Ratiometric imaging using dyes

Abstract

The disclosure relates to compounds of the formula (I), (la), (II), (Ila), (llb), (llc), (lld), and (lle), compositions comprising such compounds, and the use of such compounds in imaging.

Description

RATIOMETRIC IMAGING USING DYESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Appl. No. 63 / 526,386, filed July 12, 2023, which is incorporated by reference as if fully set forth herein.BACKGROUND

[0002] Lysosomes actively regulate their luminal pH, which is necessary for optimal enzymatic activity. Endocytic processes are involved in many diseases, including Alzheimer’s disease (AD), in which sub-optimal lysosomal function has been reported. To measure acidification, pH-sensitive probes can be delivered to endosomes and lysosomes using labeled dextran polymers or proteins. However, commercially available probes have limited sensitivity in the acidic range of lysosomes, and their fluorescence is subject to photobleaching.SUMMARY

[0003] The disclosure relates to the development of fluorescent dyes that work well in acidic environments, such as inside lysosomes. In one example, the disclosure describes the preparation, characterization, and uses of a novel pH- sensitive probe, ApHID, a green-emitting dye with a pKa of 5.4, increasing brightness with acidity, and resistance to oxidation and photobleaching. The fluorescent dyes of the disclosure (e.g., ApHID) can be used, for example, to measure lysosomal pH in macrophages, yielding virtually identical results when compared with FITC and Oregon Green. However, the fluorescent dyes of the disclosure (e.g., ApHID) circumvent limitations presented by most commercially available pH-sensitive probes, and they can be useful in demanding imaging applications, such as intravital imaging of tissues.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] FIGS. 1 A-1 E show the on-off switch and spectroscopic properties of ApHID, (A) ApHID structure and on-off fluorescence switch. ApHID contains an aniline moiety acting as an electron donor at alkaline pH, which quenches fluorescence. The group ‘R’ consists of a 10-carbon polyethylene glycol chain (PEG4), which ends in a succinimidyl ester group that confers increased solubility in water and allows for derivatization of molecules containing primary amines such as proteins or amino-dextran polymers. (B) Quantum yield of the hydrolyzed NHS ester form of ApHID measured in various pH-adjusted buffers, using fluorescein in 0.01 M NaOH (pH 12) as a standard. Quantum yield was plotted against pH, yielding a titration that was fit to a 4-component sigmoid with -log IC50 (pKa) of 5.4.The maximal extinction coefficient and quantum yield of ApHID, measured in a subsequent experiment in pH 3.0 buffer, are 0.64 and 99,710 M-1 cm1, respectively. (C-E) Absorbance (C), excitation (D), and emission (E) spectra plotted against buffer pH, measured for a 0.04 mg / mL dilution of 10 KDa aminodextran labeled with ApHID at a 1 .6:1 molar ratio, in pH-adjusted buffers. Two independent measurements were completed for each experiment. Geometrical objects and bars indicate averages ±SEM.

[0005] FIGS. 2A-2F show that ApHID fluorescence and pKa are not affected by oxidation caused by *OH radicals, protein, or various salt concentrations in solution. (A, B) ApHID, fluorescein, Oregon Green, and LysoSensor y / b absorbance (A) and emission (B) profiles vs buffer pH, measured in solution. Each dye was attached to 10 KDa amino-dextran polymers and diluted to 0.02 mg / mL in various pH-adjusted buffers. ApHID absorbance decreases monotonically with increasing pH, and emission increases strongly with increasing acidity. (C) ApHID, Oregon Green, and fluorescein (hydrolyzed NHS ester forms) fluorescence titrations against buffer pH in the presence of various amounts of hydroxyl radical (•OH) generated by mixing various amounts of ferrous perchlorate (II) and H2O2 in solution, incubated at 37 °C for 20h. (D-F) ApHID (D), Oregon Green (E), and fluorescein (F) dyes attached to 10 KDa amino-dextrans were diluted to 0.04 mg / mL in pH-adjusted buffers enriched either with 50 mg / mL BSA, 1 mM CaCL, and 1 mM MgCL or sodium acetate instead of sodium chloride and incubated at 37 °C for 20h. For all experiments, measurements were repeated twice. Normalized fluorescence intensities were plotted against buffer pH resulting in various titrations and fit to 4-component sigmoidal curves. In all panels, geometrical objects and bars indicate average ±SEM.

[0006] FIGS. 3A-3G show that ApHID is highly resistant to laser-induced photobleaching. (A-F) J774 macrophages, imaged by confocal microscopy using a 40X objective, with lysosomes labeled with 70 KDa dextran polymers derivatized with ApHID (A, D), fluorescein (B, E) or Oregon Green (C, F) at approx. 2:1 molar ratio, prior to (A, B, C) or after (D, E, F) exposure to laser irradiation using a 35 mW, 488 nm laser line for 50 half-second pulses. Cells were fixed with 0.5% PFA prior to imaging. ApHID and Oregon Green labeled cells were imaged in 50 mM TRIS maleate buffer adjusted to pH 5.0, and fluorescein was imaged in 1X PBS buffer adjusted to pH 7.4. Laser output was set to 30% (usual outputs during confocal imaging range between 0.5% and 4%), yielding 5.2 W of power at the front element of the 40X objective used for imaging. Single confocal planes were irradiated for 50 cycles, with 1 second intervals between each cycle. Pixel dwelltime was 33 ps. (G) Normalized fluorescence intensity was plotted against irradiation cycle for ApHID, fluorescein, and Oregon Green dextrans. The experiment was repeated twice; two dishes were measured per condition and 4 fields were imaged per dish. Triangles indicate the average fluorescence intensity for each irradiation cycle. The SEM error bars, based on two measurements, fit within the symbols. Scale bar: 5 pm.

[0007] FIGS. 4A-4G show that different degrees of ApHID labeling of dextrans does not alter ApHID’s fluorescence properties. Also, only high negative charge density will induce a change on ApHID’s pKa when incorporated to dextrans. (A) Representative structure of a dextran polymer containing lysine groups that can be derivatized with pH-sensitive (green spheres) and pH-independent (blue spheres) dyes. (B-E) ApHID / Alexa 405 (B, C) and ApHID / Cy5*3SO3(D, E) ratios measured for dextrans labeled with various amounts of ApHID, and a constant amount of either pH-independent dye, solubilized at 0.04 mg / mL in pH-adjusted buffers. Ratios were plotted against buffer pH. The titrations were either normalized to the ApHID / pH-independent ratio for the dextran containing the least amount of ApHID at pH 4.0 (B, D) or by the ApHID / pH-independent ratio for the dextran with the highest content in ApHID at pH 5.0 (C, E). (F, G) ApHID / Alexa405 ratios were measured for dextrans containing various amounts of Alexa 405 dye and a constant amount of ApHID. Each Alexa 405 molecule carries three negatively charged sulfate groups (F). Fluorescence ratios were plotted against buffer pH, and the various titrations were fit to a 4-component sigmoid from which -log IC5o (pKa) was calculated. pKa values are indicated next to each titration (G). Experiments were repeated three times. Geometrical objects indicate averages. The SEM error bars fit within the symbols. Abbreviations: ‘A405’: Alexa Fluor 405; ‘Cy5’: Cy5-3SO3-; ‘ApH’: ApHID.

[0008] FIGS. 5A-5P show lysosomal pH in J774 macrophages reported by ApHID matches that reported by fluorescein and Oregon Green. Also, ApHID is sensitive to changes in pH during live imaging. (A-E) Calibration confocal microscopy images of late endosomal and lysosomal (LE / Ly) compartments in J774 macrophages, loaded with dextran polymers labeled with ApHID and Alexa 647. Cells were incubated with 0.5 mg / mL dextrans overnight and chased for 3h in fresh DMEM the following morning. Following chase, cells were fixed in 0.5% PFA and incubated for 20 min in pH 4.0 and pH 4.5 buffer or 30 min in pH 5.0-6.5 buffers at 27 °C, followed by imaging by confocal microscopy (A-D). ApHID / Alexa 647 ratios were calculated for each field and plotted against buffer pH. The resulting titration was fit to a 4-component sigmoidal curve (E, blue-dotted line) and compared witha titration of the same dextrans measured in solution using a spectrofluorometer, also at 27 °C (E, red-dotted line). Two wells were imaged for each pH-adjusted buffer tested, and three fields were acquired per well. The experiment was repeated three times. Geometrical objects indicate averaged ratios. SEM was within the geometrical objects. (F-N) Confocal microscopy micrographs of J774 macrophages loaded with dextrans labeled with ApHID (F-H), fluorescein (I -K), or Oregon Green (L-N) with Alexa 647 as a pH-independent dye. The cells were either fixed in 0.5% PFA and incubated in pH 5.0 buffer for 30 min at 37 °C (F, I, L) or imaged live at 37 °C in complete DMEM medium (G, J, M). In order to alkalinize acidic compartments, 20 mM methylamine was added to the cells (H, K, N). (O-P) LE / Ly pH was calculated for each lysosome (O) or field (P), and fields were averaged for each well imaged and condition (P). Two wells were imaged for each condition, and four fields were acquired for each well. The experiment was independently replicated three times. Geometrical objects and bars indicate averaged pH ±SD (O) or ±SEM (P). Image processing was done using MetaMorph software. LE / Ly signal was dissected using an intensity threshold applied to the pH-independent channel (Alexa 647). A mask was then generated and transferred to the pH-sensitive channel. The integrated intensity was measured for each masked channel, and pH-sensitive / pH-independent ratios were calculated for each object or field imaged. Individual LE / Ly were dissected using internally thresholded objects and morphometry analysis functions in MetaMorph, and integrated intensity for all channels was measured for each object. Calculated ratios were then interpolated to pH values using calibration curves prepared for each dye pair. To prepare the curves, J774 macrophages were fixed in 0.5% PFA and incubated in pH 5.0 buffer for 30 min and then imaged. Fluorescence ratios corresponding to pH 5.0 were calculated per LE / Ly or per field as described above and used to generate all subsequent ratios corresponding to pH values 3.5 and 5.0-7.4, using titration data obtained previously for the same dextrans in solution. The resulting ratio-to-pH titrations were fit to 4-component sigmoidal curves. Scale bars: 10 pm. Abbreviations: ‘OG’: Oregon Green; ‘A647’: Alexa Fluor 647; ‘ApH’: ApHID.DESCRIPTION

[0009] Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0010] Mammalian cells utilize a variety of endocytic mechanisms to internalize small molecules, macromolecules, and particles that are delivered to specificsealed organelles. Late endosomes and lysosomes (LE / Ly) are membrane-bound vesicles containing more than 60 different hydrolases and more than 100 membrane proteins, which constitute the degradative endpoint of the endocytic system. These organelles have the capacity to tightly regulate their intraluminal pH, which is required for maintaining optimal enzymatic activity. In most cells, during the 30 minutes following internalization, ligands encounter an increasingly acidic environment ranging from about pH 6 in sorting endosomes to a pH of 4.5- 5 in lysosomes. The main regulator of vesicular pH is the V-ATPase complex, with its different subunits playing a role in the regulation of membrane potential and vesicular pH as well as organelle function.

