AU2006336403A1 - Stilbazium research assays - Google Patents

Description

WO 2007/086900 PCT/US2006/012942 STILBAZIUM RESEARCH ASSAYS RELATED APPLICATION DATA This application claims the benefit of United States Provisional Patent Application Serial No. 60/669,615 filed on April 8, 2005, United States Provisional Patent Application Serial No. 60/734,518 filed on November 8, 2005 and United States Provisional Patent Application Serial No. 60/773,366 filed on February 13, 2006. The disclosures of which are incorporated herein by reference in their entireties. FIELD OF THE INVENTION The present invention relates to compositions, methods, and kits for use in assays. More particularly, the present invention relates to the use of stilbazium compounds and analogs thereof as fluorescents, stains or tags for use in assays. BACKGROUND OF THE INVENTION One of the most frequently used molecular biological techniques for detecting homologous nucleic acid sequences is nucleic acid hybridization, i.e. DNA/DNA, RNA/RNA or RNA/DNA hybridization. In this technique, nucleic acid (DNA or RNA) used as a probe is labeled, and the labeled nucleic acid is hybridized to a nucleic acid (DNA or RNA) to be detected. When the nucleic acid used as a probe has homology to the nucleic acid to be detected, each single-stranded nucleic acid hybridizes to its complementary sequence so as to form a double-stranded sequence, and then the double-stranded sequence is detected by a labeled probe. The need to identify, mark, isolate, modify or otherwise determine analytes, microorganisms, amino acids or nucleic acid sequences (for example multiple pathogens or multiple genes or multiple genetic variants) alone, in blood or in other biological fluids has become increasingly apparent in many branches of medicine. Most multi-analyte assays, such as assays that detect multiple nucleic acid sequences, involve multiple steps, have poor sensitivity, a limited dynamic range (typically on the order of 2 to 100-fold differences) and/or often utilize sophisticated instrumentation. As the human genome is elucidated, there are numerous opportunities for performing assays to determine the presence of specific sequences, distinguishing between alleles in 1 WO 2007/086900 PCT/US2006/012942 homozygotes and heterozygotes, determining the presence of mutations, evaluating cellular expression patterns, etc. In many of these cases, one will wish to determine a number of different characteristics of the same sample in a single reaction. In many assays, there is an interest in determining the presence of specific sequences, whether genomic, synthetic, or cDNA. These sequences may be associated particularly with genes, regulatory sequences, repeats, multimeric regions, expression patterns, and the like. There is also an interest in determining the presence of one or more pathogens. The need to identify and quantify a large number of bases or sequences, potentially distributed over centimorgans of DNA, offers a major challenge. Ideally, the method is accurate, reasonably economical in limiting the amount of reagents required and/or provides for a highly multiplexed assay, which allows for differentiation and quantification of multiple genes, and/or single nucleotide polymorphisms (SNP) determination, and/or gene expression at the RNA or protein level. Radioactivity has been the dominant read-out in early drug discovery assays. However, the need for more information, higher throughput and miniaturization has caused a shift towards fluorescence detection. Fluorescence-based reagents can yield more powerful, multiple parameter assays that are higher in throughput and information content and require lower volumes of reagents and test compounds. Fluorescence is also safer and less expensive than radioactivity-based methods. Another technique for detecting biological compounds is fluorescence in-situ hybridization (FISH). Swiger et al. (1996) Environ. Mol. Mutagen. 27:245-254; Raap (1998) Mut. Res. 400:287-298; Nath et al. (1997) Biotechnic. Histol. 73:6-22. FISH allows detection of a predetermined target oligonucleotide, e.g., DNA or RNA, within a cellular or tissue preparation by, for example, microscopic visualization. Thus, FISH is an important tool in the fields of, for example, molecular cytogenetics, pathology and immunology in both clinical and research laboratories. FISH involves the fluorescent tagging of an oligonucleotide probe to detect a specific complementary DNA or RNA sequence. Specifically, FISH involves incubating an oligonucleotide probe comprising an oligonucleotide that is complementary to at least a portion of the target oligonucleotide with a cellular or tissue preparation containing or suspected of containing the target oligonucleotide. A detectable label, e.g., a fluorescent dye molecule, is bound to the oligonucleotide probe. A fluorescence signal generated at the site of hybridization is typically visualized using an epi fluorescence microscope. An alternative approach is to use an oligonucleotide probe conjugated with an antigen such as biotin or digoxygenin and a fluorescently tagged antibody directed toward that antigen to visualize the hybridization of the probe to its DNA target. A variety of FISH formats are 2 WO 2007/086900 PCT/US2006/012942 known in the art. See, e.g., Dewald et al. (1993) Bone Marrow Transplantation 12:149-154; Ward et al. (1993) Am. J. Hum. Genet. 52:854-865; Jalal et al. (1998) Mayo Clin. Proc. 73:132-137; Zahed et al. (1992) Prenat. Diagn. 12:483-493; Kitadai et al. (1995) Clin. Cancer Res. 1:1095-1102; Neuhaus et al. (1999) Human Pathol. 30:81-86; Hack et al., eds., (1980) Association of Cytogenetic Technologists Cytogenetics Laboratory Manual. (Association of Cytogenetic Technologists, San Francisco, Calif.); Buno et al. (1998) Blood 92:2315-2321; Patterson et al. (1993) Science 260:976-979; Patterson et al. (1998) Cytometry 31:265-274; Borzi et al. (1996) J. Immunol. Meth. 193:167-176; Wachtel et al. (1998) Prenat. Diagn. 18:455-463; Bianchi (1998) J. Perinat. Med. 26:175-185; and Munne (1998) Mol. Hum. Reprod. 4:863-870. Thus, an assay for the differentiation and/or quantification of single or multiple genes, and/or SNP determination and/or gene expression at the RNA or protein level, that has higher sensitivity, a large dynamic range, better fluorescence, a greater degree of multiplexing and/or fewer and more stable reagents would increase the simplicity and/or reliability of multianalyte assays, and may reduce their costs. There is also a continuing need in the assay art for labels with the following features: (i) high fluorescent intensity (for detection in small quantities), (ii) adequate separation between the absorption and emission frequencies, (iii) desirable solubility, (iv) ability to be readily linked to other molecules, (v) stability towards harsh conditions and high temperatures, (vi) a symmetric, nearly Gaussian emission lineshape for deconvolution of multiple colors, and/or (vii) compatibility with automated analysis. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A through 1L present confocal images of murine mammary carcinoma 4T1 cells incubated with a compound according to some embodiments of the present invention (Figures 1B, 1F and 1J) and co-stained with Hoechst (Figures 1A, 1E and 11, blue) to stain the nuclei and MitoTracker Deep Red (Figures IC, 1G and 1K, appears green in images) to stain the mitochondria. Figure 1D presents an overlaid image of the images presented in Figures 1A through l C. Figure 1H presents an overlaid image of the images presented in Figures 1E through 1G. Figure 1L presents an overlaid image of the images presented in Figures 11 through 1K. 3 WO 2007/086900 PCT/US2006/012942 SUMMARY OF THE INVENTION Embodiments of the invention include assays for determining the presence or absence of a cell, an analyte, a nucleic acid or a microorganism in a sample suspected of containing a cell, an analyte, a nucleic acid or a microorganism, said assay comprising combining the sample with a labeling reagent to form a labeled cell, nucleic acid or microorganism, said labeling reagent comprising a dye which directly stains the cell, analyte, nucleic acid or microorganism to provide a stained sample comprising a stained cell, analyte, nucleic acid or microorganism, wherein said dye is a compound represented by Formula I R1 N+ R N--- --- NI--N R2 RR4I wherein for Formula I, the NRI 1

R

2 and NR 3

R

4 moieties are in the ortho, meta or para positions; wherein X- is an anionic salt; wherein R 1 , R 2 , R 3 , or R 4 are independently selected from the group consisting of Cl-o 10 alkyl (linear or branched), alkenes (linear or branched), or wherein R 1 and R 2 or R 3 and R4 taken together with the nitrogen atom to which they are attached form pyrrolidino or piperidino rings; wherein R 5 is a polyalkylene glycol moiety, a

C

1

-

1 0 alkyl (linear or branched), an alkene (linear or branched), an alkyne, a substituted and unsubstituted aryl, a substituted and unsubstituted benzyl and/or an organometallic moiety.

