 3, wherein the anion comprises decanoate, ethylenediamine tetraacetate, urocanate, nicotinate, pyridoxinate, orotate, citrate, hexadecanoate, biotinate, deoxycholate, a derivative or variant thereof, or any combination thereof.

[0079] Aspect 5. The method of aspect 3 or 4, wherein the organic cation comprises choline.

[0080] Aspect 6. The method of any one of aspects 1-5, wherein the NIR dye is SO3SQ and the ionic liquid is choline deoxycholate, wherein the choline and the deoxycholate are in a ratio of 1 : 1.

[0081] Aspect 7. The method of any one of aspects 1-6, wherein molecular brightness is increased from about 350 times an original level to about 35,000 times an original level.

[0082] Aspect 8. A composition comprising an NIR dye and a biocompatible IL in water.

[0083] Aspect 9. The composition of aspect 8, wherein the NIR dye comprises SO3SQ, SQ, NMe3SQ, C5, SO3C5, NMe3C5, ICG, IR-1061 , a derivative or variant thereof, or any combination thereof.

[0084] Aspect 10. The composition of aspect 8 or 9, wherein the IL comprises an organic cation and an anion in a ratio of from about 4:1 to about 1:1.

[0085] Aspect 11. The composition of aspect 10, wherein the anion comprises decanoate, ethylenediamine tetraacetate, urocanate, nicotinate, pyridoxinate, orotate, citrate, hexadecanoate, biotinate, deoxycholate, a derivative or variant thereof, or any combination thereof.

[0086] Aspect 12. The composition of aspect 10 or 11 , wherein the organic cation comprises choline

[0087] Aspect 13. The composition of any one of aspects 8-12, wherein the NIR dye is SO3SQ and the ionic liquid is choline deoxycholate, wherein the choline and the deoxycholate are in a ratio of 1 :1.

[0088] Aspect 14. The composition of any one of aspects 8-13, wherein the composition is not cytotoxic.

[0089] Aspect 15. The composition of any one of aspects 8-14, wherein the composition causes substantially no hemolytic activity in human red blood cells.

[0090] Aspect 16. A method for performing medical imaging in a subject, the method comprising administering the composition of any one of aspects 8-15 to the subject and visualizing a signal from the NIR dye.

[0091] Aspect 17. The method of aspect 16, wherein the signal comprises a fluorescence signal.

[0092] Aspect 18. The method of aspect 16 or 17, wherein the subject is a human.

[0093] Aspect 19. A method for performing photothermal treatment for a disease or disorder in a subject, the method comprising administering the composition of any one of aspects 8-15 to a treatment location in the subject and irradiating the treatment location.

[0094] Aspect 20. The method of aspect 19, wherein irradiation is performed with NIR light.

[0095] Aspect 21. The method of aspect 19 or 20, wherein the disease or disorder comprises cancer.

[0096] Aspect 22. The method of any one of aspects 19-21 , wherein the treatment location comprises a tumor.

[0097] Aspect 23. The method of any one of aspects 19-22, wherein the subject is a human.

[0098] Aspect 24. A method for enhancing immunohistochemical staining of a cell or tissue, the method comprising applying an NIR dye conjugated to an antibody and an IL to the cell or tissue.

[0099] Aspect 25. The method of aspect 24, wherein the NIR dye comprises SO3SQ, SQ, NMe3SQ, C5, SO3C5, NMe3C5, ICG, IR-1061, Alexa Fluor 647, a derivative or variant thereof, or any combination thereof.

[0100] Aspect 26. The method of aspect 24 or 25, wherein the IL comprises an organic cation and an anion in a ratio of from about 4:1 to about 1:1.

[0101] Aspect 27. The method of aspect 26, wherein the anion comprises decanoate, ethylenediamine tetraacetate, urocanate, nicotinate, pyridoxinate, orotate, citrate, hexadecanoate, biotinate, deoxycholate, a derivative or variant thereof, or any combination thereof.

[0102] Aspect 28. The method of aspect 26 or 27, wherein the organic cation comprises choline

[0103] Aspect 29. The method of any one of aspects 24-28, wherein the NIR dye is Alexa Fluor 647 and the ionic liquid is choline deoxycholate, wherein the choline and the deoxycholate are in a ratio of 1 :1.

[0104] Aspect 30. The method of any one of aspects 24-29, further comprising using fluorescence microscopy to visualize the cell or tissue.EXAMPLES

[0105] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.Example 1 : Materials and Methods

[0106] Materials The materials required for the synthesis of ionic liquids were procured from Sigma-Aldrich, the brief details regarding the reactants are as follow: choline bicarbonate (-80% in H20 #C7519-100 mL), deoxycholic acid (>98% #D2510-100G), pyridoxine (>98% #P5669- 25G), orotic acid monohydrate (97%, #O8402-25G), citric acid monohydrate (>99.0%, C1909- 25G), ethylenediaminetetraacetic acid (EDTA) anhydrous, powder (>99%, #EDS-100G), decanoic acid (> 98.0%, #C1875-100G), palmitic acid (>99% #P0500-10G), nicotinic acid (>98%#N4126-100G), 4-imidazoleacrylic acid (99% #859796-5G). D-Biotin (#B002-5GM) is purchased from Caisson Labs. COSTAR 96-well opaque black-bottom and 96-well clear plates (#353075) were obtained from Corning. 6-well polystyrene tissue culture treated multiple well plates (#229106) obtained from Celltreat. DMEM / High glucose without sodium pyruvate and L- glutamine cell culture solution (#SH30081.FS), Mammary Epithelial Cell Growth Medium (MEGM) BulletKit (CC-3151 & CC-4136, #CC-3150) was purchased from Lonza, Dulbecco's Phosphate Buffered Saline (DPBS), Sterile, 7.0 - 7.6, without calcium and magnesium, liquid (#SH30028.02), and trypsin-EDTA (#SH30042.01) were obtained from the Cytiva life sciences. Penicillinstreptomycin (#P4458-100ML), Fetal bovine serum (FBS, #F0926-500ML), and Cholera toxin lyophilized powder from Vibrio cholerae (>90%, #C8052-1MG) were purchased from Sigma Aldrich. CellTier-Glo Luminescent cell viability assay (#G7570) was purchased from Promega Corporation. The MCF-10A and HEK-293 cell lines were obtained from ATCC. MCF-10A cell line was cultured in MEGM medium supplemented with 10% v / v FBS, 100 ng / mL cholera toxin, 1% penicillin-streptomycin (penstrap), 20 ng / mL epidermal growth factor, 500 ng / mL hydrocortisone,5% Chelex-treated horse serum, and 0.01 mg / mL human insulin. Human blood is purchased from Bio-IVT.

[0107] Nuclear Magnetic Resonance (NMR) spectroscopy.1H NMR characterization of the synthesized ILs and dyes was conducted using Bruker Ascend 400 MHz spectrometer.

[0108] Dynamic Light Scattering (PLS). Changes in the hydrodynamic diameter (Dh) and zeta potential (surface charge) during [Ch][Doc]-SO3SQ complexation was monitored using Zetasizer Pro, NanoZS (from Malvern Instruments, UK) equipped with a He-Ne laser (with avalanche photodiode detector QE > 50 % 633 nm, 4 mW). The data was recorded in polystyrene cuvette (DTS0012 cuvette, #D-51588) at scattering angle of 173° to the incident beam, at 25 °C. The measurements were performed in triplicates and each reading was averaged from 15 internal runs. Zeta potential measurements was performed using disposable zeta cell (DTS1070).

[0109] Cytotoxicity. The cytotoxicity of [Ch][Doc] was determined using HEK293 and MCF10A cells. Both MCF10A and HEK293 cells were grown in cell culture treated 96-well plates at cell density 1.5 x 104per well in MEGM and DM EM media respectively. The cells were supplemented with 1% pencillin-straptomycin and 10% FBS under standard conditions (incubation at 37 °C and 5% CO2). The MCF10A cells were added with 100 ng mL'1cholera toxin and growth hormones. At cells confluency of -80%, the [Ch][Doc] was treated to the cells from concentration 0 pmol L'1to 450 pmol L’1and then incubated for 24 h. After 24 h, the cell media present in each well was replaced by 50 pL of new media and 50 pL CellTiter-Glo(R) luminescent cell viability reagent. The prepared 96 well plate was incubated for 15 mins at 37 °C and then read for luminescence at microplate reader (Biotek H1 Synergy Hybrid Multi-mode). The negative control was used for calculating the percentage cell viability.

[0110] Hemolysis. Hemolysis assays were performed using previously reported protocol. Briefly, the red blood cells (RBC) from commercially available human whole blood were isolated and washed two times with saline. The RBC stock was prepared by diluting the acquired RBCs to 1 :50 with saline which then treated with [Ch][Doc] at concertation 20, 80, 160, 300 pmol L'1in quadruplicate in 96-clear well plate. After treatment, the 96-plate was incubated at 37 °C for 1 h, then from each well, 200 pL volume was transferred to a separate 1.5 mL plastic tube and centrifuged at 4 °C at 500g for 10 min. From each tube, 100 pL supernatant was collected to measure absorbance at 405 nm using UV-vis microplate reader. The experiment was performed with positive (20% Triton-X-100) and negative (1 x PBS pH 7.4) control under same conditions which was used to calculate the percentage hemolysis by subtracting the negative control (1 xPBS, i.e., minimum baseline) from each absorbance value and then normalized against positive control (20% Triton-X-100).

[0111] Photostabilitv Study. The photostability of SO3SQ was investigated at a concentration of 1 mol L’1by illuminating an aqueous solution of SO3SQ, both in the presence and absence of 200 mmol L-1[Ch][Doc], Control samples were kept in the dark to assess any potential degradation not induced by light exposure. For the illumination, a Thorlabs SLS201 Stabilized Fiber-Coupled Tungsten Halogen Light Source (300-2600 nm) with a universal power adapter was utilized. The samples were exposed to white LED light at a power intensity of 10 and 100 mW cm-2s“1, with a spectral range covering 400 to 900 nm. The experiments were conducted under ambient conditions, without additional precautions to remove oxygen, allowing for potential oxidative effects to occur naturally. The degradation of SO3SQ was monitored periodically using steady-state fluorescence spectroscopy, focusing on the decrease in fluorescence at A = 720 nm as an indicator of photostability. This allowed for the comparison of the compound’s behavior in light-exposed conditions with and without the presence of [Ch][Doc], as well as in control conditions kept in the dark.