[0011] Endocytic processes play several roles in many diseases, including Alzheimer’s disease (AD) and atherosclerosis, and lysosomal enzymatic deficiencies are the main culprit leading to lysosomal storage disorders such as Tay-Sachs disease and ceroid lipofuscinosis. LE / Ly membrane permeabilization can be caused by a variety of factors, and AD’s fibrillar amyloid-beta has been reported to damage LE / Lys and cause enzyme leakage. During AD pathogenesis, a deficiency in endolysosomal acidification has been shown to block autophagic flux, causing neurons to fill with undigested autophagic cargo, leading to extensive cellular damage. Also, it has been hypothesized that aging might diminish overall endolysosomal function, which could lead to neurodegenerative conditions. Microglia, the immune cells of the brain, are chronically activated in parts of the AD brain, causing damage to neurons while probably inefficiently digesting fibrillar amyloid-beta due to insufficient LE / Ly function. It is therefore important to find tools to visualize LE / Ly compartments and measure their acidification both in cell culture and in vivo models. Novel methodology in this direction would shed light on pathogenic processes for which understanding is still limited.

[0012] LE / Ly pH can be quantified in cells by fluorescence microscopy imaging of organelles labeled with pH-sensitive dyes, which can be delivered to the organelles using labeled dextrans or proteins. Once incorporated into the endocytic system, dextrans or proteins labeled with pH-sensitive probes reach endocytic compartments, where they serve as pH-sensors. Organelle pH can be determined with precision by ratiometric imaging, and ratios measured can be interpolated to pH values using a ratio to pH calibration prepared using fixed cells. In 1978, Okhuma and Poole measured LE / Ly pH in macrophages for the first time using FITC-dextrans. Others have used ratiometric pH imaging of dextrans or proteins labeled with pH-dependent and pH-independent fluorophores to measure lysosomal pH in many types of cells. The acidity of extracellular degradativecompartments -lysosomal synapses-, formed by macrophages contacting large aggregates of LDL during digestive exophagy, using ratiometric pH imaging of CypHer5E and Alexa 488 probes attached to the aggregates have also been measured.

[0013] Commercially available fluorescence probes have a limited capacity to sense LE / Ly pH. FITC, for example, which has been widely used to measure endosomal pH, has a pKa of 6.5, and its brightness decreases with acidity, which limits its sensitivity in the acidic range of LE / Lys (pH 4.0-5.5). Also, FITC undergoes rapid photobleaching, which limits its use in applications requiring extended imaging. In the past two decades, several pH-sensitive probes were developed commercially, including Oregon Green, pHrodo red, pHrodo deep red, and pHrodo green, among others, with improved fluorescence dynamic range in the acidic spectrum of endosomes. Oregon Green has a pKa of 4.7, and its dynamic range in the pH range of 4-6 is better than that of FITC. However, Oregon Green’s brightness decreases with acidity, limiting its sensitivity. pHrodo red and pHrodo green brightness increases with acidity, but the probes have a pKa of 6.5, which, like FITC, limits their sensitivity in the acidity range of LE / Lys. The pHrodo deep red probe, which was developed recently, has a pKa of 5 and a good dynamic range in the pH reach of 4.0-5.5, but its fluorescence needs to be detected in the far-red spectrum, and its brightness is somewhat modest, which limits its uses. For prolonged fluorescence excitation or intravital imaging of tissues, which can cause extensive photobleaching and light scattering, bright probes with robust fluorescence, resistance to chemical modification, and excellent fluorescence dynamic range between pH 4.0 and 5.5 are required.

[0014] The disclosure provides, among other things, examples of compounds with at least one of robust fluorescence, resistance to chemical modification, and excellent fluorescence dynamic range between pH 4.0 and 5.5. An example of such compounds includes (e.g., water-soluble) BODIPY-based fluorescent compounds / dyes of the formula (I):each R1, R3, and R4is optional substituted alkyl;R2is optionally substituted alkyl;R5and R6are each independently OR7, O-N(8)2 or N(R8)2, wherein R7is independently H or optionally substituted alkyl, and each R8is optionally substituted alkyl or each R8, together with the atom to which they are attached, form a heterocycle; and each X is independently a halogen.

[0015] An example of a compound of the formula (I) includes a compound of the formula (la):

[0016] In the compounds of the formula (I) and (la), each X can be independently a fluorine or a chlorine, such as wherein both X are fluorine. Alternatively, or in addition, at least one of R1, R3, and R4can be an optionally substituted (Ci-C4)alkyl, such as methyl. Alternatively, or in addition, R5and R6can each independently be OR7or N(R8)2. For example, R5and R6can each independently be NHR7, wherein R7is optionally substituted alkyl. Alternatively, R5is OH and R6is NHR7, wherein R7is optionally substituted alkyl, such as ethyl. R5can also be O-N(R8)2, wherein each R8is optionally substituted alkyl or each R8, together with the atom to which they are attached, form a heterocycle, such as a five-membered heterocycle, such as a heterocycle of the formula:wherein the dashed line can represent a double bond. In some embodiments, the dashed line is absent such that the heterocycle comprises a single bond between the carbon atoms connected by the dashed line. Additionally, or alternatively, to R5being O-N(R8)2, R6can be NHR7, wherein R7is optionally substituted alkyl, such as ethyl. R5can also be a five-membered heterocycle, such as a heterocycle of the formula:wherein the dashed line can represent a double bond. In some embodiments, the dashed line is absent such that the heterocycle comprises a single bond between the carbon atoms connected by the dashed line. R5can also be NHR7, wherein R7is -(alkyl-O)n-R9or alkyl substituted with a group of the formula -(alkyl-O)n-R9, wherein n is an integer from 1 -10, such as 3-8 or 4-6 and R9is H, alkyl (substituted with, e.g., halogen, such as I, Cl or Br; amino, or with any other group that can be used for conjugation, e.g., with a dextran), -alkyl-C(O)OR7, -alkyl-C(O)O-N(R8)2; and R6is N(R8)2. For example, the — (alkyl-O)n— can be a group of the formula -(CH2CH2-O)n-. R7can be -(alkyl-O)n-R9, wherein R9is -alkyl-C(O)O-(NR8)2, each R8, together with the atom to which they are attached, form a heterocycle, such as a heterocycle of the formula:

[0017] Compounds of the formula (I) and (la) include compounds of the formula:

[0018] A protein (e.g., low density lipoproteins) or antibody conjugate of any compound described herein is also contemplated. Methods of imaging using any compound described herein, including protein or antibody conjugates, are also contemplated. The methods include localizing the conjugates on a cell surface, intracellularly (e.g., in the cytoplasm), or in one or more intracellularcompartments / organelles; irradiating the conjugates with a wavelength of light that they absorb (for example, with light of from about 500 nm to about 650 nm); and measuring the emission of the conjugates (for example, measure the emission at about 650 nm to about 800 nm).

[0019] Also contemplated herein are methods for measuring changes in pH over a range of 4.2-7 using a water-soluble BODIPY-based fluorescent dye, such as the compounds of the formula (I), (la), and (II) (described below) and conjugates thereof. The pH can be measured in any environment, including endosomes (e.g., late endosomes) and lysosomes (e.g., using dextran as molecular carrier of ApHID) or endosomes (e.g., when using acetylated low-density lipoproteins as ApHID carrier). The methods for measuring changes in pH over a range of 4.2-7 using a water-soluble BODIPY-based fluorescent dye or a conjugate thereof, the method comprising (i) localizing the water-soluble BODIPY-based fluorescent dye or a conjugate thereof intracellularly in one or more intracellular compartments / organelles; (ii) irradiating the water-soluble BODIPY-based fluorescent dye or a conjugate thereof with a wavelength of light absorbed by the water-soluble BODIPY-based fluorescent dye or a conjugate thereof (for example, with light of from about 500 nm to about 650 nm); measuring the emission of the water-soluble BODIPY-based fluorescent dye or a conjugate thereof (for example, measure the emission at about 650 nm to about 800 nm); and determining the pH based on fluorescence ratios corresponding to the pH as described, for example, in the Examples section herein.

[0020] Additional examples of water-soluble BODIPY-based fluorescent dye include compounds of the formula (II):wherein:R1a, R2a, R3a, R1 b, R2b, R3b, are independently selected from a hydrogen atom, a straight- or branched-chain hydrocarbon group having one to twenty carbon atoms, wherein said straight- or branched-chain hydrocarbon group is optionallysubstituted with one or more hydrophilic moieties, and wherein at least one of R2a, and R2bis a hydrocarbon group substituted with a hydrophilic moiety;A1and A2are independently selected from a hydrogen atom, a straight- or branched-chain hydrocarbon group having one to six carbon atoms; andY1and Y2are independently selected from a hydrogen atom, an aromatic ring, a heteroaromatic ring, a carbocyclic ring, a heterocyclic ring, or a straight- or branched-chain hydrocarbon group having one to six carbon atoms, and wherein said straight- or branched-chain hydrocarbon group may optionally be taken together to form a heterocyclic or heteroaromatic ring. Examples of straight- or branched-chain hydrocarbon groups include alkyl, cycloalkyl, aryl, and heteroaryl groups. Examples of hydrophilic moieties include hydrocarbons that are interrupted by at least one heteroatom (e.g., oxygen and NH), such as alkylene oxides of the formula -(alkyl-O)n- including -(CH2CH2-O)n-. Other hydrophilic moieties include carboxylic acids, guanidines, amines, and the like.

[0021] Compounds of the formula (II) include compounds of the formula (Ila):wherein n is 0, or an integer of 1 to 6; m is 0, or an integer of 1 to 6;G1and G2are independently selected from an oxygen atom or amino group;Q1and Q2are independently selected from an oxygen atom or two hydrogen atoms (e.g., forming -CH2-); andZ1and Z2, are independently selected from a hydrogen atom, a straight- or branched-chain hydrocarbon group having one to twenty carbon atoms, wherein said straight- or branched-chain hydrocarbon group is optionally substituted with one or more hydrophilic moieties, wherein at least one of Z1, and Z2is a hydrocarbon group substituted with a hydrophilic moiety; and wherein at least one hydrophilic moiety terminates in a reactive chemical moiety (e.g., a carboxylic acid or an NHS ester) capable of forming amide bonds.

[0022] Compounds of the formula (II) and (Ila) include compounds of the formula (Hb):wherein n is an integer of 1 to 6; andR4ais selected from one of the following: a reactive chemical moiety that enables amide bond formation or a cell-targeting agent attached via an amide bond.Examples of reactive chemical moieties include moieties of the formula:Examples of cell-targeting agents include a polysaccharide attached via an amide bond, an oligosaccharide attached via an amide bond, or a cyclic oligosaccharide attached via an amide bond. The oligosaccharide can be a dextran or aminodextran and / or can have a molecular weight of at least 40,000 (e.g., from about 40,000 to 500,000 g / mol).

[0023] Compounds of the formula (II), (Ila), and (lib) include compounds of the formula (He):

[0024] Compounds of the formula (II), (Ila), and (lib) include compounds of the formula (lid)wherein n is an integer of 1 to 6.

[0025] Compounds of the formula (II), (Ila), and (lib) include compounds of thewherein n is an integer of 1 to 6.