R

5 may also be an organometallic compound such as organotin, organosilicon, or organogermanium. Additionally, R 5 may be (CH 2 )n-MR 6 , wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R 6 is a selected from the group consisting of propyl, butyl, or any alkyl compound; contacting the stained sample; and observing the accumulation of the stained cell, analyte, nucleic acid or microorganism. The assays can include cellular, chemical and biological assays. The dye can be loaded into a lipid vesicle or suitable biomaterial. In particular, the dye can be loaded into a microcapsule, liposome, micelle and/or bicelle to form a microencapsulated formulation, a liposomal formulation, a micelle formulation and/or a bicelle formulation, respectively. Further, the dye can be pegylated. 4 WO 2007/086900 PCT/US2006/012942 Embodiments of the present invention can include a probe comprising a ligand or antibody and a compound of Formula I. The probe can be used to detect cells, analytes, nucleic acids and microorganisms. Embodiments of the present invention further include methods of selecting an analyte that binds to a compound of Formula I. Embodiments of the present invention include determining the presence or absence of one or more target compounds in a sample, wherein the compounds are represented by Formula I or a fluorescent molecule and the steps include providing a plurality of electrophoretic probes specific for the one or more target compounds, each electrophoretic probe having a target-binding moiety; combining with the sample the plurality of electrophoretic probes such that in the presence of a target compound a complex is formed between each target compound and one or more electrophoretic probes specific therefor; and separating and identifying the compounds to determine the presence or absence of the one or more target compounds. Embodiments of the present invention further include kits for staining cells, analytes, nucleic acids and/or microorganisms. DETAILED DESCRIPTION The foregoing and other aspects of the present invention will now be described in more detail with respect to other embodiments described herein. It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the embodiments of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Also, as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items. As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y." As used herein, phrases such as "from about X to Y" 5 WO 2007/086900 PCT/US2006/012942 mean "from about X to about Y." Unless otherwise defined, all terms, including technical and scientific terms used in the description, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented. "Alkyl" as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2 dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. "Loweralkyl" as used herein, is a subset of alkyl, and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso butyl, tert-butyl, and the like. Alkyl and loweralkyl groups may be unsubstituted or substituted one or more times with halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, alkyl-S(O)m, haloalkyl-S(O)m, alkenyl-S(O)m, alkynyl S(O)m, cycloalkyl-S(O)m, cycloalkylalkyl-S(O)m, aryl-S(O)m, arylalkyl-S(O)m, heterocyclo S(O)m, heterocycloalkyl-S(O)m, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano where m=0,1 or 2. "Alkoxy," as used herein alone or as part of another group, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxy group. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like. "Acyl" or "Alkanoyl" as used herein alone or as part of another group, refers to a C(O)R radical, where R is any suitable substituent such as alkyl, alkenyl, alkynyl, aryl, alkylaryl, etc. "Cell" as used herein refers to a basic component of a living or fixed organism and includes organelles. Thus, detecting the presence of a cell, assaying a cell, staining a cell, etc. 6 WO 2007/086900 PCT/US2006/012942 can refer to a whole cell or at least one organelle of the cell. According to embodiments of the present invention, cells may be plant or animal cells. As recognized by one skilled in the art, "organelles" as used herein refer to cellular components or structures suspended in the cytoplasm including those providing a boundary therefor and having specialized functions. Organelles include, but are not limited to, the nucleus, smooth and/or rough endoplasmic reticulum, centrosome, cytoskeleton, cell wall, cell membrane, flagella, cilia, chloroplast, mitochondria, golgi apparatus, ribosome, lysosome, centriole, acrosome, glyoxysome, secretory vesicle, peroxisome, vacuole, melanosome, myofibril and parenthesome. "Dye" as used herein refers to a substance that imparts color and/or fluorescence and/or is quantifiable or distinguishable. The color and/or fluorescence can be temporary, semi-permanent or permanent. "Analyte" as used herein refers to the substance or chemical constituent that undergoes analysis. For example, an analyte can be a molecule, protein, chemical substance, etc. that can be detected as a result of biological, chemical or clinical testing to evaluate the same. Analytes can include, but are not limited to, ions; metabolites such as glucose and urea; trace metabolites such as hormones, drugs, steroid hormones; gases such as respiratory gases, anesthetic gases, toxic gases and flammable gases; toxic vapors; proteins and nucleic acids; antigens and antibodies and microorganisms. "Nucleic acid" as used herein refers to an oligonucleotide, nucleotide, or polynucleotide, and to DNA or RNA or chimeras thereof, single stranded or double-stranded, and can be fully or partially synthetic or naturally occurring. Nucleic acids can include modified nucleotides or nucleotide analogs. Further, the nucleic acids can be from any species of origin, including plant species or mammalian species such as human, non-human primate, mouse, rat, rabbit, cattle, goat, sheep, horse, pig, dog, cat, etc. In some embodiments, the nucleic acid is an isolated nucleic acid. As used herein, an "isolated" nucleic acid means a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism, for example, the cell structural components or other polypeptides or nucleic acids commonly found associated with the nucleic acid. "Lipid vesicle" as used herein refers to structures including amphiphiles, for example, surfactants or phospholipids, characterized by the presence of an internal void. The internal void can be filled with any appropriate material such as a liquid, aqueous solution, gas, gel, solid material or mixture thereof. Lipid vesicles include, but are not limited to, liposomes, 7 WO 2007/086900 PCT/US2006/012942 helices, discs, tubes, tori, hexagonal, phase structures, micelles, gel phases, reverse micelles, bicelles, microemulsions, emulsions and combinations thereof. "Microorganism" as used herein refers to microscopic organisms that can exist as a single cell or cell clusters. Embodiments of the present invention include a compound comprising Formula I: Ri N+ R3 R2 R5 \ R4 R 5 I or a solvate thereof, wherein X is an anionic salt, wherein R 1 , R 2 , R 3 , or R 4 are independently selected from the group consisting of methyl, ethyl, C1-10o alkyl (linear or branched), alkenes (linear or branched), or wherein when R 1 and R 2 or when R 3 and R 4 are taken together with the nitrogen atom to which they are attached, they form pyrrolidino or piperidino rings. X7 can be selected from the group including fluoride, chloride, bromide, iodide halide, mesylate, tosylate, napthylate, nosylate, para-aminobenzoate, benzenesulfonate, besylate, lauryl sulfate, 2,4-dihydroxy benzophenone, 2-(2-hydroxy-5'-methylphenyl) benzotriazole, ethyl 2-cyano 3,3-diphenyl acrylate and 5-butyl phenyl salicylate. R 5 is a polyalkylene glycol moiety, a C 1 . 10 alkyl (linear or branched), an alkene (linear or branched), an alkyne, a substituted and unsubstituted aryl, a substituted and unsubstituted benzyl and/or an organometallic moiety.