[0112] UV-Vis-NIR and Steady State Fluorescence: The UV-VIS-NIR spectra of the dye samples were recorded using Agilent Cary 5000 UV-Vis-NIR spectrometer. The fluorescence spectra were recorded with Horiba PTI QuantaMaster QM-8075-21 fluorometer in NIR region using InGaAs detector (1427C-AU Horiba). IR-1061 in DCM were used as reference standards to measure absorption and fluorescence. The FQY is calculated by using the following equation 1.where E represents the integrated emission counts, A is 1 x10-Atimes with the superscript A indicating the absorbance at the excitation wavelength. The symbol q refers to the refractive index of the solvent, and denotes the quantum yield.

[0113] Time-resolved Absorption. A 1 kHz regeneratively amplified TkSapphire laser (Coherent Astrella, Santa Clara, California) with a 7 W, 100 fs output pulse centered at 800 nm was split with a 85-15 beamsplitter to generate pump and probe beams. To generate the pump, the reflected portion of the 800 nm output was directed into a commercial optical parametric amplifier (OPerA Solo, Vilnius, Lithuania) to generate the 650 nm pump. Both the output of the OPerA Solo as well as the remainder of the originally transmitted 800 nm light were directed into a commercial transient absorption spectrometer (Ultrafast Systems Helios, Sarasota, Florida). The pump pulsewas chopped at 500 Hz before being depolarized and focused with a 350 mm focal length lens to the sample position. The remaining 800 nm light was first passed onto a mechanical delay stage before being focused onto a translating CaF2crystal to generate a visible white light continuum from 400 to 800 nm. The white light was then filtered to remove any remaining fundamental light and split into probe and reference beams. The reference beam is then reflected into a separate camera to account for jitter and intensity fluctuations. Ultrafast data for each sample were collected by averaging 3 scans with 2 s of averaging at each time delay and corrected with a polynomial to account for temporal chirp. Due to the small lasers spot size at the area of excitation, power density measurements are difficult. However, as focal length is constant throughout all scans, excitation power can be directly compared in this case. A power of 300 Wwas used for TAS spectra, however, additional power dependent studies with 35 and 500 pW were done on select samples and the lifetime trends hold, leading the inference that the lifetime trends, within reason, are not power dependent. All power measurements were made with a Coherent LaserCheck - Laser Power Meter.

[0114] For anisotropy measurements, the depolarized pump beam was passed through a polarizer. The polarization of the pump beam was then checked relative to the horizontally polarized probe beam such that “Parallel” refers horizontal polarization of both beams, IHH, and “Perpendicular” refers to a horizontal polarization of the probe beam and vertical polarization of the pump, IVH- A power of 300 pWwas used for all anisotropy experiments. To acquire anisotropy from parallel and perpendicular measurements, calculations using Equation 2,where r(A,t) is the anisotropy and AOD(A,t) is the change in optical density, both as a function of probe wavelength and delay time.

[0115] All samples were held in a 2 mm quartz cuvette (FireflySci, Inc., Staten Island, New York). Dye samples were at 100 pM in water, and IL / Dye solutions were at 0.5 M IL / Dye concentrations. Kinetic fitting at single wavelengths was performed withing the Igor Pro 8 software package. Lifetime measurements were determined by fitting the absorption maximum with biexponetial decay function, Equation 3,

[0116] where Ai and A2represent the weight of lifetimes TI and T2, respectively. From these lifetime values, an average lifetime Tavgwas calculated by equation 4.

[0117] Computational Studies: (a) Density functional theory (DFT): Quantum chemical analyses of the SO3SQ dye and the dye interacting with the anion portions of the ionic liquids (specifically deoxycholate, nicotinic anion, and ALA) are analyzed via density functional theory (DFT) in the Gaussian16 quantum chemistry program. These anions are chosen for exhibiting strong, middle, and weak intensities from experiment. All computations include implicit treatment of the water solvent. The molecular geometries are optimized with B3LYP / 6-311G(d,p), and then time- dependent-DFT computations with CAM-B3LYP / 6-311G(d,p) and PBEO / 6-311G(d,p) provide the electronically excited state transition energies and oscillator strengths. The latter correlate to the intensities observed in experiment. The sulfoxyl chains are deleted from the computational analysis as these are recognized not to contribute to the excitation energy enough to be considered and add significant computational time and complexity. These are replaced with methyl groups creating phenyl methyl ether end groups.

[0118] Molecular Dynamics: The Schrodinger Software Package, a widely used molecular modeling tool, was employed to perform computational calculations to simulate two scenarios: (1) the behavior of SO3SQ dye molecules in an aqueous solution and (2) the behavior of SO3SQ dye molecules in an aqueous solution in the presence of an ionic liquid. In practical experiments, the dye concentration in the solution was as low as 10 pM. The system that would ideally mimic this dye concentration would be too large, thus, being costly in terms of computational resources and excessively time-consuming. Therefore, the system was simplified by increasing the concentrations of both the dye and ionic liquid, which allowed reduction of the size of the system while maintaining its relevance. In the first scenario, 3 SO3SQ dye molecules, 6 Na+counter-ions, and 9991 water molecules were modeled (a total of 10,000 molecules). The second model included 3 SO3SQ dye molecules, 6 Na+ions, 300 choline anions, 300 deoxycholate anions, and 9391 water molecules (a total of 10,000 molecules). Obtained models were used to perform a 100 ns Molecular Dynamics simulation using a Desmond Module. The OPLS3e force field was utilized to perform the simulation under NPT conditions, at a target temperature of 300.0 K and pressure of 1.01325 bar, with a recording interval of 25 ps. Prior to the simulation, the model was relaxed using a default relaxation protocol. The Maestro interface was used to analyze the trajectories obtained from the simulation and performed clustering based on the dye molecules' Root Mean Square Deviation (RMSD).

[0119] Immunocytochemistry (IHC). Human embryonic kidney (HEK293) cells were used for immunocytochemistry. HEK cells were grown under standard conditions (37 °C, 5 % CO2, DMEM media with 10 % FBS and 1 % antibiotic-antimycotic) on sterile microscope cover glass or coverslip with thickness of 0.17 mm (Fisher scientific) in 6-well tissue culture plates in a sterile tissue culture hood. The cells were fixed with 4% paraformaldehyde for 20 minutes, washed 5 times with 5 minutes incubation at room temperature (RT) for each wash. Washes were done using PBS supplemented with 0.1% Triton X-100. The cells were then blocked for 30 minutes at RT in PBS supplemented with 0.1% Triton X-100 and 1 % BSA. After blocking, the primary antibody which is the Anti-a-Tubulin mouse monoclonal IgG (Merck) diluted to 1 :500 in the blocking solution was added to the cells for overnight incubation at 4 °C. Following primary antibody incubation, experimental cells were washed as before, and tubulin detection was performed using alexaFluor 647. For control, cells were washed with PBS once after staining with alexaFluor 647 and then sections were subsequently counterstained with DAPI Fluoromount-G (SouthernBiotech) and mounted under coverslips. For experiment samples, cells were incubated for 30 minutes with different concentrations of [Ch][Doc] (300 ug / mL, 600 pg mL'1, 1 pg mL'1, 3 pg mL'1, 9 pg mL'1, 12 pg mL’1) after staining with alexaFluor 647. The sections for experiment samples were washed once with PBS, counterstained with DAPI Fluoromount-G and was then mounted under coverslips. Imaging of cells was done after sealing the coverslips on microscope slide 1 .0 mm (Fisher scientific). Fluorescence of tubulin in HEK cells was observed with excitation wavelength of 653 nm using a Leica Stellaris STED confocal microscope. In the control samples, cells were not washed with [Ch][Doc], An ANOVA test with post-hoc Tukey HSD analysis was used to assess the statistical brightness intensity between control and [Ch][Doc] treated cells.

[0120] In-vitro Photothermal Treatment of live HEK293 cells. HEK293 cells near 100% confluency were treated with 500 ng of SO3SQ and incubated overnight in 5% CO2 at 37 °C. A STED laser (750 nm) at 100% intensity was used to irradiate cells for 10 min, followed by incubation for 2 h. Afterward, cell death was assessed with a LIVE / DEAD Cell Imaging KitTM Invitrogen following the manufacturer’s protocols. Confocal microscopy was then used to assess cell death through fluorescence (528 nm excitation, > 617 nm emission) and live cells (FITC) in the green channel. The number of dead cells was counted in three biological replicates. In control sample, SO3SQ is not encapsulated with [Ch][Doc] and in the treatment samples, dyes were encapsulated in 40 ug / mL [Ch][Doc], All samples were washed with 1 mL PBS before confocal imaging.

[0121] Quantification of Brightness Intensity of SO3SQ in Live Cells. 250 ng and 500 ng of SO3SQ dye with and without encapsulated in [Ch][Doc] was incubated in HEK293 cells overnight.[Ch][Doc] is not added to the control sample, and in the treatment samples, dyes were encapsulated in 40 pg mL1[Ch][Doc], All samples were washed with 1 ml_ PBS before confocal imaging. Confocal microscopy was used to assess brightness intensity in control and treatment group. An ANOVA test with post-hoc Tukey HSD analysis was used to assess the brightness intensity between control and [Ch][Doc] treated cells.

[0122] Scanning Electron Microscope (SEM): SEM samples were prepared with concentrations of 30 and 200 mmol L1[Ch][Doc] in MilliQ water. 10 pL of this solution were added onto plasma- cleaned aluminum SEM sample stubs measuring 9.5 x 9.5 mm (JEOL, #10-005110-50). After drop casting 10 pL of the AuNP solution onto the stubs, the samples were left to dry overnight at 4 °C in a sealed environment to avoid dust accumulation. The next day, all samples were sputter- coated with gold for making samples conducting for imaging (16.5 mm, 35 mA, 200 s). Imaging was performed using a JSM-7200 FLV field-emission scanning electron microscope (FESEM).

[0123] Dye Imaging Studies: To visually compare the fluorescence enhancement of the NIR dyes in the presence of [Ch][Doc], a 1 pmol L'1aqueous solution of IR-1061 was prepared, both with and without 200 mmol L‘1[Ch][Doc], Both solutions were simultaneously irradiated with a 1064 nm laser at 30% power using the PRISM in vivo SWIR imaging system, and fluorescence images were captured with the NIR camera.