[0026] Compositions are also contemplated herein wherein the compositions comprise at least one compound of the formula (I), (la), (II), (Ila), (lib), (lie), (lid), and (lie) and a second fluorophore. The fluorescence emission of the second fluorophore preferably remains pH independent over the range of pH 4-9, but need not. The second fluorophore can be a separate component or a second part of the same compound. An example of such a compound includes a compound of the formula:n is 0, or an integer of 1 to 6; m is 0, or an integer of 1 to 6; a is an integer of 1 to 6; b is an integer of 1 to 6; c is 0 or 1 ;X and Y are independently a reactive chemical moiety that enables amide bond formation (e.g., NHS esters and the like), a cell-targeting agent (e.g., cell targeting agents described in Physiological Rev. 77. 759-803 (1997), which is incorporated by reference as if fully set forth herein) attached via an amide bond, a straight- or branched-chain hydrocarbon group having one to six carbon atoms attached via and amide bond, or a straight- or branched-chain hydrocarbon group having one to six carbon atoms attached via an amide bond terminating in the second fluorophore;Z1, Z2, and Z3are each independently from a hydrogen atom, an aromatic ring, a heteroaromatic ring, a carbocyclic ring, a heterocyclic ring, a straight- or branched- chain hydrocarbon group having one to six carbon atoms, or a straight- or branched-chain hydrocarbon group terminating in the second fluorophore; and wherein at least one of X, Y, and Z1to Z3contain the second fluorophore moiety, where the second fluorophore can be Alexa405, Alexa 647, Alexa 546, Cy3, Cy5, Cy4, Cy5«3SO3, or rhodamine as non-limiting examples.

[0027] All diastereomers of the compounds of the formulae (I), (la), (lb), (Ic), and (ll)-(XII) are contemplated herein.

[0028] As used herein, the term “ratiometric imaging’’ relates to a technique used to analyze ion concentrations by measuring fluorophore shifts. It involves the use of ratiometric indicators, such as the compounds described herein, that show a shift in their emission or excitation spectra depending on the environment in which they are located. The shift in emission or excitation spectra can occur, for example, when the ratiometric indicators are in their protonated or deprotonated form.

[0029] The term “substituted” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto another group (e.g., on an aryl or an alkyl group). Examples of substituents include, but are not limited to, a halogen (e.g., F, Cl, Br, and I), OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, -(CH2)O-2P(0)(OR)2, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)O-2N(R)C(0)R, (CH2)O-2N(R)C(0)OR, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR,N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, or C(=NOR)R wherein each R can be, independently, hydrogen, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl, wherein any alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl or two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl, which can be mono- or independently multi-substituted.

[0030] The term “alkyl" as used herein refers to substituted or unsubstituted straight chain and branched mono- or divalent alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms (C1-C40), 1 to about 20 carbon atoms (C1-C20), 1 to 12 carbons (C1-C12), 1 to 8 carbon atoms (Ci-C8), or, in some embodiments, from 1 to 6 carbon atoms (Ci-C6). Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n- pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups, as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0031] The term “alkenyl” as used herein refers to substituted or unsubstituted straight chain and branched mono- or divalent alkenyl groups and cycloalkenyl groups having at least one double bond and having from 1 to 40 carbon atoms (Cr C40), 1 to about 20 carbon atoms (C1-C20), 1 to 12 carbons (C1-C12), 1 to 8 carbon atoms (Ci-Cs), or, in some embodiments, from 1 to 6 carbon atoms (Ci-Ce). Examples of straight chain alkenyl groups include those with from 1 to 8 carbon atoms, such as -CH=CH-, -CH=CHCH3, and -CH2CH=CHCH2- groups, wherein the double bonds can have an E- or Z-configuration. And when there are multiple bonds, each double bond can, independently, have an E- or a Z-configuration. Examples of branched alkenyl groups include, but are not limited to, -CH=C(CH3)- and CH2C=CH(CH3) groups. Representative substituted alkenyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0032] The terms “cycloalkyl” and “carbocyclic ring,” as used herein refers to substituted or unsubstituted cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ringmembers, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. In some embodiments, cycloalkyl groups can have 3 to 6 carbon atoms (Cs-Ce). Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like.

[0033] The term “cycloalkylalkyl” as used herein refers to substituted or unsubstituted alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a cycloalkyl group as defined herein. Representative cycloalkylalkyl groups include, but are not limited to, cyclopentylalkyl.

[0034] The term “alkylcycloalkyl” as used herein refers to substituted or unsubstituted cycloalkyl groups as defined herein in which a hydrogen of a cycloalkyl group as defined herein is replaced with a bond to an alkyl group as defined herein. Representative alkylcycloalkyl groups include, but are not limited to, alkylcyclopropyl.

[0035] The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of a substituted or unsubstituted alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. In the special case wherein the carbonyl carbon atom is bonded to a hydrogen, the group is a “formyl” group, an acyl group as the term is defined herein. An acyl group can include 0 to about 12-40, 6-10, 1 -5 or 2-5 additional carbon atoms bonded to the carbonyl group. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning here. A nicotinoyl group (pyridyl- 3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, acryloyl groups, and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl” group. An example is a trifluoroacetyl group.

[0036] The term “heterocyclylcarbonyl” is an example of an acyl group that is bonded to a substituted or unsubstituted heterocyclyl group, as the term “heterocyclyl” is defined herein. An example of a heterocyclylcarbonyl group is a prolyl group, wherein the prolyl group can be a D- or an L-prolyl group.

[0037] The term “aryl” as used herein refers to substituted or unsubstituted cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring. Thus arylgroups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons (Ce-Cu) or from 6 to 10 carbon atoms (Ce-Cio) in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed herein. An “aromatic ring” is an example of an “aryl” group.

[0038] The term “aralkyl” and “arylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylary l)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.

[0039] The terms “heteroaromatic ring,” “a heterocyclic ring,” “heterocyclyl,” “heterocyclo,” and “heterocycle” as used herein refers to substituted or unsubstituted aromatic and non-aromatic ring compounds containing 3 or more ring members, of which, one or more (e.g., 1 , 2 or 3) is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. In some embodiments, heterocyclyl groups include heterocyclyl groups that include 3 to 8 carbon atoms (C3-C8), 3 to 6 carbon atoms (C3-C8), 3 to 5 carbon atoms (C3-C5) or 6 to 8 carbon atoms (C6- C3). A heterocyclyl group designated as a C2- heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms, and so forth. Likewise, a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase “heterocyclyl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. Representative heterocyclyl groups include, but are not limited to pyrrolidinyl, azetidinyl, piperidynyl, piperazinyl, morpholinyl, chromanyl,indolinonyl, isoindolinonyl, furanyl, pyrrolidinyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl, triazyolyl, tetrazolyl, benzoxazolinyl, benzthiazolinyl, and benzimidazolinyl groups. Examples of indolinonyl groups include groups having the general formula:, wherein R is as defined herein.Examples of isoindolinonyl groups include groups having the general formula: OQ -R, wherein R is as defined herein.Examples of benzoxazolinyl groups include groups having the general formula:L £ -R, wherein R is as defined herein.Examples of benzthiazolinyl groups include groups having the general formula:, wherein R is as defined herein.In some embodiments, the group R in benzoxazolinyl and benzthiazolinyl groups is an N(R)2group. In some embodiments, each R is hydrogen or alkyl, wherein the alkyl group is substituted or unsubstituted. In some embodiments, the alkyl group is substituted with a heterocyclyl group (e.g., with a pyrrolidinyl group).

[0040] The term “heterocyclylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein. Representative heterocyclylalkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl methyl, and indol-2-yl propyl.

[0041] The term “heterocyclylalkoxy” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein, and the alkyl group is attached to an oxygen. Representative heterocyclylalkoxy groups include, but are not limited to, -O-(CH2)q- heterocyclyl, wherein q is an integer from 1 to 5. In some embodiments, heterocyclylalkoxy groups include -O-(CH2)qmorpholinyl, such as -O-CH2CH2-morpholine.

[0042] The term “heteroarylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.

[0043] The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include one to about 12-20 or about 12-40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group is an alkoxy group within the meaning herein. A methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

[0044] The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein R is defined herein, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term “amine” also includes ammonium ions as used herein.

[0045] The term “amino group” as used herein refers to a substituent of the form - NH2, -NHR, -NR2, -NR3+, wherein each R is defined herein, and protonated forms of each, except for -NR3+, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group.

[0046] An example of an “alkylamino” is -NH-alkyl and -N(alkyl)2.

[0047] An example of a “cycloalkylamino” group is -NH-cycloalkyl and -N(cycloalkyl)2.

[0048] An example of a “cycloalkyl heterocycloamino” group is -NH-(heterocyclo cycloalkyl), wherein the heterocyclo group is attached to the nitrogen and the cycloalkyl group is attached to the heterocyclo group.

[0049] An example of a “heterocyclo cycloamino” group is -NH-(cycloalkyl heterocycle), wherein the cycloalkyl group is attached to the nitrogen and the heterocyclo group is attached to the cycloalkyl group.

[0050] The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0051] The term “haloalkyl” group, as used herein, includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1 ,1 - dichloroethyl, 1 ,2-dichloroethyl, 1 ,3-dibromo-3,3-difluoropropyl, perfluorobutyl, -CF(CH3)2and the like.

[0052] As used herein, the term “salts” and “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include the conventional nontoxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.

[0053] Pharmaceutically acceptable salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. In some instances, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s PharmaceuticalSciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is hereby incorporated by reference.

[0054] The term “solvate” means a compound, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non- covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.

[0055] The term “prodrug” means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide an active compound, particularly a compound of the invention. Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound of the invention that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Specific prodrugs of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid. The carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule. Prodrugs can typically be prepared using well-known methods, such as those described by Burger’s Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001 , Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers GmbH).

[0056] Values expressed in a range format 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 sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1 % to about 5%” or “about 0.1 % to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1 % to 0.5%, 1 .1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0057] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section headingcan occur within or outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0058] In the methods described herein, the steps can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified steps can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed step of doing X and a claimed step of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0059] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1 % of a stated value or of a stated limit of a range.

[0060] The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0061] The term “substantially no” as used herein refers to less than about 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1 %, 0.5%, 0.1%, 0.05%, 0.001 %, or at less than about 0.0005% or less or about 0% or 0%.

[0062] Those skilled in the art will appreciate that many modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Thus, the description is not intended and should not be construed to be limited to the examples given but should be granted the full breadth of protection afforded by the appended claims and equivalents thereto. In addition, it is possible to use some of the features of the present disclosure without the corresponding use of other features. Accordingly, the foregoing description of or illustrative embodiments is provided for the purpose of illustrating the principles of the present disclosure and not in limitation thereof and can include modification thereto and permutations thereof.