R

5 may also be an organometallic compound such as organotin, organosilicon, or organogermanium. Additionally, R 5 may be (CH 2 )n-MR 6 , wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R 6 is a selected from the group consisting of propyl, butyl, or any alkyl compound. The compound may be a stilbazium compound. In some embodiments of the present invention, the compound has the following structure: O' C"3 'No 8 WO 2007/086900 PCT/US2006/012942 In other embodiments of the present invention, the compound has the following structure: N N In still other embodiments of the present invention, the compound has the following structure: CC 'N N Stilbazium chloride as well as other stilbazium salts, analogs and homologs thereof may exist as bright red to dark red or other colored compounds that may possess UV-visible chromophores and may further exhibit characteristic strong fluorescence. Stilbazium chloride as well as other stilbazium salts, analogs and homologs exhibit classic staining properties and can bond to cloth, paper, wood plastic, glass, metal and other substrates, as well as skin and related living tissues, while retaining its red or pink or other coloration. Stilbazium chloride as well as other stilbazium salts, analogs and homologs may stain biopolymers such as single stranded or double stranded deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by covalently bonding to nucleophilic groups on the DNA and RNA chains. One mechanism of action may include a strong covalent bond being formed between the nucleophile (Nu:) and one of the stilbazium chloride side chains. The other side chain retains the chromophore and fluorophore as shown in the scheme below. 9 WO 2007/086900 PCT/US2006/012942 Scheme 1. Nu-: N N+ Stilbazium Chloride NCl H - Interm ediate Nu N No Partially covalently bonded Stilbazium Chloride The nucleophile may be a nucleic acid, a microorganism, an amino acid, a cell, or an analyte. Additionally, in particular embodiments, the nucleic acid, amino acid, cell, or analyte may be bound to a nucleophile such as OH, 0, NH 2 , and the like before the nucleophile is covalently bonded to stilbazium chloride, salts, analogs and homologs thereof. The compounds of the present invention are capable of existing as geometric isomers. All such isomers, individually and as mixtures, are included within the scope of the present invention for their industrial uses. The E,E isomer is one configuration of the invention, and both the cisoid and transoid 2,6-conformations of the E,E-configuration are possible. Additionally, the ortho conformation of the structure can be formed in addition to the para and meta structures illustrated above. The ortho conformation structure can include the same salts and moieties as disclosed above and throughout the application. The compounds of the present invention can be fast acting, staining the object of interest in less than 10 minutes, and in some embodiments, seconds. The compounds of the present invention may be visible in bright field and/or under fluorescence. Moreover, the 10 WO 2007/086900 PCT/US2006/012942 compounds of the present invention provide a non-toxic staining option for viable cells and can provide repeatable staining of living cells. Additionally, the compounds of the present invention can be used in multiple assays of the same cultures. Embodiments of the present invention can further provide detection of live cell mitotic division. Compounds of the present invention are safe to users and user-friendly. Additionally, compounds of the present invention may exhibit stability at room temperature for a period of time sufficient to allow appropriate assays to be performed. Embodiments of the present invention can also include compounds represented by Formula I that are encapsulated, formulated in lipid vesicles such as liposomes, micelles and bicelles and/or pegylated (PEG). As used herein, "encapsulated" refers to a formulation of a compound according to the present invention confined by a material or matter. The material or matter can be synthetic or of natural origin. Accordingly, a lipid vesicle provides an exemplary mechanism for encapsulation. Moreover, as understood by one skilled in the art, compounds of the present invention can further be encapsulated by application of a coating surrounding the compound. Such coatings can include biomaterials and further include materials discussed below in reference to microcapsules. The amount of the compound encapsulated or formulated in lipid vesicles and/or pegylated can be determined by one skilled in the art based upon the method for which the compound is employed. In general, the compound can be present in an amount in a range from about 1 weight percent to about 50 weight percent or more of the formulation. Such encapsulated, lipid vesicle formulations and pegylated formulations can be water soluble. 1. Microencapsulation According to some embodiments, compounds of the present invention can be encapsulated in microcapsules. As used herein, the term "microcapsules" is intended to contemplate single molecules, encapsulated discrete particulate, multiparticulate, liquid multicore and homogeneously dissolved active components. The encapsulation method may provide either a water soluble or oil soluble active component encapsulated in a shell matrix of either a water or oil soluble material. The microencapsulated active component may be protected from oxidation (e.g., UV) and hydration, and may be released by melting, rupturing, biodegrading, or dissolving the surrounded shell matrix or by slow diffusion of the active component through the matrix. Microcapsules usually fall in the size range of between about 1 and 2000 microns, although smaller and larger sizes are known in the art. 11 WO 2007/086900 PCT/US2006/012942 Compounds of the present invention may be placed in a microcapsule or hollow fiber type used for distribution. They may also be dispersed in a polymeric material or held as a liquid. The microcapsules can be made from a wide variety of materials, including polyethylene, polypropylenes, polyesters, polyvinyl chloride, tristarch acetates, polyethylene oxides, polypropylene oxides, polyvinylidene chloride or fluoride, polyvinyl alcohols, polyvinyl acetates, urethanes, polycarbonates, and polylactones. Further details on microencapusulation are to be found in U.S. Patent Nos. 5,589,194 and 5,433,953. Microcapsules suitable for use in the base materials of the present invention have diameters from about 1.0 to 2,000 microns. No particular limitation is imposed on the shape for holding the active ingredient. In other words, there are various forms for holding the active ingredient by a holding mixture. Specific examples include microcapsules in which the surface of the active ingredient has been covered with the holding mixture; and products processed into a desired shape, each being obtained by kneading the active ingredient in the holding mixture or forming a uniform solution of the holding mixture and the active ingredient, dispersing the active ingredient in the holding mixture by the removal of the solvent or the like and then processing the dispersion into a desired shape such as single molecule, molecular chain, liquid, sphere, sheet, film, rod, pipe, thread, tape or chip. In addition, these processed products having a surface covered with a barrier layer for controlling the release of the active ingredient and those coated with an adhesive for improving applicability can be given as examples. As further examples, those obtained by filling the active ingredient in the holding mixture processed into a form of a capillary tube, heat sealing both ends of the capillary tube and then encapsulating the active ingredient therein; and those obtained by centrally cutting the above mentioned capillary tube into two pieces, thereby having each one end as an opening. The container formed of a holding mixture which container has an active ingredient enclosed therein as a liquid phase to secure uniform release ability over a long period of time. As such shape, tube-, bottle- or bag-shaped container is used generally. When the mixture is formed into a container, the sustained release layer desirably has a thickness of at least about 0.002 mm for effecting stable sustained release. There occurs no particular problem when the sustained release layer has a thickness not smaller than about 0.002 mm, but that ranging from about 0.005 mm to 5 mm can be used. When it exceeds about 5 mm, the release amount of the compound tends to become too small. 12 WO 2007/086900 PCT/US2006/012942 For solids, the release surface area of the sustained release preparation formed of such a container is desirably .001 cm 2 or larger. A range of from .01 cm 2 to 1 cm 2 may be used. When the active ingredient is enclosed and held in a container of the sustained release preparation, said container having been formed of a holding mixture, it may be enclosed in portions. The enclosed amount can be about 0.5mg to 5 mg, and may be about 1mg, 2mg, 3mg, or 4mg. As the shape of the container formed of a holding mixture, a tube, bottle and bag can be used. In the case of the tube-shaped preparation, that having an internal diameter of about 0.4 mm to 10 mm can be used. Internal diameters smaller than about 0.4 mm make it difficult to fill the active ingredient in the container, while those larger than about 10 mm make it difficult to conduct encapsulation. The bottle-shaped preparation is formed by blow molding or injection molding and generally has an internal volume of about 0.1 to 200 ml. The bottle having an internal volume less than about 0.1 ml cannot be formed easily, while that having an internal volume greater than about 200 ml is not economical because there is a large difference between the amount of the active ingredient filled therein and the internal volume. In the case of a bag-shaped preparation, the amount of the active ingredient filled in the bag is desirably about 1 mg to 100 g. In some embodiments, the entire microcapsule composition can include of about 40 90 percent of liquid fill and about 10-40 percent of shell wall, the liquid fill comprising about 5-60 percent of compound, about 25-50 percent of biological synergist and 20-40 percent of a water-immiscible organic solvent and the shell including as an integral part thereof 0.5-20 percent of photostable ultraviolet light absorbent compound (all percentages being based on the weight of the entire microcapsule composition). The compound remains inside the microcapsules while the composition is packaged and in storage, i.e., in a closed container due to the partial pressure of the pyridinium salt surrounding the microcapsules. The compound is chemically stable during storage and after application until it permeates the capsule walls. Suitable fill stabilizers absorb ultraviolet radiation in the range of about 270-350 nanometers and convert it to a harmless form. They have a high absorption coefficient in the near ultraviolet portion of the spectrum (e.g. a log molar extinction coefficient of from about 2 to 5) but only minimal absorption in the visible portion of the spectrum. They do not exhibit any substantial chemical reaction with the isocyanate groups and primary amine groups of the shell forming compounds during the microencapsulation process. Among the compounds which can be used as fill stabilizers are substituted benzophenones such as 2,4-dihydroxy 13 WO 2007/086900 PCT/US2006/012942 benzophenone, 2-hydroxy-4-methoxy benzophenone, 2-hydroxy-4-octyloxy benzophenone, etc.; the benzotriazoles such as 2-(2-hydroxy-5'-methylphenyl) benzotriazole, 2-(3',5'-diallyl 2'-hydroxylphenyl)benzotriazole, etc.; substituted acrylates such as ethyl 2-cyano-3,3 diphenyl acrylate, 2-ethylhexyl-2-cyano-3,3-diphenyl acetate, etc.; salicylates such as phenyl salicylates, 5-butyl phenyl salicylate, etc.; and nickel organic compounds such as nickel bis (octylphenol) sulfide, etc. Additional examples of each of these classes of fill stabilizers may be found in Kirk-Othmer, Encyclopedia of Chemical Technology. The fill stabilizers may comprise up to 5 percent, and are generally from about 0.01 to 2 percent, by weight of the microcapsule composition. Another embodiment of the present invention may include heat sensitive materials that facilitate preservation stability, particularly in resistance to light, and microcapsules having an ultraviolet absorber enclosed therein, which are applicable to various fields. Desirable constituents that may be present in a base material include materials that can absorb heat and protect an underlying material from overheating. Thermal energy is absorbed by the phase change of such materials without causing an increase in the temperature of these materials. Suitable phase change materials include paraffinic hydrocarbons, that is, straight chain hydrocarbons represented by the formula CHn+ 2 , where n can range from 13 to 28. Other compounds that are suitable for phase change materials are a PABA salt, 2,2-dimethyl 1,3-propane diol (DMP), 2-hydroxymethyl-2-methyl-1,3-propane diol (HMP) and similar compounds. Also useful are the fatty esters such as methyl palmitate. Phase change materials that can be used include paraffinic hydrocarbons. Heat sensitive recording materials are well known which utilize a color forming reaction between a colorless or light-colored basic dye and an organic or inorganic color acceptor to obtain record images by thermally bringing the two chromogenic substances into contact with each other. Such heat sensitive recording materials are relatively inexpensive, are adapted for use with recording devices which are compact and easy to maintain, and have therefore found wide applications as recording media for facsimile systems, various computers, etc. In order to improve light resistance of heat sensitive recording materials a finely divided ultraviolet absorber or blocker can be added to the heat sensitive recording layer or protective layer. Further embodiments of the present invention provide microcapsules that can retain an ultraviolet absorber, exhibit resistance to being ruptured at a usual pressure and/or possess suitable ultraviolet ray absorbing efficiency. 