[0124] Statistical Analysis: The statistical analysis on the obtained data was performed in Microsoft Excel 365. All the data shown herein is presented as mean value ± standard deviation of at least three independent experiments. A two-tailed critical student’s t-test was used to compare the two groups.Example 2: Results and Discussion

[0125] Synthesis and characterization of ionic liguids: 12 ionic liquids have been synthesized to evaluate the impact of the molecular structure of ionic liquid anions on the fluorescence enhancement profile of different NIR dyes (FIGs. 1A-1B). These specific structures were chosen due to the variations in their chemical functionality, which significantly affect their physicochemical properties and in turn influence the fluorescence characteristics of NIR dyes. The ionic liquids were synthesized through an acid-base neutralization reaction between choline bicarbonate and different anions listed in Examples 1 and 3. In brief, choline bicarbonate was added dropwise to the equimolar aqueous solution of acidic anions dissolved in methanol, with continuous stirring at 50 °C for a duration of 12 hours to ensure complete reaction. T 0 prevent degradation of the anionic components of ILs, the solvent was removed under reduced pressure at 50 °C for 4 hours,resulting in transparent viscous liquid except [Ch][Doc] and [Ch][Cit] 1:1 which are white amorphous powders at room temperature. Subsequently, the ILs were kept in a vacuum oven to further reduce the water content The synthesized ILs were characterized using1H and13C NMR spectroscopes, mass spectroscopy, DSC and by Karl Fisher titration to measure the water content.

[0126] Fluorescence profile of SO3SQ with ionic liquids. SO3SQ exhibits a low FQY (<t> < 1%) and MB (365) in water. The leading cause of the poor optical properties in water was identified as nonradiative decay pathways and aggregation induced quenching by thermal relaxations. To assess the capacity of ionic liquids to improve the emissive behavior of this dye, the steady state fluorescence profile of a 1 pmol L'1aqueous solution of SO3SQ was measured as a function of concentration of twelve different choline-based ILs (FIGs. 2A-2B and 10A-10F). Interestingly, a several-fold increase in the fluorescence intensity of SO3SQ was observed with the addition of ILs. Among all ILs tested, [Ch][Doc] was found to produce the maximum fluorescence enhancement 95-fold increase) followed by [Ch][Hdec] and [Ch][Dec] (FIGs. 2B and 10A-10F). It is important to note that the emission intensity can vary due to factors such as deviations in lamp intensity between measurements. Although these variations are not significant within the time span of successive measurements, they can still reflect the extent of fluorescence enhancement.

[0127] It is hypothesized that this enhancement in emissive properties is due to the ability of the [Ch][Doc] to engage in hydrophobic and electrostatic interactions with SO3SQ ions. This would limit the access of fluorescence-quenching water molecules to make contact with the surface of SO3SQ ions, and prevent aggregation of the SO3SQ ions. At this concentration range of IL (25 to 50 mmol L’1) the high molar dilution of SO3SQ in [Ch][Doc] environment i.e., 1:25000 and 1 :50000 molecules of SO3SQ to [Ch][Doc], would strongly favor the separation of hydrophobically stacked SO3SQ units and their gradual inclusion in [Ch][Doc] aggregates above its critical aggregation concentration (cac). After reaching 100 mmol L'1, the rate of fluorescence enhancement of SO3SQ begins to slow down and becomes constant after 200 mmol L'1, suggesting the solvation of almost all of the SO3SQ ions present in the solution by [Ch][Doc] aggregates.

[0128] To further understand the dynamics of complexation between [Ch][Doc] and SO3SQ, the fluorescence of [Ch][Doc]-SC>3SQ was recorded in a methanol :water binary solution at different ratios, namely 1 :0, 1:0.25, 1 :0.50 and 1 :0.75 v / v (FIGs. 2C-2D). It was found that with increasing percentage of methanol, the fluorescence intensity and FQY of [Ch][Doc]-SO3SQ solution dropssharply. Similarly, the DLS profile showed a substantial decrease in the surface charge and hydrodynamic diameter (Dh) of [Ch][Doc]-SC>3SQ aggregates with increase in methanol in the solvent mixture (FIGs. 2E-2F). These observations suggest the solvation of both [Ch][Doc] and SO3SQ by methanol molecules, resulting in collapse of [Ch][Doc] aggregates, and, in turn, fluorescence quenching of SO3SQ by methanol molecules. These findings also support the notion that the observed enhancement of emission arises from the formation of [Ch][Doc] aggregates in aqueous solution followed by the incorporation of SO3SQ molecules into the aggregates.

[0129] In the case of [Cho][Hdec] and [Cho][Dec], their amphiphilic nature indicates that they likely interact with SO3SQ via hydrophobic interactions through their anionic alkyl chains and thus solubilize SO3SQ in their hydrophobic micellar core at and above their critical micelle concentration (cmc), resulting in an increase in fluorescence of SO3SQ compared to aqueous solutions. However, the smaller surface area and greater flexibility of the single alkyl chains of [Dec] and [Hdec] cause relatively weaker hydrophobic interactions with SO3SQ compared to [Ch][Doc] and thus generate less fluorescence enhancement of SO3SQ. Contrarily, the relatively rigid curved tetracyclic skeleton of [Doc] with a hydrophilic concave face and a hydrophobic convex face allows it to interact more strongly with SO3SQ molecules by hydrophobic interactions. This is also supported by the close resemblance of deoxycholate with bile acids such as cholic acid and chenodeoxycholic acid which efficiently solubilize the lipophilic fat molecules in the biological environment.

[0130] The remaining investigated ILs were found to contribute minimally towards elevating the fluorescence of SO3SQ. It is hypothesized that this is caused by the hydrophilic nature of these ILs, leading to their relatively weaker hydrophobic interactions with SO3SQ dye. This highlights the importance of maximizing the hydrophobic interactions of ILs with SO3SQ. However, the molecular geometry of both IL and dye plays an important role in inducing maximum fluorescence enhancement. Similar fluorescence enhancement of dyes has also been observed in SO3SQ and other dyes with human serum albumin (HSA) where the inclusion of dye molecule in the different preformed hydrophobic cavities inside the protein yield intensified fluorescence. In a similar fashion, various ILs have been reported to interact with different enzymes and proteins and preferentially occupied the hydrophobic cavities of these proteins.

[0131] Complexation of FChlfDocI with different dyes. After screening the library of I Ls possessing different molecular structures with SO3SQ, the complexation of [Ch][Doc] with different NIR dyes was evaluated to understand the breadth of potential usage of this IL for fluorescenceenhancement. For this reason, two bench mark dyes, ICG and IR 1061 , squarine derivatives (SQ, NMe3SQ, SO3SQ), and cyanine derivatives (C5, SO3C5, NMe3C5) (FIGs. 3A-3F and 11A-11H) were investigated with [Ch][Doc], In case of squaraine dyes, the presence of [Ch][Doc] induced an exponential rise in the fluorescence of NMe3SQ and SO3SQ, while for SQ the fluorescence enhancement took place linearly (FIG. 3B). This is suggestive of greater aqueous partial solubility of NMe3SQ and SO3SQ. This is likely due to their ionic charges bringing the [Doc] anion and NMe3SQ / SO3SQ together. Consequently, this enhances the hydrophobic interaction between [Doc] and NMe3SQ and SO3SQ. The relative fluorescence enhancement for the squaraine dyes at 200 mmol L1of [Ch][Doc] was found to be 171 -fold > 95-fold > 88-fold for SQ, SO3SQ, and NMe3SQ respectively compared to water alone. Likewise, the MB and FQY of these dyes follows a similar trend: 64,400 > 47,600 > 34,900 and 49.7 % > 41.0 % > 38.9 % for SQ, NMe3SQ and SO3SQ respectively (FIG. 4A, Table 1). Therefore, there is a 12-to-400-fold increase in the FQY of squaraine dyes in aqueous solutions in the presence of [Ch][Doc], Based on their aqueous solubility and the presence of polar functional groups, the relative hydrophobicity of these dyes follows the order: SQ > NMe3SQ > SO3SQ. This suggests that SO3SQ exhibits a higher degree of solubility in aqueous solutions compared to its analogues. Additionally, the enhanced aqueous solubility of SO3SQ facilitates ease of use and reduces its tendency to be absorbed by biological tissues. These characteristics are attributed to its relatively lower partition coefficient values in comparison to its more hydrophobic counterparts. Therefore, the SO3SQ-[Ch][Doc] was considered as the best formulation among these dyes which is still several fold brighter than the other tested dyes.

[0132] In the case of C5 dyes, the fluorescence enhancement occurs in a similar fashion and extent among different C5 analogues (FIG. 3C) which might be due to their extended / open molecular structure and low geometric / spatial compatibility with the [Ch][Doc] aggregates. This is also reflected in the MB (in the order of 100) and FQY (10-to-20-fold increase) of C5 dye formulations with [Ch][Doc], which are relatively low compared to the squaraine dyes. ICG, which is a C7 cyanine dye, is not found to exhibit any significant change in the fluorescence in the presence of [Ch][Doc], and, therefore, was employed as a standard here. It is important to note that earlier publications used a non-aqueous standard and 5 pmol L’1of dyes to calculate the FQY, with HITCI in ethanol at FQY 28.3 % as a standard for SO3SQ and ICG in ethanol at FQY 13.2 % as a standard for SO3C5. Therefore, the FQY of the SO3SQ was previously reported as 61.1 % in presence of human serum albumin (HSA). When the FQY of 1 pmol L-1SO3SQ in the presence of [Ch][Doc] is compared using an aqueous standard (ICG in water, 2.9%), an FQY of 41.0 ± 13.3% is calculated. Thus, when comparing with an ethanol standard and concentrated dyes, the reported increase in the FQY and / or MB is comparable to the reported benchmark. In many cases, simple molecular systems like [Ch][Doc] offer advantages over proteins for stabilizing NIR dyes as proteins are often sensitive to environmental changes, exhibit limited stability, and face batch-to-batch variability. In contrast, simple molecular systems provide greater stability and consistency.

[0133] I -1061 is virtually non-emissive at 1 mol L-1in aqueous solutions. However, the addition of [Ch][Doc] significantly enhances the fluorescence intensity of IR-1061 , yielding a FQY of 0.26% in the presence of 30 mmol U1[Ch][Doc] (FIGs. 3E-3F). This enhancement is visually demonstrated in FIGs. 3E-3F, where the IR-1061-[Ch][Doc] complex fluoresces brightly, in contrast to the aqueous solution of IR-1061 without [Ch][Doc], The favorable spatial geometry of IR-1061 allows it to interact with [Ch][Doc] to maximize the hydrophobic interaction between IR- 1061 and [Doc], shielding the IR-1061 from the quenching effect of water molecules. Therefore, the formulation of NIR-I and NIR-II dyes with ionic liquids is an extremely promising avenue for obtaining maximum MB and sufficiently high FQY, enabling the improvement in the high- resolution imaging for biological applications. The relative extent of the fluorescence enhancement by [Ch][Doc] among different dyes was found to depend upon the molecular structure, aqueous solubility and nature of functional group appended to the dye. The presence of the squaraine moiety between the indozoline donors makes it relatively compact compared to the extended C5 bridge, which could explain the efficient incorporation of SQ dyes in [Ch][Doc]aggregates. The evaluation of these factors and their relative contribution in affecting the fluorescence of the NIR dye is further explored below.