[0063] The disclosure also relates to the following numbered clauses, which are presented in no particular order of importance:1 . A compound of the formula:or an enantiomer, stereoisomer, or polymorph thereof, wherein: each R1, R3, and R4is optional substituted alkyl;R2is optionally substituted alkyl;R5and R6are each independently OR7, O-N(R8)2or N(R8)2, wherein R7is independently H or optionally substituted alkyl and each R8is optionally substituted alkyl or each R8, together with the atom to which they are attached, form a heterocycle; and each X is independently a halogen. The compound of clause 1 , wherein the compound is a compound of the formula:enantiomer, stereoisomer, or polymorph thereof. The compound of clause 1 or 2, wherein each X is independently a fluorine or a chlorine, such as wherein both X are fluorine. The compound of any preceding clause, wherein: at least one of R1, R3, and R4is optional substituted (Ci-C4)alkyl, such as methyl. The compound of any preceding clause, wherein:R5and R6are each independently OR7or N(R8)2. The compound of clause 5, wherein R5and R6are each NHR7, wherein R7is optionally substituted alkyl. The compound of clause 5, wherein R5is OH and R6is NHR7, wherein R7is optionally substituted alkyl, such as ethyl.The compound of clause 1 , wherein R5is O-N(R8)2, wherein each R8is optionally substituted alkyl or each R8, together with the atom to which they are attached, form a heterocycle. The compound of clause 8, wherein R6is NHR7, wherein R7is optionally substituted alkyl, such as ethyl. The compound of clause 8 or 9, wherein R5is a five-membered heterocycle. The compound of clause 10, wherein the heterocycle is a heterocycle of the formula:wherein the dashed line can represent a double bond. The compound of clause 5, wherein R5is NHR7, wherein R7is -(alkyl-O)n-R9or alkyl substituted with a group of the formula -(alkyl-O)n-R9, wherein n is an integer from 1 -10, such as 3-8 or 4-6 and R9is H, alkyl, -alkyl-C(O)OR7or -alkyl-C(O)O- N(R8)2; and R6is N(R8)2. The compound of clause 12, wherein -(alkyl-O)n- is -(CH2CH2-O)n- The compound of clause 12, wherein R7is -(alkyl-O)n-R9, wherein R9is -alkyl- C(O)O-(NR8)2, each R8, together with the atom to which they are attached, form a heterocycle. The compound of clause 14, wherein the heterocycle is a heterocycle of the formula:wherein the dashed line can represent a double bond. The compound of any preceding clause, wherein the compound is a compound of the formula:17. A protein or antibody conjugate of a compound of any preceding clause.18. A method of imaging using the conjugate of clause 17.19. A method for measuring changes in pH over a range of 4.2-7 using a water-soluble BODIPY-based fluorescent dye or a conjugate thereof, the method comprising (i) localizing the water-soluble BODIPY-based fluorescent dye or a conjugate thereof intracellularly in one or more intracellular compartments / organelles; (ii) irradiating the water-soluble BODIPY-based fluorescent dye or a conjugate thereof with a wavelength of light absorbed by the water-soluble BODI PY-based fluorescent dye or a conjugate thereof; measuring the emission of the water-soluble BODIPY- based fluorescent dye or a conjugate thereof; and determining the pH based on fluorescence ratios corresponding to the pH.20. The method of clause 19, wherein the measured pH is that of an intracellular organelle.21 . The method of clause 20, wherein the intracellular organelle is a lysosome or an endosome.22. The method of clause 19, wherein the water-soluble BODIPY-based florescent dye is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:wherein:R1a, R2a, R3a, R1b, R2b, R3b, are independently selected from a hydrogen atom, a straight- or branched-chain hydrocarbon group having one to twenty carbon atoms, wherein said straight- or branched-chain hydrocarbon group is optionally substituted with one or more hydrophilic moieties, and wherein at least one of R2a, and R2b is a hydrocarbon group substituted with a hydrophilic moiety;A1and A2are independently selected from a hydrogen atom, a straight- or branched-chain hydrocarbon group having one to six carbon atoms; and Y1and Y2are independently selected from a hydrogen atom, an aromatic ring, a heteroaromatic ring, a carbocyclic ring, a heterocyclic ring, or a straight- or branched-chain hydrocarbon group having one to six carbon atoms, and wherein said straight- or branched-chain hydrocarbon group may optionally be taken together to form a heterocyclic or heteroaromatic ring. The method of clause 19, wherein the water-soluble BODIPY-based florescent dye is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:n is 0, or an integer of 1 to 6; m is 0, or an integer of 1 to 6;G1and G2are independently selected from an oxygen atom or amino group;Q1and Q2are independently selected from an oxygen atom or two hydrogen atoms; andZ1and Z2, are independently selected from a hydrogen atom, a straight- or branched-chain hydrocarbon group having one to twenty carbon atoms, wherein said straight- or branched-chain hydrocarbon group is optionally substituted with one or more hydrophilic moieties, wherein at least one of Z1, and Z2is a hydrocarbon group substituted with a hydrophilic moiety; and wherein at least one hydrophilic moiety terminates in a reactive chemical moiety capable of forming amide bonds. The method of clause 19, wherein the water-soluble BODIPY-based florescent dye is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:wherein n is an integer of 1 to 6; andR4ais selected from one of the following: a reactive chemical moiety that enables amide bond formation or a cell-targeting agent attached via an amide bond. The method of clause 24, wherein the reactive chemical moiety that enables amide bond formation is selected among the following:The method of clause 24, wherein the water-soluble BODIPY-based florescent dye is the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:27. The method of clause 24, wherein the cell-targeting agent is selected from one of the following: a polysaccharide attached via an amide bond, an oligosaccharide attached via an amide bond, or a cyclic oligosaccharide attached via an amide bond.28. The method of clause 27, wherein said oligosaccharide is a dextran or aminodextran.29. The method of clause 27, wherein the oligosaccharide has a molecular weight of at least 40,000.30. The method of clause 19, wherein the water-soluble BODIPY-based florescent dye is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:wherein n is an integer of 1 to 631. The method of clause 19, wherein the water-soluble BODIPY-based florescent dye is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:wherein n is an integer of 1 to 6 The method of clause 19, wherein the composition further comprises a second fluorophore. The method of clause 32, wherein the fluorescence emission of the second fluorophore remains pH independent over the range of pH 4-9. The method of clause 32, wherein said composition is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:wherein n is 0, or an integer of 1 to 6; m is 0, or an integer of 1 to 6; a is an integer of 1 to 6; b is an integer of 1 to 6; c is 0 or 1 ;X and Y are independently a reactive chemical moiety that enables amide bond formation, a cell-targeting agent attached via an amide bond, a straight- or branched-chain hydrocarbon group having one to six carbon atoms attached via and amide bond, or a straight- or branched-chain hydrocarbon group having one to six carbon atoms attached via and amide bond terminating in the second fluorophore;Z1, Z2, and Z3are each independently from a hydrogen atom, an aromatic ring, a heteroaromatic ring, a carbocyclic ring, a heterocyclic ring, a straight- orbranched-chain hydrocarbon group having one to six carbon atoms, or a straight- or branched-chain hydrocarbon group terminating in the second fluorophore; and wherein at least one of X, Y, and Z1to Z3contain the second fluorophore moiety.35. The method of clause 32, wherein the second fluorophore moiety is selected from Alexa405 and rhodamine.Examples

[0064] The disclosure can be better understood by reference to the following examples which are offered by way of illustration. The disclosure is not limited to the examples given herein.Example 1 : Synthesis of PKS8323PKS8323 was synthesized using known procedures:Example 2: Synthesis of PKS8324

[0065] PKS8323 (2.00 g, 2.98 mmol, 2TEA salt) was dissolved in DMF (20 mL) and the solution was cooled to 0 °C. 0-(7-Azabenzotriazol-1 -yl)-A / ,A / ,A / ’,A / ’- tetramethyluronium hexafluorophosphate (HATU) (1 .13 g, 2.98 mmol) was added to the solution at 0 °C. After stirring for 5 minutes at 0 °C, ethylamine (2M in THF, 9.0 mmol, 4.5 mL) was added and the mixture was allowed to warm to room temperature slowly. Reaction gave a mixture of starting material, desired product and diamide. The mixture was purified by CombiFlash [silica gel; 0-10% methanol (0.5% Et3N) in DCM (0.5% Et3N)] to give product (5 eq. triethylamine by NMR; 2.47 g, 83%) as a red solid.1H NMR (500 MHz, DMSO-cfe) 5 1 .05 (t, J = 7.2 Hz, 3H), 1 .43 (s, 3H), 1 .66 (s, 3H), 2.31 (s, 3H), 2.51 (s, 3H), 2.69 (s 3H), 2.71 (s, 6H), 3.14 - 3.22 (m, 2H), 7.08 - 7.1 1 (m, 2H), 7.17 (d, J = 8.0 Hz, 1 H), 8.12 (t, J = 5.5 Hz, 1 H), 10.21 (br, 1 H).Example 3: Synthesis of PKS8107

[0066] PKS8324 (800 mg, 916 nmol, 3.7 eq TEA) was dissolved in THF (10 mL) and the solution was cooled to 0 °C. Dipyrrolidino(W-succinimidyloxy)carbenium hexafluorophosphate (565 mg, 1.37 mmol) was added and the mixture was allowed to warm to room temperature slowly. After stirring the mixture for 3 hours, THF was evaporated, and the mixture was purified by Combi-Flash (C18 column; 5 - 100% acetonitrile in water with 0.1 % formic acid; slurry was made in filter agent). Acetonitrile was removed from the product fraction, and the resulting mixture was frozen and lyophilized to give the product (425 mg, 78%) as a brick- red solid.1H NMR (500 MHz, DMSO-c / 6) 8 1 .06 (t, J = 7.2 Hz, 3H), 1 .49 (s, 3H), 1 .66 (s, 3H), 2.32 (s, 3H), 2.58 (s 3H), 2.72 (s, 6H), 2.73 (s, 3H), 2.85 (s, 4H), 3.17 - 3.23 (m, 2H), 7.14 - 7.20 (m, 3H), 8.25 (t, J = 5.6 Hz, 1 H).13C NMR (125MHz, DMSO-cfe) § 13.1 , 13.3, 13.7, 14.3, 14.6, 18.4, 25.5, 33.7, 43.6, 114.8, 118.7, 125.7, 125.9, 130.1 , 130.2, 132.2, 132.3, 132.7, 143.7, 144.3, 146.2, 154.0, 154.6, 159.3, 159.5, 162.5, 170.5. LCMS calc for C3oH35BF2N505: 594.3; found: 594.4.Example 4: Synthesis of PKS8325PKS8324 (2.47 g, 2.42 mmol, 5 eq TEA) and HATU (971 mg, 2.55 mmol) were dissolved in DMF (15 mL) under an argon atmosphere. The solution was cooled to 0 °C, and NH2-PEG4-COOH (645 mg, 2.43 mmol) was added. After stirring for 5 minutes at 0 °C, Triethylamine (4.87 mmol, 680 piL) was added. The reaction mixture was allowed to warm to room temperature and stirred overnight. After completion of the reaction, the solvent was evaporated, and the mixture was purified by CombiFlash [silica gel; 0-30% methanol (0.5% Et3N) in DCM (0.5% Et3N)] to give the product (2.3 eq. triethylamine by NMR; 1.58 g, 67%) as a red- brown solid. An analytical sample for NMR was purified by HPLC.1H NMR (500 MHz, DMSO-c6) 5 1 .05 (t, J = 7.2 Hz, 3H), 1 .43 (s, 3H), 1 .44 (s, 3H), 2.32 (s, 3H), 2.34 (t, J = 6.6 Hz, 2H), 2.50 ( 6H), 2.71 (s, 6H), 3.15 - 3.21 (m, 2H), 3.25 - 3.50 (m, 16H), 3.56 (t, J = 6.6 Hz, 2H), 7.06 - 7.08 (m, 2H), 7.18 (d, J = 8.1 Hz, 1 H), 8.11 (t, J = 6.0 Hz, 1 H), 8.13 (t, J = 5.3 Hz, 1 H).Example 5: Synthesis of PKS8326