14 WO 2007/086900 PCT/US2006/012942 Embodiments of the present invention can include a heat sensitive recording material comprising a substrate, a recording layer formed over the substrate and containing a colorless or light-colored basic dye and a color acceptor, and a protective layer formed over the recording layer, the recording material being characterized in that microcapsules having an ultraviolet absorber enclosed therein and having substantially no color forming ability are incorporated in the protective layer. Further, the present invention provides microcapsules having an ultraviolet absorber and as required an organic solvent enclosed therein, which have capsule wall film of synthetic resin and mean particle size of about 0.1 to 3gm. The following are examples of ultraviolet absorbers that may be used in the present invention. Phenyl salicylate, p-tert-butylphenyl salicylate, p-octylphenyl salicylate and like salicylic acid type ultraviolet absorbers; 2,4-dihydroxybenzophenone, 2-hydroxy-4 methoxybenzophenone, 2-hydroxy-4-octyloxybenzophenone, 2-hydroxy-4 dodecyloxybenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone, 2,2,'-dihydroxy-4,4' dimethoxybenzophenone, 2-hydroxy-4-methoxy-5-sulfobenzophenone and like benzophenone type ultraviolet absorbers; 2-ethylhexyl 2-cyano-3,3-diphenyt-acrylate, ethyl 2-cyano-3,3-diphenylacrylate and like cyanoacrylate type ultraviolet absorbers; bis(2,2,6,6 tetramethyl-4-piperidyl) sebacate, bis(2,2,6,6-tetramethyl-4-piperidyl) succinate, bis(1,2,2,6,6-pentamethyl-4-piperidyl) 2-(3,5-di-tert-butyl-4-hydroxybenzyl)-2-n-butyl malonate and like hindered amine type ultraviolet absorbers; 2-(2' hydroxyphenyl)benzotriazole, 2-(2'-hydroxy-5'-methylphenyl)benzotriazole, 2- (2'-hydroxy-5 -tert-butylphenyl)benzotriazole, 2- (2'-hydroxy-3',5'-di-tert-butylphenyl)benzotriazole, 2- (2' hydroxy-3'-tert-butyl-5'-methylphenyl)-5-chlorobenzotriazole, 2-(2'-hydroxy-3',5'-di-tert butylphenyl)-5-chlorobenzotriazole, 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)-5-tert butylbenzotriazole, 2-(2'-hydroxy-3',5'-di-tert-amylphenyl)benzotriazole, 2-(2'-hydroxy-3',5' di-tert-amylphenyl)-5-tert-amylbenzotriazole, 2-(2'-hydroxy-3',5'-di-tert-amylphenyl)-5 methoxybenzotriazole, 2-[2'-hydroxy-3'-(3",4",5",6"-tetrahydrophthalimido-methyl)-5' methylpheny l]benzotriazole, 2-(2'-hydroxy-5'-tert-octylphenyl)benzotriazole, 2-(2'-hydroxy 3'-sec-butyl-5'-tert-butylphenyl)benzotriazole, 2-(2'-hydroxy-3'-tert-amyl-5'-phenoxyphenyl) 5-methylbenzotriazole, 2-(2'-hydroxy-5'-n-dodecylphenyl)benzotriazole, 2-(2'-hydroxy-5' sec-octyloxyphenyl)-5-phenylbenzotriazole, 2-(2'-hydroxy-3'-tert-amyl-5'-phenylphenyl)-5 methoxybenzotriazole, 2-[2'-hydroxy-3',5'-bis(oa-dimethylbenzyl)phenyl]benzotriazole and like benzotriazole type ultraviolet absorbers which are solid at ordinary temperature; 2-(2' Hydroxy-3'-dodecyl-5'-methylphenyl)-benzotriazole, 2-(2'-hydroxy-3'-undecyl-5' 15 WO 2007/086900 PCT/US2006/012942 methylphenyl)-benzotriazole, 2-(2'-hydroxy-3'-tridecyl-5'-methylphenyl)-benzotriazole, 2-(2' hydroxy-3'-tetradecyl-5'-methylphenyl)-benzotriazole, 2-(2'-hydroxy-3'-pentadecyl-5' methylphenyl)-benzotriazole, 2-(2'-hydroxy-3'-hexadecyl-5'-methylphenyl)-benzotriazole, 2 [2'-hydroxy-4'-(2"-ethylhexyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(2" ethylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(2"-ethyloctyl)oxyphenyl] benzotriazole, 2-[2'-hydroxy-4'-(2"-propyloctyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4' (2"-propylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(2"-propylhexyl)oxyphenyl] benzotriazole, 2-[2'-hydroxy-4'-(1l"-ethylhexyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4' (1 "-ethylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(l "-ethyloctyl)oxyphenyl] benzotriazole, 2-[2'-hydroxy-4'-(1l"-propyloctyl)oxyphenyl] -benzotriazole, 2-[2'-hydroxy-4' (1 "-propylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy4'-(l "-propylhexyl)oxyphenyl] benzotriazole, 2-(2'-hydroxy-3'-sec-butyl-5'-tert-butylphenyl-5-n-butylbenzotriazole, 2-(2' hydroxy-3'-sec-butyl-5'-tert-butylphenyl) -5-tert-pentyl-benzotriazole, 2-(2'-hydroxy-3'-sec butyl-5'-tert-butylphenyl)-5-n-pentyl-benzotriazole, 2-(2'-hydroxy-3'-sec-butyl-5'-tert pentylphenyl)-5-tert-butylbenzotriazole , 2-(2'-hydroxy-3'-sec-butyl-5'-tert-pentylphenyl)-5 n-butylbenzotriazole, 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)-5-sec-butylbenzotriazole, 2-(2' hydroxy-3',5'-di-tert-pentylphenyl)-5-sec-butylbenzotriazole, 2-(2'-hydroxy-3'-tert-butyl-5' tert-pentylphenyl)-5-sec-butylbenzotriazole, 2-(2'-hydroxy-3',5'-di-sec-butylphenyl)-5 chlorobenzotriazole, 2-(2'-hydroxy-3',5'-di-sec-butylphenyl)-5-methoxybenzotriazole, 2-(2' hydroxy-3',5'-di-sec-butylphenyl)-5-tert-butylbenzotriazole, 2-(2'-hydroxy-3',5'-di-sec butylphenyl)-5-n-butylbenzotriazole, octyl 5-tert-butyl-3-(5-chloro-2H-benzotriazole-2-yl)-4 hydroxybenzene-propionate, condensate of methyl 3-[3-tert-butyl-5-(2H-benzotriazole-2-yl) 4-hydroxyphenyl]propionate and polyethylene glycol (molecular weight: about 300) and like benzotriazole type ultraviolet absorbers which are liquid at ordinary temperature. Of course, the ultraviolet absorber is not limited to thereabove and can be used as required in a mixture of at least two of them. Although the amount of ultraviolet absorber to be used is not limited specifically, the amount can be adjusted to 10 to 500 parts by weight, and generally from to 20 to 250 parts by weight. The microcapsules for use in the present invention can be prepared by various known methods. They are prepared generally by emulsifying and dispersing the core material (oily liquid) comprising an ultraviolet absorber and, if necessary, an organic solvent in an aqueous medium, and forming a wall film of high-molecular-weight substance around the resulting oily droplets. 16 WO 2007/086900 PCT/US2006/012942 Examples of useful high-molecular-weight substances for forming the wall film of microcapsules are polyurethane resin, polyurea resin, polyamide resin, polyester resin, polycarbonate resin, aminoaldehyde resin, melamine resin, polystyrene resin, styrene-acrylate copolymer resin, styrene-methacrylate copolymer resin, gelatin, polyvinyl alcohol, etc. Especially, microcapsules having a wall film of a synthetic resin, particularly polyurea resin, polyurethane resin and aminoaldehyde resin among other resins have excellent retainability of an ultraviolet absorber and high heat resistance and accordingly exhibit the outstanding additional effect to serve the function of a pigment which is to be incorporated in the protective layer for preventing sticking to the thermal head. Moreover, microcapsules having a wall film of polyurea resin or polyurethane resin are lower in refractive index than microcapsules with wall films of other materials and usual pigments, are spherical in shape and are therefore usable favorably because even if present in a large quantity in the protective layer, they are unlikely to reduce the density of record images (so-called whitening) owing to irregular reflection of light. Further, polyurea resin and polyurethane resin are more elastic than aminoaldehyde resin and therefore polyurea resin and polyurethane resin are generally used as a wall film for microcapsules that are used under a condition of high pressure. On the other hand, microcapsules having a wall film made from aminoaldehyde resin have a merit that the wall film can be controlled in thickness without depending on particle size of emulsion because the microcapsules can be prepared by adding a wall-forming material after emulsification of a core material. The present invention may also include an organic solvent together with an ultraviolet absorber. The organic solvent is not particularly limited and various hydrophobic solvents can be used which are used in a field of pressure sensitive manifold papers. Examples of organic solvents are tricresyl phosphate, octyldiphenyl phosphate and like phosphates, dibutyl phthalate, dioctyl phthalate and like phthalates, butyl oleate and like carboxylates, various fatty acid amides, diethylene glycol dibenzoate, monoisopropylnaphthalene, diisopropylnaphthalene and like alkylated naphthalenes, 1-methyl-l-phenyl-l1-tolylmethane, 1-methyl-l-phenyl-1-xylylmethane, 1-phenyl-l1-tolylmethane and like alkylated benzenes, isopropylbiphenyl and like alkylated biphenyls, trimethylolpropane triacrylate and like acrylates, ester of polyols and unsaturated carboxylic acids, chlorinated paraffin and kerosene. These solvents can be used individually or in a mixture of at least two of them. Among these hydrophobic media having a high boiling point, tricresyl phosphate and 1 phenyl-1-tolylmethane are desirable since they exhibit high solubility in connection with the ultraviolet absorber to be used in the present invention. Generally, the lower the viscosity of 17 WO 2007/086900 PCT/US2006/012942 the core material, the smaller is the particle size resulting from emulsification and the narrower is the particle size distribution, so that a solvent having a low boiling point is conjointly usable to lower the viscosity of the core material. Examples of such solvents having a low boiling point are ethyl acetate, butyl acetate, methylene chloride, etc. The amount of organic solvent to be used should be suitably adjusted according to the kind and amount of ultraviolet absorber to be used and the kind of organic solvent and is not limited specifically. For example, in case of using an ultraviolet absorber that is liquid at ordinary temperature, an organic solvent is not necessarily used. However, in case of using an ultraviolet absorber which is solid at ordinary temperature, since it is desired that the ultraviolet absorber be in a fully dissolved state in the microcapsules, the amount of organic solvent, for example in case of microcapsules of polyurea resin or polyurethane resin, is adjusted generally from to usually about 10 to 60 wt. %, or from to about 20 to 60 wt. %, based on the combined amount of organic solvent, ultraviolet absorber and wall-forming material. Further, in case of microcapsules of aminoaldehyde resin, the amount of organic solvent is adjusted to usually about 50 to 2000% by weight, generally from about 100 to 1000% by weight of ultraviolet absorber. Additionally, an absorber may be utilized. An absorber should be selected which reduces the sensitivity of the microcapsule in those portions of its spectral sensitivity range which interfere with the exposure of microcapsules at other wavelengths (its inactive range) without overly reducing the sensitivity of the microcapsule in those portions of the spectral sensitivity range in which the microcapsule is intended to be exposed (its active range). In some cases it may be necessary to balance the absorption characteristics of the absorber in the active range and the inactive range to achieve optimum exposure characteristics. Generally, absorbers having an extinction coefficient greater than about 100/M cm in the inactive range and less than about 100,000/M cm in the active range of the microcapsule are used. When the absorber is directly incorporated into the photosensitive composition, ideally, it should not inhibit free radical polymerization, and it should not generate free radicals upon exposure. The absorbers used in the present invention can be selected from among those absorbers that are known in the photographic art. Examples of such compounds include dyes conventionally used as silver halide sensitizing dyes in color photography (e.g., cyanine, merocyanine, hemicyanine and styryl dyes) and ultraviolet absorbers. A number of colored dyes that absorb outside the desired sensitivity range of the microcapsules and do not absorb heavily within the range could also be used as absorbers in the present invention. Among 18 WO 2007/086900 PCT/US2006/012942 these, Sudan I, Sudan II, Sudan III, Sudan Orange G, Oil Red O, Oil Blue N, and Fast Garnet GBC are examples of potentially useful compounds. Additionally, ultraviolet absorbers that may be desirable include those selected from hydroxybenzophenones, hydroxyphenylbenzo-triazoles and formamidines. The absorbers may be used alone or in combination to achieve the spectral sensitivity characteristics that are desired. Representative examples of useful hydroxybenzophenones are 2-hydroxy-4-n octoxybenzophenone (UV-CHEK AM-300 from Ferro Chemical Division, Mark 1413 from Argus Chemical Division, Witco Chem. Corp., and Cyasorb UV-531 Light Absorber from American Cyanamid), 4-dodecyl-2-hydroxybenzophenone (Eastman Inhibitor DOBP from Eastman Kodak), 2-hydroxy-4-methoxybenzophenone (Cyasorb UV-9 Light Absorber from American Cyanamid), and 2,2'-dihydroxy-4-methoxybenzophenone (Cyasorb UV-24 Light Absorber from American Cyanamid). Representative examples of useful hydroxybenzophenyl benzotriazoles are 2-(2'-hydroxy-5'-methylphenyl)benzotriazole (Tinuvin P from Ciba-Geigy Additives Dept.), 2-(3',5'-ditert-butyl-2'hydroxyphenyl)-5 chlorobenzotriazole (Tinuvin 327 from Ciba-Geigy), and 2-(2-hydroxy-5-t octylphenyl)benzotriazole (Cyasorb UV-5411 Light Absorber from American Cyanamid). Representative examples of useful formamidines are described in U.S. Patent No. 4,021,471 and include N-(p-ethoxy-carbonylphenyl)-N'-ethyl-N'-phenylformamidine (Givsorb UV-2 from Givaudan Corp.). The optimum absorber and concentration of absorber for a particular application depends on both the absorption maximum and extinction coefficient of the absorber candidates and the spectral sensitivity characteristics of the associated photoinitiators. Additionally, the microcapsules, photosensitive compositions, image-forming agents, developers, and development techniques described in U.S. Patent Nos. 4,399,209 and 4,440,846. Particularly, formulations to be applied in spraying forms such as water dispersible concentrates or wettable powders may contain surfactants such as wetting and dispersing agents, e.g. the condensation product of formaldehyde with naphthalene sulphonate, an alkylarylsulphonate, a lignin sulphonate, a fatty alkyl sulphate, and ethoxylated alkylphenol and an ethoxylated fatty alcohol. 2. Liposomal Formulations As used herein, the term "liposome" refers to a structure including a lipid bilayer enclosing at least one aqueous compartment. The walls are prepared from lipid molecules, 19 WO 2007/086900 PCT/US2006/012942 which have the tendency both to form bilayers and to minimize their surface area. The lipid molecules that comprise the liposome have hydrophilic and lipophilic portions. Upon exposure to water, the lipid molecules form a bilayer membrane wherein the lipid ends of the molecules in each layer are directed to the center of the membrane, and the opposing polar ends form the respective inner and outer surfaces of the bilayer membrane. Thus, each side of the membrane presents a hydrophilic surface while the interior of the membrane comprises a lipophilic medium. Liposomes can be classified into several categories based on their overall size and the nature of the lamellar structure. The classifications include small unilamellar vesicles (SUV), multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and oligolamellar vesicles. SUVs range in diameter from approximately about 20 to 50 nanometers and can include a single lipid bilayer surrounding an aqueous compartment. A characteristic of SUVs is that a large amount of the total lipid, about 70%, is located in the outer layer of the bilayer. Where SUVs are single compartment vesicles of a fairly uniform size, MLVs vary greatly in diameter up to about 30,000 nanometers and are multicompartmental in their structure wherein the liposome bilayers can be typically organized as closed concentric lamellae with an aqueous layer separating each lamella from the next. Large unilamellar vesicles are so named because of their large diameter, which ranges from about 600 nanometers to 30 microns. Oligolamellar vesicles are intermediate liposomes having a larger aqueous space than MLVs and a smaller aqueous space than LUVs. Oligolamellar vesicles have more than one internal compartment and possibly several concentric lamellae, but they generally have fewer lamellae than MLVs. A variety of methods for preparing liposomes are known in the art, several of which are described in Liposome Technology (Gregoriadis, G., editor, three volumes, CRC Press, Boca Raton 1984) or have been described by Lichtenberg and Barenholz in Methods of Biochemical Analysis, Volume 33, 337-462 (1988). Further methods of preparing liposomal formulations can be found in U.S. Patent Nos. 7,022,336; 6,989,153; 6,726,924; 6,355,267; 6,110,491; 6,007,838; 5,094,785 and 4,515,736. Liposomes are also well recognized as useful for encapsulating biologically active materials. Preparation methods particularly involving the encapsulation of DNA by liposomes, and methods that have a direct application to liposome-mediated transfection, have been described by Hug and Sleight in Biochimica and Biophysica Acta, 1097, 1-17 (1991). The liposomes of the present invention can be prepared from phospholipids, but other molecules of similar molecular shape and dimensions having both a hydrophobic and a 20 WO 2007/086900 PCT/US2006/012942 hydrophilic moiety can be used. For the purposes of the present invention, all such suitable liposome-forming molecules will be referred to herein as lipids. One or more naturally occurring and/or synthetic lipid compounds may be used in the preparation of the liposomes. Representative suitable phospholipids or lipid compounds for forming initial liposomes useful in the present invention include, but are not limited to, phospholipid-related materials such as phosphatidylcholine (lecithin), lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, phosphatidylcholine, and