[0134] Transient Absorption (TA) Spectroscopy. In an attempt to probe the effects of the ILs on the excited state properties of the SO3SQ molecule, transient absorption measurements were performed on aqueous solutions of SO3SQ with and without ILs (FIGs. 4A-4C and 12-14). [Ch][Nic] is selected as the negative control, since it does not alter the emission of the dye. FIGs. 4A-4C show transient spectra of SO3SQ with and without ILs [Ch][Nic] and [Ch][Doc] (100 pmol L'1SO3SQ aqueous solution and 500 mmol L-1IL-SO3SQ solutions). Single wavelength kinetics were taken from the maximum of the excited state absorption (ESA) and fit with a biexponential decay function. These kinetics are shown in FIG. 4D, decay components and averaged lifetimes are reported in Table 2.

[0135] In the absence of ionic liquid, SO3SQ has an excited state lifetime, Tavg, of 16.6 ps. The addition of ionic liquids (ILs) increases the average excited-state lifetimes to 77 ps and 1672 ps for the SC>3SQ-[Ch][Nic] and SO3SQ-[Ch][Doc] systems, respectively. This demonstrates a positive correlation between extended excited-state lifetimes and increased fluorescence quantum yields (FQY) across the investigated systems.

[0136] The transient spectrum for the aqueous solution of SO3SQ shows an ESA ranging from approximately 425 - 600 nm with two features: 1) a broadband absorption centered at 520 nm and 2) a shoulder at 500 nm that decays into the broadband absorption within a 15 ps delay between pump and probe. This spectrum also features a ground state bleaching (GSB) region ranging from 680 - 770 nm decaying at a similar rate to the ESA.The decay of the 520 nm shoulder is assigned to internal conversion into the longer-lived excited state. The [Ch][Doc]- SO3SQ ESA features a similar broadband absorption, however, the ESA is shifted towards longer wavelengths and centered at 560 nm with no 520 nm peak assigned to population of a higher excited state. The GSB is also shifted towards longer wavelengths and features a large peakcentered at 720 nm, assigned to stimulated emission from the long-lived excited state. The SC>3SQ-[Ch][Nic] ESA is similar to the other ILs as it does not feature the 520 nm peak, yet the absorption is not as redshifted as with the other ILs investigated in this report. Based upon the increase in lifetime, the ILs are likely stabilizing and lowering the energy of the lowest lying excited state of the dye, allowing direct population of the long-lived state rather than internally converting as occurs with the SO3SQ in aqueous solution.

[0137] Time-Resolved Anisotropy. Time-resolved anisotropy measurements were performed to probe rotational diffusion within the excited state. Commonly referred to as transient absorption anisotropy, this measurement measures the overlap between transition dipole moments in the ground and excited states. Essentially, this measurement allows a visualization of rotational diffusion within an excited state as a function of both wavelength and time (r(A,t)). Here, these measurements were employed to determine the effects of ionic liquids on the rotational dynamics of the dye within the excited state. FIGs. 4E-4F (and FIGs. 16-17) feature the anisotropy of each SO3SQ-IL solution overlaid with transient spectra at 0.0 ps delay time. For a typical two level system, an initial (t = 0) anisotropy (r(0)) value of 0.4 is theoretically expected for a totally anisotropic system, however, if the signal contains contributions from several overlapping transition dipole moments, an anisotropy different from 0.4 is expected. The aqueous solution of SO3SQ shows an anisotropy within the ESA of ~0.2, demonstrating the ability of molecules to rotationally diffuse within the excited state. The [Ch][Doc]-SC>3SQ solutions that showed the greatest increase in FQY, however, have increased anisotropy within the ESA, with anisotropy ranging from -0.25 - 0.32. On the other hand, [Ch][Nic]-SO3SQ shows an anisotropy within the ESA having both high -0.4 and low -0.2 contributions. This offers evidence that [Ch][Nic] interacts weakly with SO3SQ, consistent with the lifetime and FQY trends. The increase in anisotropy for [Ch][Doc] is attributed once again to the formation of aggregates around the SO3SQ, encapsulating and limiting their ability to rotationally diffuse. This encapsulation is believed to also decrease non-radiative decay methods, leading to the increase in FQY of SO3SQ from 0.4 % to 37.9 % in 200 mmol L'1[Ch][Doc],

[0138] Photostabilitv of SOsSQ-fChlfDocI formulation. The photostability of SO3SQ and SO3SQ- [Ch][Doc] solutions was evaluated by irradiating aqueous solutions of 1 pmol L'1SO3SQ, with and without 30 mmol L'1[Ch][Doc], using white LED light at intensities of 10 mW cm'2(P10) and 100 mW cm'2(P100) for 480 minutes in range 400-900 nm (FIGs. 5A-5D). At P10, the photostability of SO3SQ remained unchanged in both the presence and absence of [Ch][Doc] up to 300 minutes (FIGs. 5A and 19A-19D). However, after 350 minutes, SO3SQ in the [Ch][Doc] solution showeda slightly higher degradation rate (1.1 times greater, p < 0.01), likely due to prolonged irradiation overwhelming the protective effect of [Ch][Doc] aggregates. At P100, the half-life of SO3SQ in the presence of [Ch][Doc] dropped to 80 minutes, compared to 175 minutes without [Ch][Doc] (FIG. 5A). The reduced photostability of the SO3SQ-[Ch][Doc] formulation arises from a balance between protective aggregation and altered excited-state dynamics. In its aggregated state, SO3SQ forms IT- IT interactions and excitonic couplings that dissipate excitation energy through non-destructive pathways and shield dye molecules from reactive species like molecular oxygen, thereby enhancing photostability. When [Ch][Doc] is introduced, it forms aggregates around SO3SQ that boost fluorescence by preventing water-induced quenching and restricting dye rotation. However, these effects also result in a higher excited-state population, making the molecules more susceptible to photochemical degradation. Additionally, the microenvironment within the [Ch][Doc] aggregates marked by changes in polarity and viscosity which can promote intersystem crossing and singlet oxygen formation, further accelerating photodegradation. Therefore, while [Ch][Doc] aggregation enhances fluorescence, it simultaneously increases the reactivity of SO3SQ in the excited state, leading to reduced photostability. Notably, lasers used in NIR imaging (650-900 nm) typically operate at power densities of 10-100 mW / cm2, which are within safe exposure limits for the SC>3SQ-[Ch][Doc] system, ensuring high-efficiency operation.

[0139] Furthermore, time- and temperature-dependent stability studies of the SO3SQ-[Ch][Doc] system were conducted over 48 hours at 25 °C, 37 °C, and 50 °C (FIGs. 5B-5D). The results indicate that SO3SQ degradation accelerates at 50 °C, is moderate at 37 °C, and remains minimal at 25 °C in the dark. Since 37 °C represents physiological temperature, the relatively high stability and retained high fluorescence of the SO3SQ-[Ch][Doc] system at this temperature for 48 hours suggests its suitability for biological applications. Interestingly, fluorescence emission decreased upon heating to 37 °C and 50 °C (FIGs. 19E-19G). However, upon cooling, the SO3SQ-[Ch][Doc] system regained its fluorescence within 15 minutes, restoring its original high fluorescence intensity. This behavior is likely due to the dynamic nature of [Ch][Doc] aggregates, which disassemble upon heating due to increased thermal motion, exposing SO3SQ to the aqueous environment and reducing fluorescence. Upon cooling, the aggregates reassemble, shielding SO3SQ and restoring its enhanced fluorescence. It is important to note that while fluorescence quenching upon heating is a reversible effect, prolonged exposure to elevated temperatures leads to gradual degradation of SO3SQ in presence of [Ch][Doc], At 37 °C, SO3SQ degradation is more pronounced than at 25 °C, but the system still retains significant fluorescence for imaging applications (FIGs. 5C-5D). Even at 50 °C after 48 hours, the SO3SQ-[Ch][Doc] system maintainshigh fluorescence emission compared to SO3SQ alone. These findings strongly support the efficacy and stability of the SO3SQ-[Ch][Doc] system, highlighting its potential for biological and imaging applications.

[0140] Morphology and Internal Structure of SO3SQ-rChirDoc1 complexes. To explore the morphology of the SO3SQ-[Ch][Doc] complexes, scanning electron microscopy (SEM) was performed on samples containing 30 and 200 mmol L’1[Ch][Doc], with and without 1 pmol L'1SO3SQ (FIGS. 6A-6C and 20A-20B). The SEM images revealed the formation of rod-like structures approximately 1 pm wide and 1-4 pm long at both concentrations, suggesting that [Ch][Doc] form self-assembled structures in aqueous solution.

[0141] Our experimental observations, supported by computational modeling, indicate that in the presence of [Ch][Doc], the SO3SQ molecules undergo de-aggregation due to dominant hydrophobic interactions with [Ch][Doc], This interaction effectively solvates the SO3SQ molecules, creating a unique microenvironment that stabilizes the excited state of SO3SQ and enhances fluorescence via a solvatochromic effect. For instance, the observed 8 nm red shift (from 716 nm to 724 nm) upon the addition of [Ch][Doc], which is indicative of subtle solvent- induced modifications rather than the large shifts typically seen in ordered J-aggregates. Moreover, the rod-like structures likely represent microphase-separated domains where [Ch][Doc] directs the spatial arrangement of SO3SQ molecules. This configuration provides a hydrophobic surface that minimizes water-induced quenching and restricts dye rotation, thereby favoring radiative decay over nonradiative pathways. These findings are consistent with observations in similar systems, where hydrophobically driven and electrostatically stabilized self-assemblies form aggregates structures.