[0067] PKS8325 (1 .46 g, 1 .50 mmol, 2.3 eq. TEA) and EDC (575 mg, 3.00 mmol) were dissolved in DCM (50 ml_) under argon atmosphere. The solution was cooled to 0 °C, and A / -hydroxysuccinimide (259 mg, 2.25 mmol) was added. The reaction mixture was slowly warmed to room temperature and stirred at room temperature overnight. The reaction mixture was diluted with water and extracted with dichloromethane. The organic layer was washed with saturated brine solution, dried over anhydrous Na2SO4, and evaporated. The crude was purified by CombiFlash (C-18 column; 0% - 100% acetonitrile in water). The fractions with product were combined, the acetonitrile was evaporated, and the mixture was frozen and lyophilized to give the product (750 mg, 59%) as a red-brown solid.1H NMR (500 MHz, DMSO-c / e) 5 1 .04 (t, J = 7.2 Hz, 3H), 1 .43 (s, 3H), 1 .44 (s, 3H), 2.31 (s, 3H), 2.50 (6H), 2.71 (s, 6H), 2.80 (s, 4H), 2.91 (t, J = 6.0 Hz, 2H), 3.15 - 3.20 (m, 2H), 3.28 - 3.33 (s, 2H), 3.44 - 3.53 (m, 14H), 3.70 (t, J = 5.9 Hz, 2H), 7.05 - 7.08 (m, 2H), 7.17 (d, J = 8.0 Hz, 1 H) 8.05 - 8.10 (m, 2H).13C NMR (125 MHz, DMSO-c / e) 5 12.6, 12.7, 13.2, 14.7, 18.4, 25.4, 31 .6, 33.6, 38.6, 43.6, 47.5, 65.2, 68.8, 69.4, 69.6, 69.7, 69.8, 1 18.6, 125.6, 126.6, 129.8, 130.0, 130.5, 132.1 , 140.5, 140.7, 144.9, 153.7, 153.8, 153.9, 163.3, 163.6, 167.3, 170.1 , 172.8.Example 6MATERIALS AND METHODSMaterials

[0068] Dextran derivatization with fluorophores. Polymers of 10 KDa (Thermo Fisher D1860) or 70 KDa amino-dextrans (Thermo Fisher D1862 or Fina Biosolutions AD70x33) were solubilized at various concentrations in sterile 0.1 M NaHCO3buffer adjusted to pH 8.3 and reacted with the following N- hydroxysuccinimidyl esters (NHS): NHS-ApHID (custom-made), NHS-5 / 6- carboxyfluorescein (NHS-fluorescein, Thermo Fisher 46410), NHS-Oregon Green (Thermo Fisher 06147), NHSAIexa 405 (Thermo Fisher A30000), NHS- Cy5«3xSO3- (AstaTech 44193) or NHS-Alexa 647 (Thermo Fisher A20106) atvarious polymer:dye molar ratios for 1 -2 h at room temperature (RT) with constant rotation. We examined the effect of various degrees of ApHID derivatization or the presence of negatively charged fluorophores on pKa. 70 KDa dextrans from Fina Biosolutions were reacted with a fixed amount of NHS-Alexa 405 orNHSCy5-3xSO3~, followed by reaction with varying molar ratios of NHS-ApHID.For studies on the effect of charge density on ApHID pKa, the polymers were reacted with NHS-ApHID first, followed by aliquoting and reaction with various amounts of NHS-Alexa 405 to add negative charge density to the polymers. For ratiometric pH imaging experiments in live cells, dextrans were reacted with NHS-ApHID, NHS-fluorescein or NHS-Oregon Green and NHS-Alexa 647 at a 4:3 molar ratio. Following reaction, dextrans were purified by extensive dialysis in 3.5 KDa or 20 KDa cutoff Side-A-Lyzer dialysis cassettes (Thermo Fisher 66330 and66003) against PBS.Table 1ApHID Oregon Green Fluorescein pH-adjusted buffers 5.02 ± 0.02 4.74 ± 0.06 6.1 1 ± 0.01 Buffers + 50 mg / mL BSA 4.87 ± 0.01 4.41 ± 0.15 6.15 ± 0.04 Buffers + 1 mM MgClz, CaCIz 5.02 ± 0.02 4.79 ± 0.02 6.17 ± 0.06 Buffers + Chloride -» Acetate 4.94 ± 0.04 4.82 ± 0.03 6.26 ± 0.08 Buffers + 0.2 mM Fe(ll) 5.32 ± 0.08 5.15 ± 0.01 6.38 ± 0.02 Buffers + 0.1 mM »OH 5.36 ± 0.08 5.14 ± 0.02 6.38 ± 0.06 Buffers + 0.2 mM «OH 5.39 ± 0.09 5.12 ± 0.06 6.34 ± 0.07

[0069] Table 1 shows the log IC5o (pKa) values ± SEM calculated from sigmoidal curves fitted to the titrations shown in FIGS. 2C-2F. Buffers with pH adjusted between 1 .5 and 8.5 (see Methods) alone or enriched in either BSA, CaCL + MgCI2, and sodium acetate (as a substitutive for chloride) were tested on fluorescent probes attached to dextrans. Oxidation by «OH was tested on the hydrolyzed succinimidyl ester form of the dyes (not attached to dextrans). Note that the pKa of the probes is generally lower when attached to dextrans. Two independent experiments were completed.

[0070] Dye incorporation was determined by absorbance. Dextrans were diluted to 0.02 mg / mL in buffers. When measuring ApHID incorporation, dextrans were diluted in 25 mM citric acid, 25 mM sodium citrate buffer adjusted to pH 3.0, and absorbance was read at 502 nm. When measuring labeling with fluorescein or Oregon Green, the dextrans were diluted in pH 7.4 PBS buffer, and absorbance was read at 490 nm. When measuring labeling with Alexa 405, Cy5-3xSOs . or Alexa 647, the dextrans were diluted in buffer, and absorbance was read at 402,666, or 655 nm, respectively. Measurements were done using the quartz cuvette reader on a SpectraMax M3 plate reader (Molecular Devices). The extent of dextran labeling was lower than the polymer:dye ratio of the reaction mixture; dye incorporation efficiencies ranged from 25 to 50%.

[0071] Buffers for measurements in solution. Dextrans were solubilized in buffers with pH adjusted between 1.5 and 8.5. To prepare the buffers, buffer salts were added to 0.66X PBS as follows: pH 1 .5 to 3.5 buffer: 25 mM citric acid plus 25 mM sodium citrate; pH 4.0 to 5.5: 50 mM TRIS maleate; pH 6.0-7.0 buffer: 50 mM sodium phosphate monobasic anhydrous; pH 7.5: 25 mM TRIS hydrochloride plus 25 mM sodium phosphate dibasic; pH 8.0-8.5: 50 mM TRIS base. The pH was determined using an Orion Star A21 1 pH meter (Thermo Fisher), and acidity was adjusted by adding 1 -3N HCI or 1 -1 ON NaOH dropwise. The resulting buffer ionic strength of the solutions was approximately 150 mM. After preparation, the solutions were filtered through a 0.4 pm filter membrane and stored at 42C.

[0072] Cell culture. J774A murine macrophages (ATCC TIB-67) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g / L glucose and 1 mM sodium pyruvate (Corning 15-013-CV), with 10% fetal bovine serum (FBS, Gemini BenchMark FBS 100-106), 4 mM L-glutamine (Gibco 25030081 ), and 1 % penicillin-streptomycin (Thermo Scientific 15140163) in an incubator at 372C with humidified atmosphere and 5% CO2. Cells were passed at a subcultivation ratio of 1 :5 every 2-3 days.

[0073] Buffers and cell media for measurements in fixed and live cells. Fixed cells were incubated and imaged in buffers prepared by adding buffer salts to 0.13X PBS with 10% FBS, 40 mM methylamine hydrochloride, 40 mM sodium acetate, and 40 pM monensin (to ensure buffer equilibration across membranes) and with pH adjustment as described above. The resulting ionic strength of the solutions was approximately 150 mM. The solutions were filtered and stored at 42C. Live cells were imaged by confocal microscopy in a 96-well plate at 372C with 5% CO2, in DMEM without phenol red (Corning 90-013-PB) with 10% FBS, 2.2 g / L sodium bicarbonate (Sigma S5761 ), 4 mM L-glutamine, 1 mM sodium pyruvate (Sigma S8636) and 1% penicillin-streptomycin.

[0074] pH-dependent absorbance measurements. To measure pH-dependent absorbance spectra for ApHID, 10 KDa amino-dextrans derivatized with the dye were diluted to 0.04 mg / mL in pH-adjusted buffers (see preparation of buffers above) and loaded into 384-well flat clear bottom black polystyrene microplates (Corning, 3746). Three wells were loaded per pH buffer condition and dye. The microplates were centrifuged at 3700 rpm for 1 min, followed by absorbancespectra acquisition between 400-600 nm using a SpectraMax M3 plate reader. Absorbance was read from the top of the plate. To record pH-dependent absorbance titrations for all probes, derivatized 10 KDa amino-dextrans were diluted to 0.02 mg / mL in pH-adjusted buffers followed by absorbance reading at their excitation maxima of 500 nm (ApHID); 495 nm (fluorescein); 505 nm (Oregon Green); and 381 nm (LysoSensor yellow / blue). For both measurements, absorbance measured from buffers alone was subtracted as blank.

[0075] pH-dependent fluorescence measurements. To record pH-dependent emission spectra for ApHID, 10 KDa amino-dextrans derivatized with the dye were diluted to 0.04 mg / mL in pH-adjusted buffers and loaded into 384-well microplates. ApHID was excited at 480 nm, and emission spectra were collected between 500 and 600 nm using a SpectraMax M3 plate reader. Fluorescence was read from the bottom of the plate. To record pH-dependent emission titrations, 10 KDa or 70 KDa amino-dextrans labeled with the various probes were diluted to 0.02 mg / mL in pH- adjusted buffers, followed by excitation and fluorescence measurement at their excitation and emission maxima (ApHID, ex / em 500 / 520 nm; fluorescein ex / em490 / 525 nm; Oregon Green ex / em 505 / 525 nm; LysoSensor yellow / blue ex / em 381 / 521 nm; Alexa 405 ex / em 402 / 425 nm; Cy5*3xSO3ex / em 620 / 666 nm; Alexa 647 ex / em 620 / 655 nm). Emission measured from buffers alone was subtracted as blank. Measurements were repeated at least twice.

[0076] Quantum yield and extinction coefficient measurements. ApHID quantum yield was determined following a previously published protocol. Stock solutions of NHS-ApHID and NHSfluorescein were prepared in 1X PBS and allowed to hydrolyze overnight at RT. Four series of dilutions of ApHID, each in a different buffer, were prepared in buffers adjusted to pH 3.5 to 6.0. Four dilutions of fluorescein, with a known quantum yield of 0.95, were prepared in 0.1 M NaOH as a reference. For each dilution and buffer, absorbance and emission spectra were recorded in the range of 400-550 nm and 500-600 nm, respectively, using 1 cm quartz cuvettes and the cuvette reader in a SpectraMax M3 plate reader. Measurements in buffers alone were subtracted as blank. Integrated fluorescence was plotted against integrated absorbance for each dilution and buffer, and the curves were fit to a linear trend. Measurements were repeated twice. Quantum yields for ApHID, for each buffer pH, were calculated using the equation below:where munknown and mstandard are the slopes of the resulting linear regression for ApHID and fluorescein dilutions, respectively.

[0077] To determine the extinction coefficient (s) of ApHiD, several dilutions of a hydrolyzed NHS-ApHID stock were prepared in pH 3.0 citric acid, sodium citrate buffer, and their absorbance at 492 nm was measured in quartz cuvettes using a SpectraMax M3 plate reader. Absorbance measurements in buffers alone were subtracted as blank. The corrected absorbance was fit to Beer’s law, A = s-b c, where A is the absorbance, e is the molar extinction coefficient, b is the path length of the cuvette, and c is the concentration. Measurements were repeated twice.