dipalmitoyl-phosphatidylglycerol. Additional nonphosphorous containing lipids include, but are not limited to, stearylamine, dodecylamine, hexadecyl amine, acetyl palmitate, glycerol ricinoleate, hexadecyl sterate, isopropyl myristate, amphoteric acrylic polymers, fatty acid, fatty acid amides, cholesterol, cholesterol ester, diacylglycerol, diacylglycerolsuccinate, and the like. As understood by one skilled in the art, different lipids can be used with different properties, cationic, anionic or neutral, but the preparation method can remain the same regardless of which lipid combination is used. More specifically, once lipids have been selected for use in the liposome, they can be dissolved in an organic solvent to ensure complete mixing. The organic solvent can be removed by evaporation followed by drying and a lipid film remains of the homogenous lipid mixture. The lipid mixture can be frozen in cakes and dried. The lipid cakes can be stored frozen until hydration. The addition of an aqueous medium and agitation of the container hydrate the lipid cake. The resulting product is a large, multilamellar vesicle. This structure can include concentric rings of lipid bilayers separated by water. The large, multilamellar vesicles can be downsized by. the application of energy, either in the form of mechanical energy in the process of extrusion or by sonic energy in sonication. The hydrated lipid can be forced though a polycarbonate filter with progressively smaller pores to produce particles with a diameter of similar size to the pore. Before the final pore size is used, the lipid suspension may be subjected to several freeze-thaw cycles to ensure the final particles are homogenous in size. Final particle size is partly dependent on the lipid combination used. The mean particle size is reproducible from batch to batch. This process can produce large, unilamellar vesicles that can be reduced to small, unilamellar vesicles by the application of sonic energy from a sonicator. The particles in the test tube being sonicated can be removed by centrifugation. Mean size of the resulting vesicles can be influenced by composition, 21 WO 2007/086900 PCT/US2006/012942 concentration, volume and temperature of the lipid mixture and duration, power, and tuning of the sonicator. I Specific liposome preparation methods include, but are not limited to, the hand shaken method, sonication method, reverse-phase evaporation method, freeze-dried rehydration method, and the detergent depletion method. According to the hand-shaken method, in order to produce liposomes, lipid molecules are introduced into an aqueous environment. When dry lipid film is hydrated the lamellae swell and grow into myelin figures. Mechanical agitation, for example, vortexing, shaking, swirling or pipetting, causes myelin figures (thin lipid tubules) to break and reseal the exposed hydrophobic edges resulting in the formation of liposomes. The sonication method can be used to prepare small unilamellar vesicles. Two exemplary sonication techniques include probe sonication and bath sonication. During probe sonication, the tip of a sonicator is directly immersed into the liposome dispersion. The dissipation of energy at the tip can result in local overheating, and therefore, the vessel may be immersed into an ice/water bath. During bath sonication, the liposome dispersion in a tube is placed into a bath sonicator. Material being sonicated can be kept in a sterile container, unlike the probe units, or under an inert atmosphere. The reverse-phase evaporation method is based on the formation of inverted micelles. More specifically, inverted micelles are formed upon sonication of a mixture of a buffered aqueous phase, which includes contains the water soluble molecules to be encapsulated into the liposomes and an organic phase in which the amphiphilic molecules are solubilized. The slow removal of the organic solvent leads to transformation of these inverted micelles into a gel-like and viscous state. During some point in this procedure, the gel state collapses and some of the inverted micelles disintegrate. The excess of phospholipids in the environment contributes to the formation of a complete bilayer around the remaining micelles, which results in formation of liposomes. Liposomes made by reverse phase evaporation method can be made from various lipid formulations and have a tendency to possess aqueous volume-to lipid ratios that are four times higher than multilamellar liposomes or hand-shaken liposomes. During the freeze-dried rehydration method, freeze-dried liposomes are formed from preformed liposomes. During dehydration, the lipid bilayers and the materials to be encapsulated into the liposomes are brought into close contact. Upon reswelling, the chances for encapsulation of the adhered molecules increases. The aqueous phase is generally added in small portions to the dried materials. As a general rule, the total volume used for rehydration is less than the starting volume of the liposome dispersion. 22 WO 2007/086900 PCT/US2006/012942 The detergent depletion method can be used for preparation of a variety of liposomes and proteoliposome formulations. Detergents can be depleted from a mixed detergent-lipid micelles by various techniques that lead to the formation of homogeneous liposomes. In practice, lipids below their phase transition temperature can be used with this preparation method. However, not all detergents are suited for this method. Exemplary detergents include, but are not limited to, sodium cholate, alkyl(thio)glucoside, and alkyloxypolyethylenes. Mixed micelles are prepared by adding the concentrated detergent solution to multilamellar liposomes (the final concentration of the detergent should be well above the critical micelle concentration (CMC) of the detergent). The use of different detergents can result in different size distributions of the vesicles formed. Faster depletion rates can produce smaller size liposomes. The use of different detergents may also result in different ratios of large unilamellar vesicles/ oligolamellar vesicles/multilamellar vesicles. 3. Micelles and Bicelles As used herein, "micelle" refers to an aggregate of surfactant molecules dispersed in a liquid colloid. Micelles can be globular in shape, but may exist in other shapes including, but not limited to, ellipsoids, cylinders, bilayers, and vesicles. The shape of a micelle can be controlled largely by the molecular geometry of its surfactant molecules; however, shape also depends on the conditions (such as temperature or pH, and the type and concentration of any added salt). In a micelle, the hydrophobic tails of several surfactant molecules assemble into an oil-like core that has less contact with water. In contrast, surfactant monomers are surrounded by water molecules that create a "cage" of molecules connected by hydrogen bonds. In a nonpolar solvent, the hydrophilic groups form the core of the micelle, and the hydrophobic groups remain on the surface of the micelle (so-called reverse micelle). Micelles may form when the concentration of surfactant is greater than the critical micelle concentration (CMC), and the temperature of the system is greater than the critical micelle temperature, or Krafft temperature. In water, the hydrophobic effect is the driving force for micelle formation, despite the fact that assembling surfactant molecules together reduces their entropy. Generally, above the CMC, the entropic penalty of assembling the surfactant molecules is less than the entropic penalty of the caging water molecules. As used herein, the term "bicelle" refers to a bilayered mixed micelle. Bicelles can be characterized as a mixture of long-chain bilayer forming phospholipids and short-chain micelle forming lipids of detergents. 23 WO 2007/086900 PCT/US2006/012942 The preparation of micelles and bicelles are well known in the art. Further details regarding preparation of these structures can be found in US. Patent Nos. 6,897,297; 6,696,081; 6,586,559; 6,444,793;and 5,534,259. 4. PEGylation Attachment of polyalkylene moieties as described herein can be employed to increase water solubility or solubility in aqueous solutions and/or extend the half-life of the native compounds discussed herein. Any conventional pegylation method can be employed, provided that the pegylated agent retains the desired activity. See also Schacht, E.H. et al. Poly(ethylene glycol) Chemistry and Biological Applications, American Chemical Society, San Francisco, CA 297-315 (1997). Polyalkylene glycol is a biocompatible polymer where, as used herein, polyalkylene glycol refers to straight or branched polyalkylene glycol polymers such as polyethylene glycol, polypropylene glycol, and polybutylene glycol, and further includes the monoalkylether of the polyalkylene glycol. In some embodiments of the present invention, the polyalkylene glycol polymer is a lower alkyl polyalkylene glycol moiety such as a polyethylene glycol moiety (PEG), a polypropylene glycol moiety, or a polybutylene glycol moiety. PEG has the formula -H(CH2CH20)nH, where n can range from about 1 to about 4000 or more. In some embodiments, n is 1 to 100, and in other embodiments, n is 5 to 30. PEG can range from average molecular weight of about 1 to about 22,000. For example, an average molecular weight of about 300 can correspond to n is 5, an average molecular weight of about 2,300 can correspond to n is 50, an average molecular weight of about 13,300 can correspond to n is 300 and an average molecular weight of about 22,000 can correspond to n is 500. In some embodiments, the PEG moiety can be linear or branched. In further embodiments, PEG can be attached to groups such as hydroxyl, alkyl, aryl, acyl or ester. In some embodiments, PEG can be an alkoxy PEG, such as methoxy-PEG (or mPEG), where one terminus is a relatively inert alkoxy group, while the other terminus is a hydroxyl group. PEG can be synthesized or is a commercially available product that can be readily obtained. According to some embodiments of the present invention, the pegylated compounds of the present invention can be water soluble, soluble in isopropyl alcohol (IPA), ethanol (ETOH), dimethyl sulfoxide (DMSO) and methanol (MTOH), less sensitive to UV light than a non-pegylated counterpart and/or economical to synthesize. The compounds of the present invention can be pegylated at at least four sites and/or can be pegylated in many differing PEG lengths and molecular weights. In some 24 WO 2007/086900 PCT/US2006/012942 embodiments, the PEG moiety is PEG 20 0 through PEGsoo 000 . Pegylated compounds of the present invention can further exhibit improved solubility, improved stability, lower toxicity, decreased degradation and chemical sensitivities and/or increased conjugation potential to like molecules and other molecules such as known drugs, biological tags, labels, fluorescents, radioisotopes and the like. Embodiments of the present invention include assays in which a sample is combined with a labeling reagent. "Labeling reagents" include, but are not limited to, luminescently labeled macromolecules including fluorescent protein analogs and biosensors, luminescent macromolecular chimeras including those formed with the green fluorescent protein and mutants thereof, luminescently labeled primary or secondary antibodies that react with cellular antigens involved in a physiological response, luminescent stains, dyes, and other small molecules. "Biosensors" refer to macromolecules consisting of a biological functional domain and a luminescent probe or probes that report the environmental changes that occur either internally or on their surface. A class of luminescently labeled macromolecules designed to sense and report these changes have been termed "fluorescent-protein biosensors". The protein component of the biosensor provides an evolved molecular recognition moiety. A fluorescent molecule attached to the protein component in the proximity of an active site transduces environmental changes into fluorescence signals that can be detected using a system with an appropriate temporal and spatial resolution. Because the modulation of native protein activity within the living cell is reversible, and because fluorescent-protein biosensors can be designed to sense reversible changes in protein activity, these biosensors are essentially reusable. "High content screening (HCS)" can be used to measure the effects of drugs on complex molecular events such as signal transduction pathways, as well as cell functions including, but not limited to, apoptosis, cell division, cell adhesion, locomotion, exocytosis, cell growth and metabolism, protein synthesis, enzymatic functions, cell-cell communication and the detection of healthy and/or diseased cells. Multicolor fluorescence permits multiple targets and cell processes to be assayed in a single screen. Cross-correlation of cellular responses will yield a wealth of information required for target validation and lead optimization. Methods of screening cells treated with dyes and fluorescent reagents is well known in the art. There is a considerable body of literature related to genetic engineering of cells to produce fluorescent proteins, such as modified green fluorescent protein (GFP), as a reporter 25 WO 2007/086900 PCT/US2006/012942 molecule. Some properties of wild-type GFP are disclosed by Morise et al. (Biochemistry 13 (1974), p. 2656-2662), and Ward et al. (Photochem. Photobiol. 31 (1980), p. 611-615). The GFP of the jellyfish Aequorea Victoria has an excitation maximum at 395 nm and an emission maximum at 510 nm, and does not require an exogenous factor for fluorescence activity. Uses for GFP disclosed in the literature are widespread and include the study of gene expression and protein localization (Chalfie et al., Science 263 (1994), p. 12501-12504)), as a tool for visualizing subcellular organelles (Rizzuto et al., Curr. Biology 5 (1995), p. 635 642)), visualization of protein transport along the secretory pathway (Kaether and Gerdes, FEBS Letters 369 (1995), p. 267-271)), expression in plant cells (Hu and Cheng, FEBS Letters 369 (1995), p. 331-334)) and Drosophila embryos (Davis et al., Dev. Biology 170 (1995), p. 726-729)), and as a reporter molecule fused to another protein of interest (U.S. Patent No. 5,491,084). Similarly, WO 96/23898 relates to methods of detecting biologically active substances affecting intracellular processes by utilizing a GFP construct having a protein kinase activation site. This patent, and all other patents referenced in this application are incorporated by reference in their entirety. Numerous references are related to GFP proteins in biological systems. For example, WO 96/09598 describes a system for isolating cells of interest utilizing the expression of a GFP like protein. WO 96/27675 describes the expression of GFP in plants. WO 95/21191 describes modified GFP protein expressed in transformed organisms to detect mutagenesis. U.S. Patent Nos. 5,401,629 and 5,436,128 describe assays and compositions for detecting and evaluating the intracellular transduction of an extracellular signal using recombinant cells that express cell surface receptors and contain reporter gene constructs that include transcriptional regulatory elements that are responsive to the activity of cell surface receptors. There are also numerous journals discussing the use of GFP proteins. Performing a screen on many thousands of compounds requires parallel handling and processing of many compounds and assay component reagents. Standard high throughput screens ("HTS") use mixtures of compounds and biological reagents along with some indicator compound loaded into arrays of wells in standard microtiter plates with 96 or 384 wells. The signal measured from each well, either fluorescence emission, optical density, or radioactivity, integrates the signal from all the material in the well giving an overall population average of all the molecules in the well. Another system fluorescence imaging plate reader (FLIPR) uses a low angle laser scanning illumination and a mask to selectively excite fluorescence within approximately 200 microns of the bottoms of the wells in standard 96 well plates in order to reduce background 26 WO 2007/086900 PCT/US2006/012942 when imaging cell monolayers. This system uses a charge couple device (CCD) camera to image the whole area of the plate bottom. Although this system measures signals originating from a cell monolayer at the bottom of the well, the signal measured is averaged over the area of the well and is therefore still considered a measurement of the average response of a population of cells. The image is analyzed to calculate the total fluorescence per well for cell based assays. Fluid delivery devices have also been incorporated into cell based screening systems, such as the FLIPR system, in order to initiate a response, which is then observed as a whole cell population average response using a macro-imaging system. In contrast to high throughput screens, various high-content screens (HCS) have been developed to address the need for more detailed information about the temporal-spatial dynamics of cell constituents and processes. High-content screens automate the extraction of multicolor fluorescence information derived from specific fluorescence-based reagents incorporated into cells (Giuliano and Taylor (1995), Curr. Op. Cell Biol. 7:4; Giuliano et al. (1995) Ann. Rev. Biophys. Biomol. Struct. 