[0142] Dynamic light scattering (DLS) data (FIG. 6D) further support these results, showing a hydrodynamic size peak of approximately 450 nm. This suggests that while smaller aggregates exist in solution, they coalesce into micron-sized structures upon drying, as seen in the SEM images. Higher magnification SEM images (FIG. 6B) reveal layered sheet structures on the surfaces of these complexes, providing additional evidence of the intricate morphology of the aggregates. Importantly, titration experiments with [Ch][Doc] (ranging from 0 to 220 mmol L-1) showed that the aggregate size remains in the micron range, regardless of concentration, due to the unique molecular architecture of [Ch][Doc], Unlike conventional surfactants, [Ch][Doc] has both hydrophilic and hydrophobic planes that promote self-association and stable aggregate formation even at low concentrations (as low as 5 mmol L'1). Conductivity measurements indicatethatthe critical aggregation concentration (CAC) of [Ch][Doc] remains unchanged in the presence of SO3SQ, and zeta potential values are nearly identical with or without the dye (FIGs. 21A-21B). These results confirm that micromolar concentrations of SO3SQ do not significantly influence the aggregation behavior of [Ch][Doc],

[0143] Transition Analysis. To investigate the electronic properties and interactions of molecular anion complexes with ionic liquids, computations investigation were conducted. Both CAM- B3LYP and PBEO produce similar excitation energies differing by less than 0.03 eV and similar oscillator strengths rounding to the same values for one decimal place. All of the analyzed molecular anion complexes exhibit large oscillator strengths on the order of ~1 .0. All of the excited state transitions are exhibiting the same oscillator strength value. None produce a decrease in the oscillator strength for the different ionic liquid anions in contrast to the experiment. Furthermore, increasing the number of anions interacting with the SO3SQ dye has little effect on this HO MO- LU MO transition. Therefore, molecular dynamics simulations have been employed in order to better understand role that the ionic liquid may play in influencing the oscillator strengths and, hence, intensities.

[0144] Molecular Dynamics. To investigate the impact of the ionic liquid on the fluorescence of SO3SQ, 100 ns Molecular Dynamics (MD) simulations were performed on two distinct disordered systems, SO3SQ solution in water without and with [Ch][Doc] (FIGs. 7A-7B). The trajectory analysis revealed that in the absence of the ionic liquid, the dye molecules formed aggregates in water (FIG. 7A). In the initial stages of the simulation, only two of the three SO3SQ molecules formed a sandwich-type dimer. The third SO3SQ molecule exhibited a variable conformation, with its hydrophilic tails adopting a trans conformation upon frequent solvent exposure. After 20 ns of simulation, all three SO3SQ molecules formed an aggregate, which persisted until the end of the trajectory. In contrast, when the [Ch][Doc] was present, all three SO3SQ molecules remained separated, and no aggregation was observed throughout the simulation (FIG. 7B). The hydrophobic indolizine rings of the SO3SQ molecules were surrounded by bulky [Doc] anions. At the same time, the hydrophilic sulfonyl tails were exposed to the solvent and formed hydrogen bonds with water molecules. To gain further insights into the geometries of the SO3SQ in the water solution, the trajectories obtained from the simulations have been clustered based on the dye molecules' root mean square deviations (RMSD). The most prevalent geometry of the SO3SQ dye in water is illustrated in FIG. 7C. The hydrophobic indolizine rings of the dye molecules formed a sandwich-like structure with plane-to-plane stacking. The hydrophobic region of the cluster was stabilized by three TT-TT interactions between the indolizine and phenyl rings (five in the case ofthe central molecule), two n-cation interactions, and two salt bridges (three in the case of the central molecule). The hydrophilic tails of the SO3SQ molecules were oriented equally on both sides of the aggregate and formed hydrogen bonds with water molecules. The hydrophobic region of the aggregate was concentrated at its center, while the hydrophilic region had a mushroom shape from the top and bottom, as shown in the surface map (FIG. 7E). The open hydrophobic areas formed by the stacking of indolizine and phenyl rings suggest the possibility of the formation of larger aggregates beyond the observed dimers and trimers. Considering the sandwich-like stacking, the formation of an H-aggregate can be confirmed, typically characterized by a hypsochromic (blue) shift in absorption and a large Stokes shift for weaker fluorescence. These findings are consistent with experimental results, showing shorter absorption and emission wavelengths for SO3SQ in water solution. Adding [Ch][Doc] effectively prevented the aggregation of SO3SQ in the water solution by mediating weak intermolecular interactions between the SO3SQ and components of the [Ch][Doc], as illustrated in FIG. 7D.

[0145] Specifically, the hydrophobic indolizine rings of the dye were surrounded by bulky [Doc] anions, which kept them in a maximally planar orientation. Meanwhile, the sulfonyl groups of the dye molecules were exposed to the solvent and formed hydrogen bonds with water molecules. Importantly, no significant interactions, other than hydrophobic ones, were detected between the [Doc] anions and the SO3SQ dye molecules. This steric effect allowed the ionic liquid to prevent aggregation and preserve the original optical properties of the monomeric dye. Therefore, the favorable molecular geometry of [Ch][Doc] is crucial in maximizing interactions with SO3SQ, resulting in an optimized orientation and the highest emission efficiency in the investigated system. Interestingly, it was also observed that both phenyl rings of the dye formed ir-cation interactions with [Cho] cation. In addition to the analyses described above, the aggregation behavior of the dye molecules in water solution was also investigated by analyzing the radius of gyration during the 100 ns simulation time. As shown in FIG. 7F, the gyration radius for the dye in the presence of the [Ch][Doc] varied from 30 to 40 A, indicating a degree of flexibility and motility of the individual dye molecules. In contrast, in the absence of the ionic liquid, the gyration radius stabilized at around 8-9 A after 20 ns of simulation time, implying the formation of a stable aggregate. Similar observations can be made by examining the solvent’s assessable surface area (SASA) (FIG. 7G). The SASA of the dye molecules was significantly reduced in the presence of the ionic liquid, suggesting a decrease in their exposure to the solvent, inhibiting its aggregation and allowing the dye to maintain its solubility in water. These simulation results provide valuable insights into the solvation and aggregation behavior of dye molecules in aqueous solutions,highlighting the critical role of [Ch][Doc] in modulating these behaviors. Therefore, [Ch][Doc] plays a crucial role in preventing dye aggregation in water, thereby preserving their optical properties.

[0146] Biocompatibilitv of [ChUDocl. The biocompatibility of [Ch][Doc] was evaluated by measuring cell viability of MCF10A and HEK293 cells and assessing the hemolysis of human red blood cells (RBCs). [Ch][Doc] was found to be non-cytotoxic (viability > 75 %) towards both MCF10A and HEK293 cells until 160 pmol L1(FIGs. 8A-8B). At higher concentrations i.e. , 320 pmol L’1, the cell viability drops down to 70% and 20% in HEK293 and MCF10A respectively. Finally, at 450 pmol L-1, [Ch][Doc] becomes cytotoxic with approximately 20% cell viability for both HEK293 and MCF10A. The cytotoxicity profile of SO3SQ has previously been documented in studies involving HEK293 and Drosophila (S2) cells. Even at a high drug loading concentration of 10 mmol L’1, SO3SQ resulted in only 30% and 50% cell death in HEK293 and MCF10A cells, respectively. These findings underscore its remarkable biosafety profile. Furthermore, the hemolysis of human RBCs was assessed at different concentrations [Ch][Doc] ranging from 20 to 300 pmol L'1(FIG. 8B). Up until 300 mmol L'1, hemolysis was observed to be less than 5% with respect to the positive control, Triton X-100. These studies indicate the high biocompatibility of [Ch][Doc] towards living cells and suggests that it safe for use in biological applications within this range of concentrations.

[0147] SO3SQTCh1[Doc1 emission in a biological setting. The fluorescence intensity of SO3SQ and SC>3SQ-[Ch][Doc] complex was measured in live HEK293 cells by confocal microscopy (FIGs. 22A-22C). In the experimental setup, 250 ng and 500 ng of SO3SQ dye with and without 40 pg mL’1[Ch][Doc] was treated to HEK293 cells for 24 hours, followed by analysis of PBS buffer- washed samples. It was observed that the relative brightness of SC>3SQ-[Ch][Doc] was significantly higher (FIGs. 22A-22C) than SO3SQ-only samples in the cellular environment, suggesting that the [Ch][Doc] ionic liquid enhances the fluorescence intensity. This likely results from the improved stability of SO3SQ-[Ch][Doc] complexes against degradation by cellular enzymes, making this complex a promising material for bioimaging applications.

[0148] Immunohistochemistry. Immunohistochemistry (IHC) is a powerful tool for identifying and visualizing specific proteins in tissue samples. In the experiment, HEK293 cells were stained to detect human a-tubulin protein using Alexa Fluor 647 conjugated antibody dye with and without [Ch][Doc] (FIG. 8C).

[0149] It was observed that without [Ch][Doc], the brightness of the stained samples was very low compared to the [Ch][Doc]-added samples. The brightness increased with the concentrationof [Ch][Doc], becoming almost constant after 600 pg mb1(FIG. 8D). This signifies that [Ch][Doc] helps illuminate the stained samples even at low concentrations of dye, resulting in high-resolution images. Additionally, the stability of the Dye-[Ch][Doc] complex during the immunohistochemistry procedure where detergents are used to prepare tissues and wash off unbound antibodies further supports its robustness. Despite these harsh conditions, the Dye-[Ch][Doc] complex remains stable in immunohistochemistry and related bioassay procedures.

[0150] The brighter dye-[Ch][Doc] complexes enables the detection of trace proteins that might otherwise fall below the equipment's detection limit due to low brightness, facilitating early detection of disease-related biomarkers even at low concentrations. It is important to note that Alexa Fluor 647 (a cyanine dye) and SO3SQ (a squaraine dye) are chemically distinct, yet both exhibit enhanced fluorescence in the presence of [Ch][Doc], The stabilization of Alexa Fluor 647 through predominant hydrophobic interactions prevents quenching by water molecules, thereby improving its emission profile. In imaging studies, Alexa Fluor 647 shows increased brightness in cellular environments where traditional NIR dye-stabilizing surfactants often fail.

[0151] Photothermal Treatment (PTT). Photothermal therapy (PTT) relies on the rapid conversion of absorbed light energy into heat via nonradiative decay. In this study, HEK293 cells treated with SO3SQ (600 ng / mL) showed localized heating sufficient to induce selective cell death, whereas cells treated with the SC>3SQ-[Ch][Doc] complex (40 pg / mL) exhibited minimal temperature increase and no significant cell death (FIGs. 9A-9B). This difference indicates that the photothermal effect of SO3SQ is substantially reduced when it is encapsulated within [Ch][Doc] aggregates.

[0152] Upon light absorption, SO3SQ is excited from its ground state (So) to its excited singlet state (Si). In an aqueous environment, water molecules facilitate efficient vibrational relaxation (internal conversion), converting the absorbed energy into heat which is essential for a strong photothermal effect. However, when SO3SQ is encapsulated by [Ch][Doc] aggregates, a hydrophobic microenvironment is created around the dye molecules. This encapsulation restricts the access of water molecules, thereby reducing the vibrational coupling necessary for nonradiative decay. As a result, less energy is dissipated as heat, and more energy may be emitted as fluorescence or retained in the excited state. Thus, while the [Ch][Doc] aggregation enhances fluorescence of SO3SQ by reducing water-induced quenching and restricting dye rotation, it simultaneously suppresses the nonradiative decay pathways that are critical for effective heat generation. This dual effect explains the poor photothermal performance of theSC>3SQ-[Ch][Doc] complex compared to free SO3SQ, and suggests a potential strategy for protecting healthy tissues during PTT by modulating heat release.