[0078] Hydroxyl radical generation and oxidation assay. 500 pM NHS-ApHID, NHS-fluorescein, and NHS-Oregon Green solutions were prepared in 1 X PBS and allowed to hydrolyze overnight. The probes were then diluted into 10 pM aliquots in 1 X PBS and refrigerated in ice. Stock solutions of 50 mM Fe(CIO4)2 (Sigma- Aldrich 334081 ) and 50 mM H2O2 (VWR, 30% aqueous stock, BDH7690-3) were prepared in ultra-pure water and immediately added to the fluorophore aliquots to a final concentration of 100 or 200 pM. Plastic tubes containing the aliquots were vortexed for 10 seconds, sealed in paraffin, and incubated at 372C in a convection oven with constant rotation for 20h. Following the reaction, the aliquots were diluted 1 :200 in buffers with pH adjusted between 3.5 and 7.5 and loaded into 384- well microplates. Fluorescence was measured for each probe and buffer at their ex / em maxima using a SpectraMax M3 plate reader. The experiment was repeated twice.

[0079] Effect of salt and protein on probe fluorescence and pKa. 10 KDa amino-dextrans derivatized with the various probes were diluted to 0.02 mg / mL in pH-adjusted buffers additionally supplemented with either 1 mM CaCI2and 1 mM MgCI2, sodium acetate (replacing sodium chloride), or 50 mg / mL bovine serum albumin (BSA). pH-adjusted buffers lacking sodium chloride were prepared using a 0.66X PBS base for which sodium chloride had been replaced by sodium acetate. Buffers containing 50 mg / mL BSA were prepared in 1X PBS and adjusted to pH 2.5-7.0. Dextran dilutions in buffers were vortexed and loaded into 384-well microplates. The microplates were sealed using paraffin and incubated at 37SC in a convection oven for 20h. After incubation, the fluorescence of the various probes was measured at their ex / em maxima using a SpectraMax M3 plate reader. The experiment was repeated twice.

[0080] Photobleaching study in fixed J774 macrophages using confocal microscopy and digital image analysis. J774A.1 murine macrophages were seeded in 35-mm dishes with central 7-mm diameter glass bottom imaging chambers coated with poly-D lysine at 10,000 cells per chamber and allowed to settle for 1 -2 h in an incubator at 37SC. Once settled, cells were incubated with 70KDa dextrans from Thermo Fisher previously labeled with NHS-ApHID, NHS- fluorescein, or NHSOregon Green at a final concentration of 1 mg / mL in complete DMEM overnight. The following morning, cells were washed once and chased for 3h in fresh DMEM, followed by 5 min fixation in 0.5% paraformaldehyde (PFA) and three washes in 1X PBS. Immediately prior to imaging, cells labeled with ApHID or Oregon Green dextrans were incubated in TRIS maleate pH 5.0 buffer, and cells labeled with fluorescein dextran were incubated in pH 7.4 1 X PBS buffer, for 5 min at 37SC inside the confocal microscope incubation chamber. The buffers were supplemented with 40 mM methylamine hydrochloride, 40 mM sodium acetate, 2.5 pM nigericin, and 2.5 pM monensin to facilitate buffer equilibration across cell membranes. Following buffer equilibration, cells were immediately imaged by confocal microscopy. 2 dishes per fluorophore condition were imaged, and 4 fields were acquired for each dish. The experiment was independently replicated twice.

[0081] Ratiometric pH titration of fixed J774 macrophage lysosomes. J774A.1 murine macrophages were seeded in 96-well plates with glass-like polymer bottom (Cellvis, P96-1 .5P) at 40,000 cells per well and allowed to settle for 1 -2 h in the incubator at 373C. Once settled, cells were incubated with 70 KDa amino-dextrans from Thermo Fisher labeled with NHS-ApHID (pH-sensitive) and NHS-Alexa 647 (pH-independent) at a 4:3 molar ratio, with a final concentration of 0.5 mg / mL in complete DMEM overnight. The following morning, cells were washed twice and chased in fresh DMEM for at least 3h. Next, the cells were fixed in 0.5% PFA for 5 mins and washed 3x in 1 X PBS, and the plate was then transferred to an incubation chamber and allowed to equilibrate to 37SC for at least 20 mins, followed by image acquisition (time 0). After that, the PBS buffer was carefully removed from the wells using a 200 pL pipette, and 200 pL of buffers with pH adjusted to 4.0, 4.5, 5.0, 5.5, and 6.0 (see main Methods section) were added. The buffers were supplemented with 10% FBS, 40 mM methylamine, 40 mM sodium acetate, and 40 pM monensin and had been pre-warmed to 37SC prior to addition to cells. The cells were allowed to equilibrate in the buffers for 20 min (pH 4.0 and pH 4.5) or at least 30 min (pH 5.0-6.0) followed by image stack acquisition. Two wells were imaged for each buffer condition, and 3 fields were acquired per well. The experiment was independently replicated three times.

[0082] Confocal microscopy.Photobleaching study in fixed J774 macrophages.

[0083] J774 macrophages were imaged with an LSM 880 confocal microscope (Zeiss) at 37SC. Fluorophores were excited using a 35 mW 488 nm laser with a digital power output adjusted to 30%, yielding 5.2 pW of power at the front elementof theof the 40X objective (1 .30 NA) used for imaging, as determined with a power meter. Single cell planes were irradiated for 0.5 seconds per cycle (50 cycles in total) with 1 second intervals between irradiation pulses. Fluorescence was detected using a high-sensitivity Zeiss GaAs detector with a spectral window adjusted to collect light at 500-550 nm. Pixel dwell time was 0.33 ps.Ratiometric pH imaging of J774 macrophage lysosomes.

[0084] Imaging of fixed cells or live cells was done with a Leica Stellaris confocal microscope using a 40X air objective (0.95 NA) with pinhole adjusted to 1 Airy unit. ApHID, fluorescein, and Oregon Green were excited using a white light solid-state laser adjusted to 495 nm, and Alexa 647 was excited with the laser adjusted to 650 nm. Fluorescence was detected using high-sensitivity silicon-based HyD detectors with a spectral window adjusted to collect light between 500-550 nm and 660-720 nm for all green-emitting dyes and Alexa 647, respectively. Stacks of images with 1 .5 pm separation in the vertical axis were acquired for each field imaged.

[0085] Digital image analysis.

[0086] Digital image analysis was performed using MetaMorph software (Molecular Devices), and figure panels were prepared using FIJI software.

[0087] Photobleaching study in fixed J774 macrophages. Stacks of images for each acquired field were corrected for background intensity by subtracting the 5thpercentile intensity value for each image in the stack. To quantify fluorescence signal per field, a sum projection for each stack (sum of each image in the stack) was generated, and total integrated intensity (F) per field was measured. Fluorescence was normalized to the intensity corresponding to the first irradiation cycle (Fo) and plotted as F / Foagainst cycle time.

[0088] Ratiometric pH imaging ofJ774 macrophage lysosomes. Stacks of images for each acquired field were corrected for background intensity by subtracting ’he 5th percentile intensity value for each image in the stack. To quantify pH per field, a sum projection for each was generated, and fluorescence signal from organelles was selected using an intensity threshold applied to the pH-independent channel (Alexa 647). A mask was then generated and transferred to the pH-dependent channel. Integrated intensity was measured for each masked channel, and pH- sensitive / pH-independent ratios were calculated for each field. To quantify pH per lysosome, the fluorescence signal from organelles was selected using an intensity threshold applied to the pH-independent channel, for each individual plane. The internally thresholded objects function in MetaMorph was used to identify individual objects corresponding to labeled organelles. Two adjacent objects would beseparated when the peak intensities of their Gaussian distribution differed by at least 50%. Dissected objects were analyzed using the integrated morphometry analysis function with a circularity filter set to 90%. Finally, integrated intensity of the pH-sensitive and pH-independent channels was measured for each dissected object, and their ratio values were calculated. Ratios calculated for each field or object imaged were interpolated to pH values using ratio-to-pH calibration curves. To prepare the curves, fixed J774 macrophages loaded with derivatized dextrans were incubated in 50 mM TRIS maleate pH 5.0 buffer and imaged as described earlier. The pH 5.0 ratios were determined per field or per object and used to generate pH-dependent ratio values corresponding to pH 3.5-7.4 buffers, using titration data for the same dextrans in solution. The resulting ratio-to-pH calibrations were fit to 4-component sigmoidal curves.

[0089] Statistical data analysis. Experiments in solution were repeated independently at least twice, and averages ± SEM are shown. Photobleaching measurements in fixed cells were repeated twice; two dishes were imaged per condition and experiment, and 4 fields were acquired per dish. Averaged fluorescence intensity per irradiation cycle per field (16 fields in total) ± SEM is presented. For ratiometric pH imaging in fixed or live cells, experiments were repeated three times; two wells were imaged per condition and probe, and three to four fields were acquired per well. Statistical data processing was done using GraphPad Prism 9. Averaged pH per well ±SEM or averaged pH per lysosome ±SD are presented. Differences in averaged lysosomal pH per well between conditions (six wells imaged throughout three independent experiments, for each dye pair) were assessed using One-way ANOVA with correction for multiple comparison using Dunnett’s T3 and 95% confidence interval. Brown-Forsythe’s and Bartlett’s tests were used to assess differences in data’s standard deviations between conditions. P-values are shown as p>0.05 (ns), p<0.05 (*), p<0.01 (**),

[0090] Design of a pH-sensitive probe to measure acidity in endosomes and lysosomes.

[0091] Fluorescence probes are commonly used to measure endolysosomal pH in different cell types. Sorting endosomes have a luminal pH of 5.9-6.0, whereas late endosomes and lysosomes (LE / Lys) have a pH of 4.5-5.5 1 ,27. To accurately measure pH in these organelles, a probe must be most sensitive to the pH range of 4.0-6.0. With that consideration in mind, we developed a new pH-sensitive fluorescent probe which we have called ApHID (Acidic pH Indicator Dye). Themolecule is composed of a BODIPY core with two flanking amide substitutions and an electrophilic nature (FIG. 1 A). The BODIPY core is attached to an aniline moiety acting as an electron donor. The electron donor propensity of the aniline moiety determines the range of pH sensitivity of the molecule, and it can be conveniently modulated by attaching different alkyl groups to the nitrogen of the N, A / -dialkyl-o- toluidine moiety. Based on a previous study, two methyl groups were attached to the nitrogen of N, A / -dialkyl-o-toluidine moieties (FIG. 1 A). To increase water solubility of the otherwise insoluble structure, we attached a 10-carbon polyethylene glycol chain (PEG4) to one of the amide groups. The distal end of the PEG4 chain was derivatized with an N-hydroxysuccinimidyl ester group, allowing for the labeling of proteins and other molecules such as amino-dextrans via reaction with primary amines (group R in FIG. 1 A).

[0092] The quantum yield (QY) for ApHID is 0.64 [measured relative to 5 / 6- carboxyfluorescein carboxylic acid (fluorescein) as a standard in 0.1 M NaOH], and its extinction coefficient is 99,700 M1cm1, both measured in citrate buffer at pH 3.0. The pKa value of ApHID (hydrolyzed NHS ester form) in solution is 5.4, based on its pH-dependent QY profile (FIG. 1 B). The UV-visible absorption spectra of ApHID remain similar between pH 4.0 to pH 6.0 (FIG. 1 C), but fluorescence emission spectra increase sharply in amplitude with increasing acidity (FIG. 1 D). Excitation spectra were also pH-dependent, with an excitation maximum at 506 nm (FIG. 1 E).

[0093] ApHID fluorescence and pKa remain stable in the presence of «OH radicals, protein, or salts in solution.