24:405). Cells are analyzed using an optical system that can measure spatial, as well as temporal dynamics. (Farkas et al. (1993) Ann. Rev. Physiol. 55:785; Giuliano et al. (1990) In Optical Microscopy for Biology. B. Herman and K. Jacobson (eds.), pp. 543-557. Wiley-Liss, New York; Hahn et al (1992) Nature 359:736; Waggoner et al. (1996)*Hum. Pathol. 27:494). The concept is to treat each cell as a "well" that has spatial and temporal information on the activities of the labeled constituents. The types of biochemical and molecular information now accessible through fluorescence-based reagents applied to cells include ion concentrations, membrane potential, specific translocations, enzyme activities, gene expression, as well as the presence, amounts and patterns of metabolites, proteins, lipids, carbohydrates, and nucleic acid sequences (DeBiasio et al., (1996) Mol. Biol. Cell. 7:1259; Giuliano et al., (1995) Ann. Rev. Biophys. Biomol. Struct. 24:405; Heim and Tsien, (1996) Curr. Biol. 6:178). High-content screens can be performed on either fixed cells, using fluorescently labeled antibodies, biological ligands, and/or nucleic acid hybridization probes, or live cells using multicolor fluorescent indicators and "biosensors." The choice of fixed or live cell screens depends on the specific cell-based assay required. As understood by one of skill in the art, any of the methods and screens discussed above can be utilized with the compounds of the present invention. Generally, fixed cell assays are the simplest, since an array of initially living cells in a microtiter plate format can be treated with various compounds and doses, then the cells can be fixed, labeled with specific reagents, and quantified. No environmental control of the cells 27 WO 2007/086900 PCT/US2006/012942 is required after fixation. Spatial information is acquired, but only at one time point. The availability of thousands of antibodies, ligands and nucleic acid hybridization probes that can be applied to cells makes this an attractive approach for many types of cell-based screens. The fixation and labeling steps can be automated, allowing efficient processing of assays. Live cell assays are more sophisticated and powerful, since an array of living cells containing the desired reagents can be screened over time, as well as space. Environmental control of the cells (temperature, humidity, and carbon dioxide) should be maintained during measurement, since the physiological health of the cells should be maintained for multiple fluorescence measurements over time. There is a growing list of fluorescent physiological indicators and "biosensors" that can report changes in biochemical and molecular activities within cells (Giuliano et al., (1995) Ann. Rev. Biophys. Biomol. Struct. 24:405; Hahn et al., (1993) In Fluorescent and Luminescent Probes for Biological Activity. W. T. Mason, (ed.), pp. 349-359, Academic Press, San Diego). The availability and use of fluorescence-based reagents has helped to advance the development of both fixed and live cell high-content screens. Advances in instrumentation to automatically extract multicolor, high-content information has made it possible to develop HCS into an automated tool. An article by Taylor, et al. (American Scientist 80 (1992), p. 322-335) describes many of these methods and their applications. For example, Proffitt et. al. (Cytometry 24: 204-213 (1996)) describe a semi-automated fluorescence digital imaging system for quantifying relative cell numbers in situ in a variety of tissue culture plate formats, especially 96-well microtiter plates. The system consists of an epifluorescence inverted microscope with a motorized stage, video camera, image intensifier, and a microcomputer with a PC-Vision digitizer. Turbo Pascal software controls the stage and scans the plate taking multiple images per well. The software calculates total fluorescence per well, provides for daily calibration, and configures easily for a variety of tissue culture plate formats. Thresholding of digital images and reagents which fluoresce only when taken up by living cells are used to reduce background fluorescence without removing excess fluorescent reagent. Scanning confocal microscope imaging (Go et al., (1997) Analytical Biochemistry 247:210-215; Goldman et al., (1995) Experimental Cell Research 221:311-319) and multiphoton microscope imaging (Denk et al., (1990) Science 248:73; Gratton et al., (1994) Proc. of the Microscopical Society of America, pp. 154-155) are also well established methods for acquiring high resolution images of microscopic samples. The principle advantage of these optical systems is the shallow depth of focus, which allows features of 28 WO 2007/086900 PCT/US2006/012942 limited axial extent to be resolved against the background. For example, it is possible to resolve internal cytoplasmic features of adherent cells from the features on the cell surface. Because scanning multiphoton imaging utilize short duration pulsed laser systems to achieve the high photon flux required, fluorescence lifetimes can also be measured in these systems (Lakowicz et al., (1992) Anal. Biochem. 202:316-330; Gerrittsen et al. (1997), J. of Fluorescence 7:11-15)), providing additional capability for different detection modes. Small, reliable and relatively inexpensive laser systems, such as laser diode pumped lasers, are now available to allow multiphoton confocal microscopy to be applied in a fairly routine fashion. A major component of the new drug discovery paradigm is a continually growing family of fluorescent and luminescent reagents that are used to measure the temporal and spatial distribution, content, and activity of intracellular ions, metabolites, macromolecules, and organelles. Classes of these reagents include labeling reagents that measure the distribution and amount of molecules in living and fixed cells, environmental indicators to report signal transduction events in time and space, and fluorescent protein biosensors to measure target molecular activities within living cells. A multiparameter approach that combines several reagents in a single cell can be a powerful tool for drug discovery. Methods of the present invention include high affinity of fluorescent or luminescent stilbazium molecules and analogs for specific cellular components. The affinity for specific components is governed by forces such as ionic interactions, covalent bonding (which includes chimeric fusion with protein-based chromophores, fluorophores, and lumiphores), as well as hydrophobic interactions, electrical potential, and, in some cases, simple entrapment within a cellular component. Those skilled in this art will recognize a wide variety of fluorescent reporter molecules that can be used in addition to the compounds of the present invention for multiplexing, including, but not limited to, fluorescently labeled biomolecules such as proteins, phospholipids, cellular organelles and DNA hybridizing probes. The luminescent probes can be synthesized within the living cell or can be transported into the cell via several non-mechanical modes including diffusion, facilitated or active transport, signal-sequence-mediated transport, and endocytotic or pinocytotic uptake. Mechanical bulk loading methods, which are well known in the art, can also be used to load luminescent probes into living cells (Barber et al. (1996), Neuroscience Letters 207:17-20; Bright et al. (1996), Cytometry 24:226-233; McNeil (1989) in Methods in Cell Biology, Vol. 29, Taylor and Wang (eds.), pp. 153-173). These methods include electroporation and other mechanical methods such as scrape-loading, bead-loading, impact-loading, syringe-loading, hypertonic and hypotonic loading. Additionally, cells can be genetically engineered to 29 WO 2007/086900 PCT/US2006/012942 express reporter molecules, such as GFP, coupled to a protein of interest as previously described (Chalfie and Prasher U.S. Pat. No. 5,491,084; Cubitt et al. (1995), Trends in Biochemical Science 20:448-455). Furthermore, certain cell types within an organism may contain components that can be specifically labeled with a formulation of stilbazium or an analog thereof that may not occur in other cell types. For example, epithelial cells often contain polarized membrane components. That is, these cells asymmetrically distribute macromolecules along their plasma membrane. Connective or supporting tissue cells often contain granules in which are trapped molecules specific to that cell type (e.g., heparin, histamine, serotonin, etc.). Most muscular tissue cells contain a sarcoplasmic reticulum, a specialized organelle whose function is to regulate the concentration of calcium ions within the cell cytoplasm. Many nervous tissue cells contain secretory granules and vesicles in which are trapped neurohormones or neurotransmitters. Therefore, fluorescent molecules can be designed to label not only specific components within specific cells, but also specific cells within a population of mixed cell types. Those skilled in the art will recognize a wide variety of ways to measure fluorescence. For example, some fluorescent reporter molecules exhibit a change in excitation or emission spectra, some exhibit resonance energy transfer where one fluorescent reporter loses fluorescence, while a second gains in fluorescence, some exhibit a loss (quenching) or appearance of fluorescence, while some report rotational movements (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomol. Structure 24:405-434; Giuliano et al. (1995), Methods in Neuroscience 27:1-16). In biochemistry, molecular biology and medical diagnostics, it is often desirable to add a fluorescent label to a protein so that the protein can be easily tracked and quantified. The normal procedures for labeling requires that the protein be covalently reacted in vitro with fluorescent dyes, then repurified to remove excess dye and any damaged protein. If the labeled protein is to be used inside cells, it is usually microinjected; however, this can be a difficult and/or time-consuming operation that may not be realistically performed on large numbers of cells. The compounds of the present invention can be utilized as staining agents for DNA/RNA in their isolated state or for isolated cells and organelles. Also, the compounds may be utilized as nuclear staining agents for cell based assays. Further applications include using the compounds of the present invention to stain tissues and/or organs in whole animals as well as embryos, larvae, nematodes, insects and other parasites. In some embodiments, 30 WO 2007/086900 PCT/US2006/012942 compounds of the present invention could be complexed with specific antibodies for imaging of specific organ tissues in the whole animal model systems. The term "antibody" or "antibody molecule" in the various grammatical forms as used herein refers to an immunoglobulin molecule (including IgG, IgE, IgA, IgM, IgD) and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope and can bind antigen. An "antibody combining site" or "antigen binding site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable (CDR) regions that specifically binds antigen. As is known in the art, particular properties of antibodies relate to immunoglobulin isotype. In representative embodiments, the antibody or antigen-binding fragment is an IgG2a, an IgG1 or an IgG2b isotype molecule. The antibody or fragment can further be from any species of origin including avian (e.g., chicken, turkey, duck, geese, quail, etc.) and mammalian (e.g., human, non-human primate, mouse, rat, rabbit, cattle, goat, sheep, horse, pig, dog, cat, etc.) species. The present invention can provide systems, methods, and screens that combine high throughput screening (HTS) and high content screening (HCS) that may improve target validation and candidate optimization at least by combining many cell screening formats with fluorescence-based molecular reagents and computer-based feature extraction, data analysis, and automation, resulting in increased quantity and speed of data collection, shortened cycle times, and, ultimately, faster evaluation. Accordingly, in some embodiments, the invention features a technique for determining the presence, location, or quantity of a cell, and thus, cellular organelles such as the nucleus, smooth and/or rough endoplasmic reticulum, centrosome, cytoskeleton, cell wall, cell membrane, flagella, cilia, chloroplast, mitochondria, golgi apparatus, ribosome, lysosome, centriole, acrosome, glyoxysome, secretory vesicle, peroxisome, vacuole, melanosome, myofibril and parenthesome. In other embodiments, the invention features a technique for determining the presence, location, quantity and/or health of analytes and microorganisms. In other embodiments, the invention features a technique for determining the presence, location, or quantity of an RNA molecule of interest in a cell or an in vitro sample. This method involves (a) expressing, in the cell or sample, a fusion RNA molecule including the RNA molecule of interest covalently linked to an RNA aptameir, (b) contacting the cell or sample with a fluorophore under conditions that allow the RNA aptamer to bind the 31 WO 2007/086900 PCT/US2006/012942 fluorophore and thereby increase or decrease its fluorescence, and (c) visualizing the fluorescence of the fluorophore. In still other embodiments, the invention provides methods for determining the presence, location, or quantity of a DNA molecule of interest in a cell or an in vitro sample. This method involves (a) expressing, in the cell or the sample, a fusion DNA molecule including the DNA molecule of interest covalently linked to a DNA aptamer, (b) contacting the cell or the sample with a fluorophore under conditions that allow the DNA aptamner to bind the fluorophore and thereby increase or decrease its fluorescence, and (c) visualizing the fluorescence of the fluorophore. Embodiments of the invention also include assays for determining the presence or absence of a cell, analyte, nucleic acid or microorganism in a sample, such as a fluid or aqueous sample, suspected of containing a microorganism, said assay comprising combining the sample with a labeling reagent to form a labeled cell, nucleic acid or microorganism, said labeling reagent comprising a dye which directly stains the cell, analyte, nucleic acid or microorganism to provide a stained sample comprising a stained cell, analyte, nucleic acid or microorganism, wherein said dye is a compound represented by Formula I R N -- I_ --- R2 R,5 R4I wherein for Formula I, the NR 1