[0153] Conclusions. In conclusion, this study demonstrates that biocompatible ionic liquids (ILs), particularly choline deoxycholate ([Ch][Doc]), can be strategically combined with near-infrared (NIR) dyes to create formulations with enhanced optical properties, including higher molecular brightness (MB) and fluorescence quantum yield (FQY), along with excellent biocompatibility. The comprehensive experimental and computational investigations described herein reveal that, in aqueous solution, SO3SQ tends to aggregate in a way that promotes nonradiative decay, dissipating energy as heat via water-mediated vibrational coupling. However, when formulated with [Ch][Doc], the IL’s amphiphilic and ionic character drives the formation of well-defined aggregates that encapsulate or “cage” the SO3SQ molecules. This encapsulation modifies the local microenvironment around the dye: by reducing direct contact with water, it suppresses nonradiative vibrational relaxation and shifts the energy dissipation pathway from thermal loss to radiative decay. The framework of Jablonski diagram provides a useful illustration of these changes: in the presence of [Ch][Doc], the excited SO3SQ molecules are less likely to undergo internal conversion (and thus heat generation), favoring fluorescence instead. Conversely, the addition of methanol destabilizes these aggregates by solvating [Ch][Doc] molecules, which reintroduces solvent interactions and aggregation quenching, thereby reducing the overall fluorescence intensity. These findings underscore the potential of using biocompatible ILs to tailor the photophysical behavior of NIR dyes. By precisely controlling the dye’s aggregation state and the corresponding energy dissipation pathways, it is possible to enhance fluorescence while mitigating undesirable thermal effects, an approach that holds significant promise for advancing NIR bioimaging technologies and improving the safety and efficacy of photothermal therapy.Example 3: Characterization of the Ionic Liquids Synthesized and Starting Materials Used Herein

[0154] Choline Deoxycholic acid, [ChlfDod 1 :1 :1H NMR (400 MHz, D2O) 54.00 - 3.97 (m, 2H), 3.44 (dd, J = 6.4, 3.4 Hz, 2H), 3.12 (s, 9H), 2.23 - 0.80 (m, 30H), 0.64 (s, 3H); ESI-HRMS positive ions m / z (for C5H14NO+): 104.1229;13C NMR (101 MHz, D2O) 5 184.17, 73.19, 71.41 , 67.37, 55.58, 53.89, 47.80, 46.50, 46.19, 42.07, 35.99, 35.82, 35.25, 35.14, 34.45, 33.92, 33.39, 32.30, 29.17, 28.28, 27.58, 27.11 , 26.10, 23.73, 22.98, 16.70, 12.59; ESI-HRMS negative ions m / z (for C24H39O4-): 391 .3134; IR (ATR, cm'1): 3350 (br, O-H), 2930-2850 (s, C-H), 1585 (s, COO” asym. stretch), 1470 (m, CH2bending), 1380 (w, COO” sym. stretch), 1250-1050 (s, C-O / C-N), 1040 (m, steroid skeletal); KF: 3.6%; Purity : 98.1%; Melting point by DSC 240 °C.

[0155] Choline Decanoic. rChlfDecl 1 :1 :1H NMR (400 MHz, DMSO) 6 3.85 (td, J = 5.8, 2.7 Hz, 2H), 3.44 (d, J = 5.3 Hz, 2H), 3.13 (s, 9H), 1.82 (t, J = 7.4 Hz, 2H), 1.39 (t, J = 7.2 Hz, 2H), 1.23 (d, J = 8.8 Hz, 12H), 0.92 - 0.80 (m, 3H). ESI-HRMS positive ions m / z (for C5H14NO+): 104.122913C NMR (101 MHz, DMSO) 6 178.02, 66.92, 57.61 , 54.39, 31.81, 30.00, 29.68, 29.58, 29.25, 27.11 , 22.59, 15.73. ESI-HRMS negative ions m / z (for Ci0Hi9O2-): 171.1582 IR (ATR, cm1): 3350 (br, O-H), 2950-2850 (s, C-H), 1585 (s, COO’ asym. stretch), 1470 (m, CH2bending), 1390 (w, COO“ sym. stretch), 1250-1050 (s, C-0 / C-N). KF: 3.8%. Purity: 96.2%. Melting point: 67 °C

[0156] Choline Orotic acid, (Ch Orol 1 :1 :1H NMR (400 MHz, DMSO) 5 10.90 (s, 1 H), 5.71 (s, 1 H), 3.85 (q, J = 4.8 Hz, 2H), 3.42 (q, J = 4.3 Hz, 2H), 3.12 (s, 9H). ESI-HRMS positive ions m / z (for C5H14NO+): 104.122913C NMR (101 MHz, DMSO) 6 165.90, 160.98, 152.96, 150.17, 100.79, 73.87, 59.85, 53.59. ESI-HRMS negative ions m / z (for C5H3N2O4-): 155.0278 IR (ATR, cm’1): 3350 (br, O-H / N-H), 2950-2850 (m, C-H), 1720 (s, C=O), 1600 (m, COO’ asym. stretch), 1465 (m, CH2bending), 1370 (w, COO“ sym. stretch), 1250-1050 (s, C-0 / C-N). KF: 3.4%. Purity: 95.6%. Melting point: 120 °C

[0157] Choline Nicotinic. [ChlFNicI 1 :1 :1H NMR (400 MHz, DMSO) 6 10.90 (s, 1 H), 9.46 (s, 1 H), 5.71 (s, 1 H), 3.85 (q, J = 4.8 Hz, 2H), 3.42 (q, J = 4.3 Hz, 2H), 3.12 (s, 9H). ESI-HRMS positive ions m / z (for C5Hi4NO+): 104.122913C NMR (101 MHz, DMSO) 5 168.53, 152.94, 149.71 , 138.25, 135.79, 123.05, 67.67, 55.15, 52.27. ESI-HRMS negative ions m / z (for C6H4NO2-): 122.0384 IR (ATR, cm'1): 1602 (s, C=C I C=N stretch from pyridine), 1559 (m, COO“ asym. stretch), 1479 (m, CH2bending / aromatic skeletal). KF: 6.7%. Purity: 91.2% Melting point: < -40 °C.

[0158] Choline Biotin. (ChKBiol 1 :1 :1H NMR (400 MHz, DMSO) 5 6.66 (d, J - 2.2 Hz, 1 H), 6.34 (s, 1 H), 4.30 (dd, J = 7.7, 5.0 Hz, 1 H), 4.13 (ddd, J = 7.8, 4.5, 1.8 Hz, 1 H), 3.86 (dq, J = 5.5, 2.7 Hz, 2H), 3.50 - 3.36 (m, 3H), 3.17 (s, 9H), 3.08 (dt, J = 7.3, 3.6 Hz, 1 H), 2.82 (dd, J = 12.4, 5.0 Hz, 1 H), 2.58 (d, J = 12.4 Hz, 1H), 1.93 - 1.78 (m, 2H), 1.51 - 1.36 (m, 3H), 1 .36 - 1 .19 (m, 2H). ESI-HRMS positive ions m / z (for C5H14NO+): 104.122913C NMR (101 MHz, DMSO) 5 176.36, 163.24, 67.84, 61.47, 59.67, 57.52, 54.95, 53.54, 47.57, 38.36, 30.94, 28.02, 26.87. ESI-HRMS negative ions m / z (for CIOHI5N203S-): 243.1012 IR (ATR, cm1): 3300 (br, N-H / O-H), 2925 (m, C-H), 1680 (s, C=O), 1580 (m, N-H bend / C=C), 1460 (m, CH2bending), 1380 (w, CH3bending), 1250-1050 (s, C-01 C-N), 1020 (m, S-C stretch). KF: 4.1%. Purity: 95.1%

[0159] Choline Citric, [ChirCitl 1 :1 :1H NMR (400 MHz, DMSO) 6 3.84 (qd, J = 5.0, 2.9 Hz, 2H), 3.41 (q, J = 4.3 Hz, 2H), 3.11 (s, 9H), 2.58 - 2.43 (m, 4H). ESI-HRMS positive ions m / z (for C5H14NO+): 104.122913C NMR (101 MHz, DMSO) 6 177.69, 173.23, 71.69, 66.15, 56.27, 53.67, 46.60. ESI-HRMS negative ions m / z (for C6H7O7-): 191.0434 IR (ATR, cm'1): 3350 (br, O-H),3000-2900 (m, C-H), 1710 (s, C=O), 1588 (m, COO“ asym. stretch), 1479 (m, CH2bending), 1380 (w, COO“ sym. stretch), 1250-1050 (s, C-O / C-N). KF: 2.5%. Purity: 96.7%. Melting point: 98 °C

[0160] Choline Citric, [ChirCif] 2: 1 :1H NMR (400 MHz, D2O) 5 3.84 (td, J = 4.9, 2.6 Hz, 4H), 3.46- 3.37 (m, 4H), 3.17 (s, 18H), 2.37 - 2.22 (m, 4H). ESI-HRMS positive ions m / z (for C5H14NO+): 104.122913C NMR (101 MHz, D2O) 0 177.02, 74.41 , 67.75, 55.57, 53.80, 47.29. ESI-HRMS negative ions m / z (for C6H7O7-): 191.0385 IR (ATR, cm’1): 3350 (br, O-H), 2980-2880 (m, C-H), 1720 (s, C=O), 1595 (m, COO“ asym. stretch), 1465 (m, CH2bending), 1375 (w, COO“ sym. stretch), 1250-1050 (s, C-0 I C-N). KF: 7.6%. Purity : 92.2%. Melting point: nd.

[0161] Choline Citric, [ChirCitl 3:1 :1H NMR (400 MHz, D2O) 5 3.98 (dt, J = 6.4, 3.6 Hz, 4H), 3.43 (p, J = 2.9 Hz, 4H), 3.12 (d, J = 3.4 Hz, 21 H), 2.51 (q, J = 15.0 Hz, 4H). ESI-HRMS positive ions m / z (for C5H14NO+): 104.122913C NMR (101 MHz, D2O) 5 188.26, 179.21 , 74.85, 68.10, 55.57, 54.32, 45.60.ESI-HRMS negative ions m / z (for C6H7O7-): 191.0434 IR (ATR, cm-1): 3350 (br, O- H), 2985-2880 (m, C-H), 1730 (s, C=O), 1605 (m, COO“ asym. stretch), 1468 (m, CH2bending), 1385 (w, COO“ sym. stretch), 1250-1050 (s, C-O / C-N). KF: 7.3%. Purity: 91.5%. Melting point: nd.