[0094] We compared the spectroscopic properties of ApHID with those of commercially available pH-sensitive probes, namely LysoSensor yellow / blue, fluorescein, and Oregon Green. Since LysoSensor is commercially available attached to 10 KDa dextran polymers, and since we will be using derivatized dextrans in subsequent experiments, we carried out our measurements using probes attached to dextran polymers. Dextran labeling can be achieved by reacting the fluorophores, attached to an A / -hydroxysuccinimidyl ester (NHS) group, with polymers previously derivatized with primary amines (amino-dextrans).

[0095] Fluorophores attached to 10 KDa dextrans were solubilized in buffers with pH adjusted between 1.5 and 8.5, and fluorescence and absorbance were measured and plotted against buffer pH. The resulting absorbance profiles for LysoSensor, Oregon Green and fluorescein could be fit to a sigmoidal curve, but ApHID absorbance increased monotonically with increasing acidity (FIG. 2A). The fluorescence emission profiles for all probes, including ApHID, fit a sigmoidal curve(FIG. 2B). ApHID fluorescence at pH 4.0 is almost 12 times greater relative to pH 6.0, while LysoSensor is 7 times brighter. Fluorescein and Oregon Green fluorescence decreases with acidity, being 3 and 6 times brighter at pH 6.0 relative to pH 4.0, respectively (FIG. 2B). By this analysis, ApHID shows the greatest dynamic range at the pH range of LE / Lys.

[0096] Dextrans derivatized with fluorophores can be used to label and track LE / Lys in different cell types. Depending on the cell type, the probes may be subjected to reactive oxygen species generated intracellularly, which could cause chemical modifications that affect spectroscopic properties. The most reactive ROS species is the hydroxyl radical (*OH). To test the effects of OH on ApHID, fluorescein, and Oregon Green fluorescence, hydrolyzed NHS ester forms of the probes were added to PBS solutions containing 100 or 200 pM OH and incubated for 24h at 37°C. The OH radicals were generated by the Fenton Reaction by mixing ferrous perchlorate, Fe(ll), with H2O2 in solution. As a control, we incubated the probes with ferrous perchlorate in the absence of H2O2. The intracellular concentration of H2O2 in various cell types, under physiological conditions, is within the range of 1 -10 pM. However, ROS concentrations in some cancer microenvironments can be as high as 100 pM, depending on the activation status and metabolic state of the cells. We conducted our assay with conditions that would ensure «OH concentrations above these reported levels. Following reaction, the mixtures were diluted in buffers with pH ranging between 4 and 7.5, and fluorescence was measured and plotted against buffer pH (FIG. 2C). Only small variations in pKa were observed, indicating that the probes were resistant to oxidation by *OH for at least 24h (Table 1 ).

[0097] Fluorophores reaching acidic compartments might also be sensitive to the high protein 2 and salt concentration present in the organelles. To test for this possibility, 10 KDa dextrans labeled with the probes were incubated in buffers containing 50 mg / mL BSA, 1 mM MgCL and CaCL, or sodium acetate in the absence of sodium chloride for 24h at 37SC, and fluorescence was quantified and plotted against buffer pH thereafter. The probes were generally stable in the presence of salts, but their fluorescence decreased in the presence of BSA (FIGS. 2D-2F). pKa values calculated from fit sigmoidal curves are summarized in Table 1 . The pKa of the probes did not register large variations over the various conditions tested, but it was generally lower when attached to dextrans, relative to its unattached molecular form, which has been reported in the literature for fluorescein.

[0098] ApHID is resistant to photobleaching.

[0099] Live cell and intravital fluorescence imaging techniques conducted for extended periods of time benefit from probes that can withstand prolonged, intense photoexcitation while maintaining their fluorescence properties. We determined ApHID resistance to photobleaching, induced by a 488 nm laser, over time and compared it to that of fluorescein and Oregon Green. J774 macrophages were loaded with 70 KDa dextrans derivatized with either probe at 2:1 molar ratio followed by a 4-h chase to ensure localization in LE / LY. Cells were then fixed in PFA and incubated in 50 mM TRIS maleate pH 5.0 buffer (for ApHID and Oregon Green) or 1 X PBS pH 7.4 buffer (for fluorescein) containing methylamine hydrochloride, sodium acetate, nigericin and monensin for 5 mins at 37SC to ensure buffer equilibration across membranes. Once equilibrated, cells were imaged in a confocal microscope while irradiated with a 488 nm laser for 0.5 seconds per cycle (50 cycles in total) with 1 -second intervals between irradiation pulses, and images were acquired after each cycle. The laser was adjusted to yield 5.2 pW power at the front element of the 40X objective. Fluorescence intensity for each dye and cycle (F) was normalized to fluorescence on cycle 1 (Fo) and plotted as F / Fo against irradiation time (FIG. 3G). At the end of the photobleaching experiment, ApHID fluorescence intensity in LE / Lys (FGS. 3B, 3E) had decreased by 12%, whereas that of fluorescein (FIGS. 3B, 3E) and Oregon Green (FIGS. 3C, 3F) had decreased by 83% and 82%, respectively (FIG. 3G). These results indicate that ApHID is highly resistant to photobleaching.

[0100] ApHID fluorescence and pKa remain stable with different degrees of amino-dextran derivatization and net charge.

[0101] Amino-dextrans can be derivatized with a variety of pH-sensitive and pH-independent dyes (FIG. 4A, schematic representation, green and blue spheres, respectively). Increasing dextran derivatization should increase overall brightness, which would be beneficial for applications with taxing light scattering or when extended imaging times and low laser powers are required. To test for the effect of dextran derivatization on ApHID fluorescence and pKa, we labeled 70 KDa polymers with different amounts of ApHID and a constant amount of Alexa 405 or Cy5*3xSO3 dyes (pH-independent) and measured fluorescence against buffer pH in solution. Increasing derivatization with ApHID increases ApHID / Alexa405 and ApHID / Cy5«3xSO3 ratios proportionally to their molar ratios (FIGS. 4B, 4D). However, when normalized to pH 5.0 ratio, the resulting sigmoidal curves are not significantly different from each other (FIGS. 4C, 4E). This indicates that ApHID fluorescence and pKa remain stable at a range of probe concentrations on the dextran.

[0102] We also tested the effect of dextran net charge on ApHID pKa. To do so, we derivatized dextrans with a constant amount of ApHID and various amounts of Alexa 405, which carries three negatively charged sulfate groups (FIG. 4F). Increasing dextran negative charge with Alexa 405 increased ApHID pKa, but only at high negative charge density (FIG 4G). These results indicate that ApHID fluorescence and pKa remain stable in a wide range of dextran derivatizations.

[0103] Lysosomal pH measured in J774 macrophages using ApHID coincides with measurements done using fluorescein and Oregon Green.

[0104] Many cell types endocytose dextrans and deliver them to their endosomal system. We wanted to test whether ApHID, once incorporated in LE / Lys, is able to report lysosomal pH as reliably as fluorescein, a well- characterized probe that has been extensively used to measure pH in multiple cell types. Oregon Green was also tested for comparative purposes.

[0105] J774 macrophages were loaded overnight with 70 KDa dextrans labeled with either probe as well as Alexa 647 (pH-independent), followed by a 3h chase in fresh DMEM. After fixation, cells incubated in pH 4.5 buffer showed strong ApHID fluorescence, which decreased as the cells were equilibrated with increasing buffer alkalinity (FIGS. 5A-D). To quantify pH-sensitive / Alexa 647 ratios in lysosomes, an intensity threshold was applied to the pH-independent channel (Alexa 647). A mask was then generated and transferred to the pH-dependent channel. Integrated intensity was measured for each masked channel, and pH ratios were calculated for each LE / Ly or field imaged. ApHID / Alexa 647 ratios measured in fixed cells were plotted against buffer pH, and the resulting titration was fit to a 4-component sigmoidal curve and compared with a titration of the same dextran, measured in solution (FIG 5E). The sigmoidal curve corresponding to the titration in lysosomes was practically identical to that in solution, indicating that ApHID and Alexa 647 are stable in lysosomes for extended times.

[0106] Fluorescein signal at pH 5.0 (FIG. 5I) or in living cells (FIG. 5J) was substantially weaker relative to that of ApHID (Figs. 5F-5G), whereas that of Oregon Green (FIGS. 5L-5M) was comparable. When 20 mM methylamine was added to the media, ApHID signal decreased dramatically (FIG. 5H), but fluorescein and Oregon Green became substantially brighter (FIGS. 5K, 5N). This is consistent with the pH-dependent fluorescence properties reported for these probes, which reflect the acidic nature of LE / Lys. Most importantly, these results indicate that, once in the lysosome, ApHID continues to be sensitive to changes in pH.

[0107] Next, we compared LE / Ly pH reported by each dye in living cells. pH-sensitive / Alexa 647 ratios were calculated and interpolated to pH for each imaged lysosome (FIG. 50) or well (FIG. 5P, six wells imaged throughout three independent experiments) using a ratio-to-pH calibration curve for each probe. To prepare these curves, fluorescence ratios were determined for cells incubated in pH 5.0 buffer and thereafter used to generate all subsequent ratios (pH 3.5 and 5.0-7.4), using titration data in solution from the same dextrans. This approach is appropriate because the pH-dependent dynamic range of dextrans measured in cell culture is identical to that measured in solution (FIGS. 5A-5E). It is important to note that, in fixed cells, 50 mM TRIS maleate pH 5.0 buffer equilibrated well across cell membranes at 37aC without causing significant membrane or cell swelling. However, membrane permeation was required for efficient buffer equilibration. Titration data obtained in solution was used to prepare the calibration curves for cell culture, and the data were fit to 4-component sigmoidal curves. The averaged interpolated lysosomal pH reported by ApHID after 1 h incubation in DMEM at 37SC was within 0.1 pH units of that reported by fluorescein and Oregon Green, and differences in pH between conditions were not statistically significant (Table 2). Lysosomal pH was stable for the 1 h time period during which the experiments were performed, irrespective of the pH-sensitive probe used.DISCUSSION

[0108] We report the preparation and characterization of the novel pH- sensitive probe ApHID. The chemical design of ApHID confers the molecule with robust photostability and optimal spectroscopic properties to measure pH in the acidic range of endosomes and lysosomes. ApHID contains a PEG4 chain terminated in an A / -hydroxysuccinimidyl ester reactive group, which increases solubility and allows for the derivatization of proteins and other molecules containing primary amines. In addition, ApHID is a weak base, whereas fluorescein, Oregon Green, and many other pH-sensitive probes are carboxylic acids, which is a novel feature. ApHID’s fluorescence increases with increasing acidity, whereas fluorescein and Oregon Green become dimmer. ApHID’s dynamic range between pH 4.0 and pH 6.0 was almost twice that of LysoSensor yellow / blue and Oregon Green, and four times greater than that of fluorescein. This allows for more accurate pH measurements in the lysosome. Importantly, the pKa of ApHID and its pH-dependent fluorescence remain stable when exposed to high amounts of salt and protein as well as to «OH radicals at concentrations substantially higher than those found in endosomes and lysosomes. Once endocytosed by J774 macrophages, ApHID fluorescence remains stable for an extended time.Altogether, the probe is robust to chemical modification, and it is much more photostable than the other pH indicator dyes we tested.