R

2 and NR 3

R

4 moieties are in the ortho, meta or para positions; wherein X is an anionic salt and X- can be selected from the group including fluoride, chloride, bromide, iodide halide, mesylate, tosylate, napthylate, nosylate, para aminobenzoate, benzenesulfonate, besylate, lauryl sulfate, 2,4-dihydroxy benzophenone, 2 (2-hydroxy-5'-methylphenyl) benzotriazole, ethyl 2-cyano-3,3-diphenyl acrylate and 5-butyl phenyl salicylate.; wherein RI, R 2 , R 3 , or R4 are independently selected from the group consisting of methyl, ethyl, C1-1 0 alkyl (linear or branched), alkenes (linear or branched), or wherein R 1 and R 2 or R 3 and R 4 taken together with the nitrogen atom to which they are attached form pyrrolidino or piperidino rings; wherein R 5 is a polyalkylene glycol moiety, a C1- 1 0 alkyl (linear or branched), an alkene (linear or branched), an alkyne, a substituted and unsubstituted aryl, a substituted and unsubstituted benzyl and/or an organometallic moiety. 32 WO 2007/086900 PCT/US2006/012942

R

5 may also be an organometallic compound such as organotin, organosilicon, or organogermanium. Additionally, R 5 may be (CH 2 )n-M 6 , wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R 6 is a selected from the group consisting of propyl, butyl, or any alkyl compound; contacting the stained sample; and observing the accumulation of the stained cell, analyte, nucleic acid or microorganism. The dye can be encapsulated. In some embodiments, the dye can be loaded into a microcapsule or a lipid vesicle such as a liposome, micelle and/or bicelle, to form a microencapsulated formulation or a lipid vesicle formulation, respectively. The dye can also be pegylated. In various embodiments of the invention, a cell may be bound by one of the compounds according to Formula I. In some embodiments, the cell is a prokaryotic cell, such as a gram-negative or gram-positive bacterial cell. In other embodiments, the cell is a eukaryotic cell. For example, the cell may be a yeast, Caenorhabditis, Xenopus, Drosophila, zebrafish, squid, plant, mammalian, embryonic, or human cell. In yet other embodiments, the cell or the sample is contacted with the fluorophore by incubating the cell or the sample with the fluorophore. In still other embodiments, the fluorophore is injected into the cell or administered to a plant, embryo, mammal, transgenic animal, or human including the cell. In other embodiments, the population of nucleic acids contains more than one DNA molecule or more than one RNA molecule. The nucleic acids may have naturally-occurring or non-naturally-occurring polynucleotide sequences. Regions of the nucleic acids; such as all or part of a loop, tetraloop, or helix; contain random sequences that differ between some or all of the members of the population. In other embodiments, the sequence of a loop, tetraloop, or helix is the same in all of the members of the population. The lengths of any of the loops or helices may be the same or may differ between members of the population. The populations of nucleic acids may contain any number of unique molecules. For example, the population may contain as few as about 10, 102, 109, or 1011 unique molecules or as many as about 101 3 , 101 s , 1020, or more unique molecules. In some embodiments, at least one of the polynucleotide sequences is a non-naturally-occurring sequence. The nucleic acids may either all have the same length or some of the molecules may differ in length. By "fluorophore" is meant a compound that is capable of emitting a fluorescent signal. As described herein, fluorophores of use in the invention have a higher fluorescence intensity when bound to a nucleic acid or protein than when unbound in solution. The fluorescence intensity of the bound fluorophore can be at least about 1, 5, 10, 50, 100, 500, or 1000 times that of the unbound fluorophore in an aqueous solution. Examples of conditions 33 WO 2007/086900 PCT/US2006/012942 that may enhance the fluorescence of the bound fluorophore include rigidification, conformational restriction, and sequestration from solvent. In one embodiment, the fluorophore does not covalently bind the aptamer. Other fluorophores have a lower fluorescence intensity when bound to a nucleic acid or protein than when unbound in solution. The fluorescence intensity of the bound fluorophore can be at least about 2, 5, 10, 50, 100, 500, or 1000 less than that of the unbound fluorophore in an aqueous solution. An example of a condition that may decrease the fluorescence of the bound fluorophore is a change in the conformation of the fluorophore that decreases its fluorescence intensity. In one embodiment, the fluorophore does not covalently bind the aptamer. Other fluorophores for use in multiplexing, such as calcium-sensing dyes, may adopt at least two different conformational states that result in different fluorescence intensities. An aptamer of the invention may modulate the fluorescence of the fluorophore by increasing the percentage of the fluorophore in a particular conformational state with increased or decreased fluorescence. The fluorophore is soluble in an aqueous solution at a concentration of about 0.11M, 1pM, 10/M, and 50M. Incubating a cell with these concentrations of the fluorophore may not effect the viability of the cell. In another embodiment, incubating a cell with the fluorophore at these concentrations for as few as about I or 2 hours to as many as about 8, 12, 24, 36, or more hours does not require the presence of another compound to prevent toxic effects of the fluorophore, such as the inactivation of proteins in the cell; inhibition of replication, transcription, or translation; or the induction of cell death. By "cell permeable" is meant capable of migrating through a cell membrane or cell wall into the cytoplasm or periplasm of a cell by active or passive diffusion. The fluorophore can migrate through both the outer and inner membranes of gram-negative bacteria or both the cell wall and plasma membrane of plant cells. Additionally, the fluorophore can be used to visualize a cell, analyte and/or nucleic acids in an in vitro sample. Embodiments of the present invention may include nucleic acid probes comprising a single-stranded oligonucleotide and a compound represented by Formula I 34 WO 2007/086900 PCT/US2006/012942 R, N+ R3 R _ X_ RNR wherein for Formula I, the NR 1

R

2 and NR 3

R

4 moieties are in the ortho, meta or para positions; wherein X is an anionic salt; wherein R 1 , R 2 , R 3 , or R 4 are independently selected from the group consisting of methyl, ethyl, C-lo 0 alkyl (linear or branched), alkenes (linear or branched), or wherein R 1 and R2 or R 3 and R 4 taken together with the nitrogen atom to which they are attached form pyrrolidino or piperidino rings; wherein R 5 is a polyalkylene glycol moiety, a C- 1 o alkyl (linear or branched), an alkene (linear or branched), an alkyne, a substituted and unsubstituted aryl, a substituted and unsubstituted benzyl and/or an organometallic moiety. R 5 may also be an organometallic compound such as organotin, organosilicon, or organogermanium. Additionally, R 5 may be