[0162] Choline Ethylenediaminetetraacetic acid, [ChlfEDTAI 4:1 :1H NMR (400 MHz, D2O) 64.02- 3.92 (m, 8H), 3.50 - 3.38 (m, 8H), 3.24 (s, 8H), 3.12 (s, 36H), 2.78 (s, 4H). ESI-HRMS positive ions m / z (for C5H14NO+): 104.122913C NMR (101 MHz, D2O) 5 178.39, 67.37, 62.50, 58.00, 55.57, 53.84, 48.89. ESI-HRMS negative ions m / z (for CioH2084-): 291.1113 IR (ATR, cm’1): 3350 (br, O-H / N-H), 2930-2850 (m, C-H), 1605 (s, COO" asym. stretch), 1400 (m, COO" sym. stretch), 1250-1050 (s, C-01 C-N), 1020 (m, EDTA skeletal vibrations). KF: 8.2%. Purity: 90.1%. Melting point: nd.

[0163] Choline Urocanic acid, [ChlfUrol 1 :1 :1H NMR (400 MHz, D2O) 5 7.72 (s, 1 H), 7.31 - 7.16 (m, 2H), 6.28 (d, J = 16.0 Hz, 1 H), 3.95 (dq, J = 5.5, 2.7 Hz, 2H), 3.44 - 3.37 (m, 2H), 3.09 (s, 9H). ESI-HRMS positive ions m / z (for C5H14NO+): 104.122913C NMR (101 MHz, DMSO) 5 171.91 , 138.30, 127.07, 70.05, 55.92, 53.55, 49.52. ESI-HRMS negative ions m / z (for C6H5N2O2): 137.0495 IR (ATR, cm’1): 3350 (br, O-H / N-H), 2950-2850 (m, C-H), 1650 (s, C=O), 1585 (m, C=N / C=C), 1465 (m, CH2bending), 1380 (w, COO’ sym. stretch), 1250-1050 (s, C-0 I C-N). KF: 3.7%. Purity: 94.2%. Melting point: nd.

[0164] Choline Pyridoxine, fChlfPyrl 1:1 :1H NMR (400 MHz, D2O) 5 7.43 (s, 1 H), 4.52 (s, 2H), 3.96 (tt, J = 5.4, 2.7 Hz, 2H), 3.56 (q, J = 7.0 Hz, 2H), 3.41 (dd, J = 7.1 , 3.4 Hz, 2H), 3.09 (s, 9H), 2.23 (s, 3H). ESI-HRMS positive ions m / z (for C5H14NO+): 104.122913C NMR (101 MHz, DMSO)6 149.85, 130.56, 67.48, 60.10, 56.27, 53.63, 49.05, 20.13. ESI-HRMS negative ions m / z (for CioH2084-): 291.1113 IR (ATR, cm-1): 3350 (br, O-H / N-H), 2950-2850 (m, C-H), 1650 (s, C=O), 1585 (m, C=N / C=C), 1465 (m, CH2bending), 1380 (w, COO’ sym. stretch), 1250-1050 (s, C-0 I C-N). KF: 3.6%. Purity: 97.3%. Melting point: nd.

[0165] Choline Hexadecanoic, fChlfHdecl 1 :1 :1H NMR (400 MHz, D2O) 6 3.96 (tq, J = 4.8, 2.0 Hz, 2H), 3.44 (td, J = 4.8, 1.4 Hz, 2H), 3.13 (d, J = 1.4 Hz, 9H), 2.07 (t, J = 7.9 Hz, 2H), 1.48 (q, J = 7.2 Hz, 2H), 1 .23 (s, 24H), 0.85 - 0.77 (m, 3H). ESI-HRMS positive ions m / z (for C5H14NO+): 104.1229.13C NMR (101 MHz, D2O) 5 182.49, 72.62, 59.75, 53.94, 40.89, 32.05, 29.96, 26.44, 22.67, 13.27. ESI-HRMS negative ions m / z (for CI6H3IO2-): 255.2535 IR (ATR, cm-1): 3340 (br, O-H), 2950-2850 (s, C-H), 1587 (s, COO” asym. stretch), 1475 (m, CH2bending), 1380 (w, COO sym. stretch), 1250-1040 (s, C-0 I C-N). KF: 4.2%. Purity: 95.1%. Melting point: 47 °C.

[0166] Choline bicarbonate:1H NMR (400 MHz, D2O) 5 3.97 - 3.90 (m, 2H), 3.43 - 3.37 (m, 2H), 3.08 (s, 9H).13C NMR (101 MHz, D2O) 6 160.28, 67.31 , 55.54, 53.35. IR (ATR, cm’1): 3350 (br, O-H), 2950-2850 (m, C-H), 1650 (s, HCO3“ asym. stretch), 1400 (m, COO“ sym. stretch), 1250- 1050 (s, C-0 / C-N), 1015 (m, bicarbonate skeletal).

[0167] Urocanic acid1H NMR (400 MHz, DMSO) 6 12.35 (s, 1 H), 12.10 (s, 1 H), 7.48 (s, 1 H), 7.44 (s, 1H), 6.30 (s, 1 H), 6.26 (s, 1 H).13C NMR (101 MHz, DMSO) 5 168.71 , 138.93, 115.20, 105.60, 44.37, 30.94. IR (ATR, cm’1): 3200-3100 (br, O-H / N-H), 2950-2850 (m, C-H), 1690 (s, C=O), 1590 (m, C=C / C=N), 1465 (m, CH2bending), 1380 (w, COO’ sym. stretch), 1250-1050 (s, C-0 I C-N).

[0168] Biotin1H NMR (400 MHz, DMSO) 6 12.00 (s, 1 H), 6.44 (t, J = 1.8 Hz, 1H), 6.37 (s, 1 H), 4.31 (ddt, J = 7.7, 5.2, 1.2 Hz, 1 H), 4.14 (ddd, J = 7.8, 4.4, 1.9 Hz, 1 H), 3.11 (ddd, J = 8.6, 6.1 , 4.4 Hz, 1 H), 2.83 (dd, J = 12.5, 5.1 Hz, 1H), 2.58 (d, J = 12.4 Hz, 1 H), 2.21 (t, J = 7.4 Hz, 2H), 1.69 - 1.24 (m, 6H).13C NMR (101 MHz, DMSO) 5 174.92, 163.18, 61.52, 59.64, 55.87, 33.95, 28.58, 28.51 , 25.01. IR (ATR, cm-1): 3300 (br, O-H / N-H), 2950-2850 (m, C-H), 1700 (s, C=O), 1580 (m, N-H bending), 1465 (m, CH2bending), 1380 (w, COO“ sym. stretch), 1250-1050 (s, C- O / C-N), 1020 (m, S-C stretch).

[0169] Citric acid1H NMR (400 MHz, DMSO) 5 12.40 (s, 2H), 5.17 (s, 1 H), 2.78 (s, 1H), 2.74 (s, 1 H), 2.68 (s, 1 H), 2.64 (s, 1 H).13C NMR (101 MHz, DMSO) 5 175.00, 171.76, 72.91 , 43.14. IR (ATR, cm-1): 3300 (br, O-H), 2985-2850 (m, C-H), 1688 (s, C=O), 1422 (m, COO’ asym. stretch), 1380 (w, COO“ sym. stretch), 1250-1000 (s, C-O).

[0170] Decanoic acid1H NMR (400 MHz, DMSO) 6 11.96 (s, 1 H), 2.19 (t, J = 7.4 Hz, 2H), 1.49 (p, J = 7.0 Hz, 2H), 1.25 (s, 12H), 0.91 - 0.82 (m, 3H).13C NMR (101 MHz, DMSO) 5 174.95,34.12, 31.75, 29.36, 24.96, 22.57, 14.41. IR (ATR, cm’1): 3300 (br, O-H), 2950-2850 (s, C-H), 1700 (s, C=O), 1465 (m, CH2bending), 1415 (w, O-H bending), 1290-1200 (m, C-O stretch), 950-900 (w, O-H out-of-plane bending).

[0171] Deoxycholic acid1H NMR (400 MHz, DMSO) 5 11.94 (s, 1H), 4.47 (d, J = 4.3 Hz, 1 H), 4.21 (d, J = 4.1 Hz, 1 H), 3.79 (q, J = 3.2 Hz, 1H), 2.17 (dddd, J = 53.4, 15.7, 9.3, 6.0 Hz, 2H), 1.86 - 0.82 (m, 31 H), 0.60 (s, 3H).13C NMR (101 MHz, DMSO) 0 175.42, 73.25, 63.92, 57.52, 47.92, 46.46, 44.72, 42.49, 36.37, 34.29, 31.17, 28.02, 26.77, 23.84, 17.44, 12.64. IR (ATR, cm-1): 3400 (br, O-H), 2950-2850 (s, C-H), 1700 (s, C=O), 1600-1550 (m, COO“ asym. stretch), 1460 (m, CH2bending), 1250-1050 (s, C-O), 1020 (m, steroid skeletal).

[0172] EDTA1H NMR (400 MHz, DMSO) 53.47 (s, 8H), 2.77 (s, 4H).13C NMR (101 MHz, DMSO) 5 173.23, 54.95, 47.92. IR (ATR, cm’1): 3300 (br, O-H / N-H), 2950-2850 (m, C-H), 1700-1600 (s, C=O), 1450 (m, COO’ sym. stretch), 1350 (w, C-N stretch), 1250-1050 (s, C-O), 950-900 (w, N-H bending).

[0173] Hexadecanoic acid1H NMR (400 MHz, DMSO) 5 11.96 (s, 1 H), 2.18 (t, J = 7.4 Hz, 2H), 1.48 (t, J = 7.2 Hz, 2H), 1.24 (s, 24H), 0.91 - 0.81 (m, 3H).13C NMR (101 MHz, DMSO) 5 174.93, 34.12, 31.79, 29.54, 29.40, 29.21 , 29.04, 24.97, 22.94, 14.41. IR (ATR, cm-1): 3310 (br, O-H), 2950-2850 (s, C-H), 1705 (s, C=O), 1468 (m, CH2bending), 1417 (w, O-H bending), 1290-1210 (m, C-O stretch), 950-900 (w, O-H out-of-plane bending).