[0109] It is possible to derivatize amino-dextrans with various degrees of ApHID along with pH-independent fluorophores without altering ApHID’s fluorescence properties. This is advantageous when working with difficult imaging applications such as intravital imaging, which benefits from bright markers that can withstand sustained excitation. Interestingly, in derivatized dextrans, the pKa of ApHID can be modulated by altering the ionic environment on the polymers. We found that increasing the dextran’s negative charge by derivatizing it with anionic molecules such as Alexa 405 increased ApHID’s pKa. This is most likely due to the stabilizing effect played by the nearby negative charge, which favors protonation of the aniline group in the ApHID core. By the same principle, adding a positive charge to the polymer backbone should disfavor aniline protonation, thus requiring higher acidity to activate ApHID fluorescence, and ultimately lowering ApHID’s pKa. This demonstrates that ApHID pH sensitivity can be finely tuned by modulating amino-dextran net charge, which might be advantageous when pH sensing of different acidity ranges is required.We demonstrate that ApHID can be used to measure the pH of acidic compartments in cultured cells. We validated our measurements by multiple comparisons with other pH-sensitive probes, fluorescein and Oregon Green, which are commonly used to track lysosomes and measure their pH. For ratiometric pH imaging, we derivatized dextrans with the various pH-sensitive probes and a second, pH-independent dye (Alexa 647) and loaded J774 macrophages with the polymers. Once endocytosed, dextrans reach late endosomes and lysosomes, allowing for ratiometric imaging of the organelles. As expected, once in acidic compartments, all probes tested were sensitive to changes in acidity induced by the addition of 20 mM methylamine to the DMEM medium, which alkalinized lysosomes. pH-sensitive / Alexa 647 ratios measured per field or for individual lysosomes were interpolated to pH values using ratio-to-pH calibration curves. The accuracy of the ratio-to-pH calibration will determine the precision of the pH measurements. We have developed and tested a procedure for J774.A1 macrophages, but the calibration procedure should be tested for each cell type and experimental condition.

[0110] The pH distribution of individual lysosomes reported by ApHID was tighter than that of Oregon Green, and similar to that of fluorescein. Differences between lysosomal pH measured with ApHID, fluorescein, and Oregon Green were minimal and not statistically significant. The pH reported by ApHID stayedwithin 0.1 pH units of that reported by the other dyes, indicating that they are effectively equivalent.CONCLUSIONS

[0111] We have developed a novel pH-sensitive dye, ApHID. We believe that ApHID can be useful in demanding fluorescence imaging applications such as intravital imaging. Its spectroscopic properties, pH-dependence between pH 4.0- 6.0, as well as its resistance to oxidation and photobleaching make it optimal for measuring LE / Ly pH in a variety of cell types. We believe ApHID properties circumvent a number of limitations presented by most commercially available pH- sensitive probes such as fluorescein or Oregon Green, and we are confident thatApHID will prove useful in demanding imaging applications such as intravital imaging of tissues.Table 2Descriptive StatisticsFluorescein +ApHID ApHID + MeNH2Fluorescein MeNH2OG OG + MeNH2Average pH ±SEM 4.99 ± 0.03 6.42 ± 0.01 5.06 ± 0.01 6.28 ± 0.01 4.98 ± 0.01 6.56 ± 0.13One-way ANOVA (Dunnett's multiple comparison test)Comparison Mean 1 Mean 2 Mean Diff. p value SignificanceApHID vs. Fluorescein 4.99 5.06 -0.07 0.3 nsApHID vs. OG 4.99 4.98 0.02 0.97 nsApHID vs ApHID +MeNH24.99 6.42 -1.43 <0.0001Fluorescein vsFluorescein + MeNH25.06 6.28 -1.21 <0.0001OG vs. OG + MeNH24.98 6.56 -1.6 <0.001

[0112] Table 2 shows Lysosomal pH ± SEM reported by ratiometric pH imaging using ApHID, fluorescein and Oregon Green dextrans, also labeled with Alexa 647 (pH-independent) with statistical comparisons. Average pH values were calculated using a total of six wells imaged in three independent experiments. Two wells were imaged for each condition, and four fields were imaged per dish.Differences in averaged pH between conditions were assessed using One-way ANOVA with Dunnett’s multiple comparison test using 95% confidence interval. P- values shown as p>0.05 (ns), p<0.05 (*), p<0.01 (**), p<0.001 (***), and p<0.0001 (****). Abbreviations: ‘OG’: Oregon Green.

Claims

CLAIMSWhat is claimed is:

1. A compound of the formula:or an enantiomer, stereoisomer, or polymorph thereof, wherein: each R1, R3, and R4is optional substituted alkyl;R2is optionally substituted alkyl;R5and R6are each independently OR7, O-N(R8)2 or N(R8)2, wherein R7is independently H or optionally substituted alkyl and each R8is optionally substituted alkyl or each R8, together with the atom to which they are attached, form a heterocycle; and each X is independently a halogen.

2. The compound of claim 1 , wherein the compound is a compound of the formula:enantiomer, stereoisomer, or polymorph thereof.

3. The compound of claim 1 or 2, wherein each X is independently a fluorine or a chlorine, such as wherein both X are fluorine.

4. The compound of claim 1 , wherein: at least one of R1, R3, and R4is optional substituted (Ci-C4)alkyl, such as methyl.

5. The compound of claim 1 , wherein:R5and R6are each independently OR7or N(R8)2.

6. The compound of claim 5, wherein R5and R6are each NHR7, wherein R7is optionally substituted alkyl.

7. The compound of claim 5, wherein R5is OH and R6is NHR7, wherein R7is optionally substituted alkyl, such as ethyl.

8. The compound of claim 1 , wherein R5is O-N(R8)2, wherein each R8is optionally substituted alkyl or each R8, together with the atom to which they are attached, form a heterocycle.

9. The compound of claim 8, wherein R6is NHR7, wherein R7is optionally substituted alkyl, such as ethyl.

10. The compound of claim 8 or 9, wherein R5is a five-membered heterocycle.11 . The compound of claim 10, wherein the heterocycle is a heterocycle of the formula:wherein the dashed line can represent a double bond.

12. The compound of claim 5, wherein R5is NHR7, wherein R7is -(alkyl-O)n-R9or alkyl substituted with a group of the formula -(a|ky!-O)n-R9, wherein n is an integer from 1 -10, such as 3-8 or 4-6 and R9is H, alkyl, -alkyl-C(O)OR7or -alkyl-C(O)O-N(R8)2; and R6is N(R8)2.

13. The compound of claim 12, wherein -(alkyl-O)n- is -(CH2CH2-O)n-.

14. The compound of claim 12, wherein R7is -(alkyl-O)n-R9, wherein R9is -alkyl-C(O)O- (NR8)2, each R8, together with the atom to which they are attached, form a heterocycle.

15. The compound of claim 14, wherein the heterocycle is a heterocycle of the formula:wherein the dashed line can represent a double bond.

16. The compound of claim 1 , wherein the compound is a compound of the formula:

17. A protein or antibody conjugate of a compound of claim 1 or 16.

18. A method of imaging using the conjugate of claim 17.

19. A method for measuring changes in pH over a range of 4.2-7 using a water-soluble BODIPY-based fluorescent dye ora conjugate thereof, the method comprising (i) localizing the water-soluble BODIPY-based fluorescent dye or a conjugate thereof intracellularly in one or more intracellular compartments / organelles; (ii) irradiating the water-soluble BODIPY-based fluorescent dye or a conjugate thereof with a wavelength of light absorbed by the water-soluble BODIPY-based fluorescent dye or a conjugate thereof; measuring the emission of the water-soluble BODIPY-based fluorescent dye or a conjugate thereof; and determining the pH based on fluorescence ratios corresponding to the pH.

20. The method of claim 19, wherein the measured pH is that of an intracellular organelle.

21. The method of claim 20, wherein the intracellular organelle is a lysosome or an endosome.

22. The method of claim 19, wherein the water-soluble BODIPY-based florescent dye is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:wherein:R1a, R2a, R3a, R1b, R2b, R3b, are independently selected from a hydrogen atom, a straight- or branched-chain hydrocarbon group having one to twenty carbon atoms, wherein said straight- or branched-chain hydrocarbon group is optionally substituted with one or more hydrophilic moieties, and wherein at least one of R2a, and R2b is a hydrocarbon group substituted with a hydrophilic moiety;A1and A2are independently selected from a hydrogen atom, a straight- or branched- chain hydrocarbon group having one to six carbon atoms; andY1and Y2are independently selected from a hydrogen atom, an aromatic ring, a heteroaromatic ring, a carbocyclic ring, a heterocyclic ring, or a straight- or branched-chain hydrocarbon group having one to six carbon atoms, and wherein said straight- or branched-chain hydrocarbon group may optionally be taken together to form a heterocyclic or heteroaromatic ring.

23. The method of claim 19, wherein the water-soluble BODIPY-based florescent dye is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:m is 0, or an integer of 1 to 6;G1and G2are independently selected from an oxygen atom or amino group;Q1and Q2are independently selected from an oxygen atom or two hydrogen atoms; andZ1and Z2, are independently selected from a hydrogen atom, a straight- or branched-chain hydrocarbon group having one to twenty carbon atoms, wherein said straight- or branched- chain hydrocarbon group is optionally substituted with one or more hydrophilic moieties, wherein at least one of Z1, and Z2is a hydrocarbon group substituted with a hydrophilic moiety; and wherein at least one hydrophilic moiety terminates in a reactive chemical moiety capable of forming amide bonds.

24. The method of claim 19, wherein the water-soluble BODIPY-based florescent dye is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:wherein n is an integer of 1 to 6; andR4ais selected from one of the following: a reactive chemical moiety that enables amide bond formation or a cell-targeting agent attached via an amide bond.

25. The method of claim 24, wherein the reactive chemical moiety that enables amide bond formation is selected among the following:

26. The method of claim 24, wherein the water-soluble BODIPY-based florescent dye is the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:

27. The method of claim 24, wherein the cell-targeting agent is selected from one of the following: a polysaccharide attached via an amide bond, an oligosaccharide attached via an amide bond, or a cyclic oligosaccharide attached via an amide bond.

28. The method of claim 27, wherein said oligosaccharide is a dextran or aminodextran.

29. The method of claim 27, wherein the oligosaccharide has a molecular weight of at least 40,000.

30. The method of claim 19, wherein the water-soluble BODIPY-based florescent dye is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:wherein n is an integer of 1 to 631. The method of claim 19, wherein the water-soluble BODIPY-based florescent dye is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:wherein n is an integer of 1 to 632. The method of claim 19, wherein the composition further comprises a second fluorophore.

33. The method of claim 32, wherein the fluorescence emission of the second fluorophore remains pH independent over the range of pH 4-9.

34. The method of claim 32, wherein said composition is comprised of the following structure, or salt, enantiomer, stereoisomer, or polymorph thereof:wherein n is 0, or an integer of 1 to 6; m is 0, or an integer of 1 to 6; a is an integer of 1 to 6; b is an integer of 1 to 6; c is 0 or 1 ;X and Y are independently a reactive chemical moiety that enables amide bond formation, a cell-targeting agent attached via an amide bond, a straight- or branched-chain hydrocarbon group having one to six carbon atoms attached via and amide bond, or a straight- or branched-chain hydrocarbon group having one to six carbon atoms attached via and amide bond terminating in the second fluorophore;Z1, Z2, and Z3are each independently from a hydrogen atom, an aromatic ring, a heteroaromatic ring, a carbocyclic ring, a heterocyclic ring, a straight- or branched-chain hydrocarbon group having one to six carbon atoms, or a straight- or branched-chain hydrocarbon group terminating in the second fluorophore; and wherein at least one of X, Y, and Z1to Z3contain the second fluorophore moiety.

35. The method of claim 32, wherein the second fluorophore moiety is selected from Alexa405 and rhodamine.

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