[0174] Nicotinic acid1H NMR (400 MHz, DMSO) 5 13.44 (s, 1 H), 9.08 (dd, J = 2.2, 0.9 Hz, 1 H), 8.80 (dd, J = 4.8, 1 .7 Hz, 1 H), 8.28 (dt, J = 7.9, 2.0 Hz, 1 H), 7.55 (ddd, J = 7.9, 4.8, 0.9 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 167.11 , 153.79, 151.11, 137.43, 127.38, 124.28. IR (ATR, cm’1): 3300 (br, O-H), 3050 (w, aromatic C-H), 2950-2850 (m, aliphatic C-H), 1710 (s, C=O), 1585 (m, C=C / C=N), 1460 (m, CH bending), 1400-1380 (w, O-H bending), 1250-1050 (s, C-O), 1020- 980 (m, C-N).

[0175] Orotic acid1H NMR (400 MHz, DMSO) 5 11.34 (s, 1 H), 10.91 (s, 1 H), 6.01 (t, J = 1.8 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 164.49, 162.31 , 151.30, 142.76, 103.63, 31.17. IR (ATR, CRY1): 3450-3200 (br, O-H / N-H), 2831 (w, C-H), 1657 (s, C=O from COOH and CONH), 1435 (m, C=C / N-H bending), 1280-1050 (s, C-O / C-N).

[0176] Pyridoxine1H NMR (400 MHz, DMSO) 5 7.88 (s, 1 H), 4.74 (s, 2H), 4.49 (s, 2H), 2.33 (s, 3H).13C NMR (101 MHz, DMSO) 5 150.10, 146.39, 139.33, 133.56, 131.61 , 59.29, 56.98, 19.79. IR (ATR, cm-1): 3350 (br, O-H / N-H), 2950-2850 (m, C-H), 1650 (s, C=O), 1585 (m, C=N / C=C), 1465 (m, CH2bending), 1380 (w, O-H bending), 1250-1050 (s, C-O / C-N).

[0177] It should be emphasized that the above-described embodiments of the present disclosureare merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the abovedescribed embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.REFERENCES1. Amde, M et al, Environ. Sci. Technol. 2015, 49, 12611.2. Antaris, A. L; et al. Biomaterials 2016, 15, 235-242.3. Antaris, A. L; et al. Nat. Commun. 2017, 8, 15269.4. Azevedo, AM et al, Anal. Chim. Acta 2020, 1133, 180.5. Azevedo, AM et al, Chemosphere 2021 , 277, 130227.6. Barone, V et al. Chem. Phys. 1997, 107, 3210.7. Becke, AJ, J. Chem. Phys. 1993, 98, 5648.8. Bricks, J. L; et al. Methods Appl. Fluoresc. 2017, 6, 012001.9. Bwambok, DK et al, ACS Nano 2009, 3, 3854.10. Chen, J.; et al. Chem. Commun. 2016, 52, 4025-4028.11. Cosco, AD et al., Angew. Chem. Int. Ed. 2017, 56, 13126.12. Cosco, ED et al, J. Chem. Phys. 2021, 5, 727.13. Cossi, M et al, Chem. Phys. Lett. 1996, 255, 327.14. Davies, K. S.; et al. Bioorg. Med. Chem. 2016, 24, 3908-3917.15. Detty, M. R.; et al. J. Med. Chem. 2004, 47, 3897-3915.16. Egorova, KS et al, Coord. Chem. Rev. 2017, 117, 7132.17. Escobedo, J. O.; et al. Curr. Opin. Chem. Biol. 2010, 14, 64-70.18. G. Singh, G. Singh, S. Kancharla, T. S. Kang, J. Phys. Chem. B 2019, 123, 2169.19. G. Singh, G. Singh, T. S. Kang, Phys. Chem. Chem. Phys. 2016, 18, 25993.20. G. Singh, M. Kaur, V. K. Aswal, T. S. Kang, RSC Adv. 2020, 10, 7073.04-1014. , 2206-2217. 4, 15, 9380-9387. 1764. 16-29. 23, 1. , 2257. , 194-213. Sensitive Drug Delivery Systems 2018, 567. 9, 3745-3754. 41-3050. , 4730. 4, 2050-2055. , 8, 033001. 59. , 449. -7138. . Kang, Phys. Chem. Chem. Phys. 2021 , 23, 320. , 107. . 2017, 23, 12494-12501. 22, 6, e202200198. 85, 4089-4095. 8, e202300212.Ndaleh, D.; et al. J. Org. Chem. 2021 , 86, 15376-15386. Ni, Y.; et al. Chem. Commun. 2014, 12, 3774-3791. Patidar, P et al, J. Colloid Interface Sci. 2018, 555, 691. Perdew, JP et al, Phys. Rev. Lett. 1996, 77, 3865. Reichardt, A et al, Solvents and Solvent Effects in Organic Chemistry, John Wiley & Sons, 2011. Revision, C et al., Gaussian, Inc., Wallingford CT 2016. Runge, A et al, Phys. Rev. Lett. 1984, 52, 997. S. J. Shiffka, M. A. Kane, P. W. Swaan, Biochem. Biophys. Acta 2017, 1859, 2269. Sarcan, E. T.; et al. J. Inorg. Organomet. Polym. Mater. 2018, 551 , 329-338. Shindy, H. Dyes Pigm. 2017, 145, 505-513. Singh, G et al, J. Mol. Liq. 2021 , 325, 115156. Singh, G et al, J. Phys. Chem. B 2018, 122, 12227. Singh, G et al, J. Phys. Chem. C 2020, 124, 3791. Su, L. et al, ACS Appl. Mater. Interfaces 2021 , 13, 43458. Tanner, EE et al, J. Control. Release 2018, 286, 137. Tanner, EE, Adv. Drug Deliv. Rev. 2019, 31, 1901103. Usama, S. M.; et al. Curr. Opin. Chem. Biol. 2021 , 63, 38-45. Wang, J.; et al. Coord. Chem. Rev. 2018, 354, 135-154. Wasserscheid, P. et al, Ionic Liquids in Synthesis, Vol. 1, Wiley Online Library, 2008. Widengren, J et al, Bioimaging 1996, 4, 149. Wu, X et al, Chem. Soc. Rev. 2015, 44, 4179. Yanai, T et al, Chem. Phys. Lett. 2004, 393, 51. Yang, M et al, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2024, 16, e1960. Yang, W et al, J. Phys. Chem. A 1986, 34, 4586. Zhang, X. D.; et al. Adv. Mater. 2016, 28, 6872-6879.71. Zhao, Xetal, Chem. Sci.2022, 13, 11280.72. Zhu, S.; etal. Adv. Mater.2019, 31, 1900321.

Claims

CLAIMSWhat is claimed is:

1. A method for enhancing molecular brightness of a near infrared (NIR) dye in aqueous solution, the method comprising contacting the dye with a biocompatible ionic liquid (IL).

2. The method of claim 1 , wherein the NIR dye comprises SO3SQ, SQ, NMe3SQ, C5, SO3C5, NMe3C5, ICG, IR-1061 , a derivative or variant thereof, or any combination thereof.

3. The method of claim 1, wherein the IL comprises an organic cation and an anion in a ratio of from about 4: 1 to about 1 :1.

4. The method of claim 3, wherein the anion comprises decanoate, ethylenediamine tetraacetate, urocanate, nicotinate, pyridoxinate, orotate, citrate, hexadecanoate, biotinate, deoxycholate, a derivative or variant thereof, or any combination thereof.

5. The method of claim 3, wherein the organic cation comprises choline.

6. The method of claim 1 , wherein the NIR dye is SO3SQ and the ionic liquid is choline deoxycholate, wherein the choline and the deoxycholate are in a ratio of 1 :1.

7. The method of claim 1 , wherein molecular brightness is increased from about 350 times an original level to about 35,000 times an original level.

8. A composition comprising an NIR dye and a biocompatible IL in water.

9. The composition of claim 8, wherein the NIR dye comprises SO3SQ, SQ, NMe3SQ, C5, SO3C5, NMe3C5, ICG, IR-1061 , a derivative or variant thereof, or any combination thereof.

10. The composition of claim 8, wherein the IL comprises an organic cation and an anion in a ratio of from about 4: 1 to about 1 :1.

11. The composition of claim 10, wherein the anion comprises decanoate, ethylenediamine tetraacetate, urocanate, nicotinate, pyridoxinate, orotate, citrate, hexadecanoate, biotinate, deoxycholate, a derivative or variant thereof, or any combination thereof.

12. The composition of claim 10, wherein the organic cation comprises choline13. The composition of claim 8, wherein the NIR dye is SO3SQ and the ionic liquid is choline deoxycholate, wherein the choline and the deoxycholate are in a ratio of 1 :1.

14. The composition of claim 8, wherein the composition is not cytotoxic.

15. The composition of any one of claims 8-14, wherein the composition causes substantially no hemolytic activity in human red blood cells.

16. A method for performing medical imaging in a subject, the method comprising administering the composition of any one of claims 8-15 to the subject and visualizing a signal from the NIR dye.

17. The method of claim 16, wherein the signal comprises a fluorescence signal.

18. The method of claim 16, wherein the subject is a human.

19. A method for performing photothermal treatment for a disease or disorder in a subject, the method comprising administering the composition of any one of claims 8-15 to a treatment location in the subject and irradiating the treatment location.

20. The method of claim 19, wherein irradiation is performed with NIR light.

21. The method of claim 19, wherein the disease or disorder comprises cancer.

22. The method of claim 19, wherein the treatment location comprises a tumor.

23. The method of claim 19, wherein the subject is a human.

24. A method for enhancing immunohistochemical staining of a cell or tissue, the method comprising applying an NI dye conjugated to an antibody and an IL to the cell or tissue.

25. The method of claim 24, wherein the NIR dye comprises SO3SQ, SQ, NMe3SQ, C5, SO3C5, NMe3C5, ICG, IR-1061 , Alexa Fluor 647, a derivative or variant thereof, or any combination thereof.

26. The method of claim 24, wherein the IL comprises an organic cation and an anion in a ratio of from about 4: 1 to about 1 :1.

27. The method of claim 26, wherein the anion comprises decanoate, ethylenediamine tetraacetate, urocanate, nicotinate, pyridoxinate, orotate, citrate, hexadecanoate, biotinate, deoxycholate, a derivative or variant thereof, or any combination thereof.

28. The method of claim 26, wherein the organic cation comprises choline29. The method of claim 24, wherein the NIR dye is Alexa Fluor 647 and the ionic liquid is choline deoxycholate, wherein the choline and the deoxycholate are in a ratio of 1 :1.

30. The method of claim 24, further comprising using fluorescence microscopy to visualize the cell or tissue.

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