(CH

2 )n-MRt, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R 6 is a selected from the group consisting of propyl, butyl, or any alkyl compound. The single-stranded oligonucleotide can be a DNA oligomer. A phosphorus atom in the DNA oligomer can be linked by the chemical bond via a linker. The single-stranded oligonucleotide can have a sequence complementary to a specific sequence in a target nucleic acid containing the specific sequence Embodiments of the present invention may include methods of selecting a nucleic acid molecule which binds to Formula I, wherein said binding increases the fluorescence intensity of said Formula I, said method comprising the steps of: (a) providing a population of candidate nucleic acid molecules; (b) selecting said candidate nucleic acid molecules which bind said Formula I; (c) contacting said candidate nucleic acid molecules which bind said Formula I with said Formula I; and (d) selecting said nucleic acid molecules which, upon binding said Formula I or, increase its fluorescence intensity. The nucleic acid can be a DNA or an RNA. 35 WO 2007/086900 PCT/US2006/012942 Another embodiment of the present invention includes methods of determining the presence, location, or quantity of a nucleic acid of interest in a cell or an in vitro sample, said method comprising the steps of: (a) expressing in said cell or said sample a nucleic acid of interest; (b) contacting said cell or said sample with Formula I; whereby said compound binds to said Formula I and increases its fluorescence intensity; and (c) visualizing or measuring the fluorescence of said Formula I, thereby determining the presence, location, or quantity of said nucleic acid of interest in said cell or said in vitro sample. Embodiments of the present invention include methods of determining whether Formula I is capable of modulating the transcription of a nucleic acid of interest, said method further comprising the steps of: (a) expressing in a cell or an in vitro sample a nucleic acid of interest; (b) contacting said cell or said sample with said Formula I or with said Formula I alone, whereby said compound binds to said Formula I and increases its fluorescence intensity; and (c) measuring said fluorescence intensity in the presence and absence of said compound, whereby said compound is determined to modulate said transcription if said compound effects a change in said fluorescence intensity. Other embodiments include kits for staining nucleic or amino acids in a sample, comprising: (a) a staining mixture that contains one or more dyes to form a combined mixture; wherein each dye independently has the Formula I; b) instructions for combining said dye or dyes with a sample containing or thought to contain nucleic or amino acids, said instructions comprising i) combining a sample that is thought to contain nucleic or amino acids with a staining mixture that contains said dye or dyes to form a combined mixture; and ii) incubating the combined mixture for a time sufficient for the dye in the staining mixture to associate with the nucleic or amino acids in the sample mixture to form a dye-amino acid or dye-nucleic acid complex that gives a detectable optical response upon illumination. Additional embodiments include methods of detecting a target analyte in a sample containing or suspected of containing one or more analytes, comprising the steps of: (a) providing the sample on a solid support wherein the analyte is a nucleic acid molecule; (b) combining with said sample a specific-binding molecule, wherein (i) said specific-binding molecule is a polymerase chain reaction amplification product comprising biotin as a detectable label, and (ii) said combining is performed under conditions that allow formation of a first complex comprising said specific-binding molecule and said analyte, when present; (c) removing any unbound specific-binding molecule; (d) providing a compound having the Formula I; and (e) detecting an optical response based upon the binding of the compound. 36 WO 2007/086900 PCT/US2006/012942 Embodiments of the present invention include methods for analyzing cells comprising providing an array of locations which contain multiple cells wherein the cells contain one or more fluorescent reporter molecules; scanning multiple cells in each of the locations containing cells to obtain fluorescent signals from the fluorescent reporter molecule in the cells; converting the fluorescent signals into digital data; and utilizing the digital data to determine the distribution, environment or activity of the fluorescent reporter molecule within the cells. Embodiments of the present invention may also be utilized within cell arrays. Cell arrays are used for screening large numbers of compounds for activity with respect to a particular biological function and involves preparing arrays of cells for parallel handling of cells and reagents. Standard 96 well microtiter plates which are 86 mm by 129 mm, with 6 mm diameter wells on a 9 nm pitch, are used for compatibility with current automated loading and robotic handling systems. The microplate is typically 20 mm by 30 mm, with cell locations that are 100-200 microns in dimension on a pitch of about 500 microns. Methods for making microplates are described in U.S. Patent No. 6,103,479. Microplates may consist of coplanar layers of materials to which cells adhere, patterned with materials to which cells will not adhere, or etched 3-dimensional surfaces of similarly pattered materials. The terms "well" and "microwell" refer to a location in an array of any construction to which cells adhere and within which the cells are imaged. Microplates may also include fluid delivery channels in the spaces between the wells. The smaller format of a microplate increases the overall efficiency of the system by minimizing the quantities of the reagents, storage and handling during preparation and the overall movement required for the scanning operation. In addition, the whole area of the microplate can be imaged more efficiently, allowing a second mode of operation for the microplate reader as described later in this document. As described above, according to the present invention, it is possible to provide a novel compound which is an unfamiliar fluorescent dye which may show a large fluorescent enhancement upon intercalation into a double-stranded nucleic acid when used in detection of the nucleic acid, and shows a great difference between the excitation wavelength and the emission wavelength (i.e., has a large Stokes shift). The compound is used for conventional nucleic acid assays by contacting it with a double-stranded nucleic acid or by linking it with a single-stranded oligonucleotide to from a nucleic acid probe. The compounds of the present invention may be further characterized in that their fluorescent spectrum does not overlap with that of any known fluorescent intercalative dye. Therefore, combined use of at least two nucleic acid probes using the compounds of the 37 WO 2007/086900 PCT/US2006/012942 present invention and conventionally known fluorescent intercalative dye(s) makes it possible to measure the amplification products from at least two target nucleic acids in a sample in a closed vessel without separation while amplifying the target nucleic acids, i.e., measure at least target nucleic acids simultaneously. The present invention is explained in greater detail in the Example that follows. This example is intended as illustrative of the invention and is not to be taken are limiting thereof. EXAMPLE Spectra were obtained on solutions at ambient temperature (220C) in methacrylate cuvettes with a 1cm path length in a Beckman DU-640 spectrometer (Beckman-Coulter, Palo Alto CA). The scan rate was 10nnm/min from 800nm to 250nm. Blank scans were run in the same diluent as used for the analyte and subtracted from the compound spectra. Both stilbazium chloride (as a red powder) and a liposomal formulation contain stilbazium (as a turbid, colored solution) were provided. Ethidium bromide, DMSO and Ethanol were purchased from Sigma-Aldrich (St. Louis, Mo). Methacrylate, 1.5 mL cuvettes were purchased from Fisher Scientific. The molar absorption (E) was calculated by dividing the observed optical density by the number of moles of analytes ([molar]*volume) and presents the theoretical absorption of 1.0 mole of the test substance. Stilbazium chloride, when dissolved in absolute ethanol, resulted in a deep red colored solution. A second dilution in either absolute or 70% (aq.) ethanol was performed prior to performing the analyses. In both cases, a single, broad peak of absorption was observed with an apparent maxima of 509-511 nm and a single, broad peak with a molar extinction coefficient, E = 6638400. The molecule was similarly soluble in DMSO with deep, red coloration at the 4.1 mg/mL initial concentration. Spectra were obtained after a secondary dilution in the same solvent to a final concentration of 50 pM which yielded a single, broad peak with e = 4401574 at 507nm. The stilbazium chloride was found to be mostly insoluble in water. Thus, spectra were performed in solutions of ethanol and DMSO. Among the 2 solvents, greater absorption was observed for the ethanolic solutions than for compound dissolved in DMSO (6 = 6638400 Vs. 4401574). Secondary dilution into 70% aqueous ethanol was identical to the results with absolute ethanol. 38 WO 2007/086900 PCT/US2006/012942 The liposomal formulation provided as a yellow to pink colored solution was added directly to a cuvette containing water (50pgL in 1.0 mL) yielded apparent light scattering turbidity. The resulting spectrum had a maxima at 585nm with E = 302400. When formulated as a water soluble liposome complex, the formulation was slightly turbid on addition to water in a 1:20 dilution. The resulting spectrum showed a shift in the wavelength with maximal optical density at 585 nm and c = 302400. The reduced molar absorption is not surprising since the analyte is contained in the liposome suspension. Light, scattering on the surface of the liposomes likely hindered interaction with the compound. For comparison, the DNA binding dye, ethidium bromide, dissolved in water, had its maximal optical density at 284 nm with E = 6602400 and a much smaller peak at 478 nm E = 700460. Compounds of the present invention can fluoresce in a range from about 400 nm to 700 nm. Cells Staining Murine mammary carcinoma 4TI cell populations were individually incubated with three compounds according to the present invention for about 30 minutes, and subsequently co-stained with HOECHST (blue) and MitoTracker Deep Red (green). Confocal images were taken with a Zeiss Meta 510 LSM. The dyes were excited by laser at a wavelength of 540nm (red). Figure 1A through 1L present confocal images of 4T1 cells incubated with a compound according to some embodiments of the present invention (Figures lB, 1F and 1J) and co-stained with Hoechst (Figures 1A, 1E and lI, blue) to stain the nuclei and MitoTracker Deep Red (Figures 1C, 1G and 1K, appears green in images) to stain the mitochondria. Figure 1D presents an overlaid image of the images presented in Figures 1A through 1C. Figure 1H presents an overlaid image of the images presented in Figures lE through 1G. Figure 1L presents an overlaid image of the images presented in Figures 11 through 1K. The overlaid images indicate that the compounds of the present invention may stain cells, in particular, the mitochondria, in a viable or fixed cell population. The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. Although exemplary embodiments of this invention have been described, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included 39 WO 2007/086900 PCT/US2006/012942 within the scope of this invention as defined in the claims. The invention is defined by the following claims, with equivalents of the claims to be included therein. 40

Claims (25)

1. An assay for determining the presence, absence or health of a cell, analyte, nucleic acid or microorganism in a sample, said assay comprising combining the sample with a labeling reagent to form a labeled cell, analyte, nucleic acid or microorganism, said labeling reagent comprising a dye that stains the cell, analyte, nucleic acid or microorganism to provide a stained sample comprising a stained cell, analyte, nucleic acid or microorganism, wherein said dye is a compound of Formula I: Ri .. R 3 N--- --- I)C--N R2 Rs R4 wherein for Formula I, the NR 1 R 2 and NR 3 R 4 moieties are in the ortho, meta or para positions; X- is an anionic salt; R 1 , R 2 , R 3 , or R 4 are independently selected from the group consisting of methyl, ethyl, C1- 10 alkyl (linear or branched), alkenes (linear or branched), or wherein R 1 and R 2 or R 3 and R4 taken together with the nitrogen atom to which they are attached form pyrrolidino or piperidino rings; R 5 is selected from the group consisting of methyl, ethyl, C1-o 10 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, an organometallic compound, a polyalkylene glycol moiety, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; and observing the accumulation of the stained cell, analyte, nucleic acid or microorganism.

2. The assay of claim 1, wherein said dye is encapsulated or pegylated. 41 WO 2007/086900 PCT/US2006/012942

3. The assay of claim 1, wherein R 5 is (CH 2 )n-MR 6 and n is a number from 1 to 6, M is an organometallic compound selected from the group consisting of tin, silicon, and germanium, and wherein R6 is selected from the group consisting of propyl, butyl, and alkyl, substituted or unsubstituted.

4. The assay of claim 1, wherein the compound of Formula I has the following structure: CH3 N]

5. The assay of claim 1, wherein the compound of Formula I has the following structure: N N

6. The assay of claim 1, wherein the compound of Formula I has the following structure: N NCI N N 42 WO 2007/086900 PCT/US2006/012942

7. A probe comprising a ligand or antibody and a compound of Formula I: N----X- - --- N R54 R2 RSR4 wherein for Formula I, the NRI 1 R 2 and NR 3 R 4 moieties are in the ortho, meta or para positions; X is an anionic salt; R 1 , R 2 , R 3 , or R 4 are independently selected from the group consisting of methyl, ethyl, C 1 - 10 alkyl (linear or branched), alkenes (linear or branched), or wherein R 1 and R 2 or R 3 and R 4 taken together with the nitrogen atom to which they are attached form pyrrolidino or piperidino rings; R 5 is selected from the group consisting of methyl, ethyl, C 1 -o 10 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, an organometallic compound, a polyalkylene glycol moiety, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; and wherein said compound is linked to the ligand or antibody by a chemical bond.

8. The probe according to claim 7, wherein the ligand is an oligonucleotide.

9. The probe according to claim 8, wherein the oligonucleotide is a DNA oligomer.

10. The probe according to claim 9, wherein a phosphorus atom in the DNA oligomer is attached to the dye.

11. The probe according to claim 8, wherein said oligonucleotide has a sequence complementary to a specific sequence in a target nucleic acid containing the specific sequence. 43 WO 2007/086900 PCT/US2006/012942

12. A method of selecting an analyte that binds to a compound of Formula I: NRI --- N R2 X- R5 R wherein for Formula I, the NR 1 R 2 and NR 3 R 4 moieties are in the ortho, meta or para positions; wherein X- is an anionic salt; R 1 , R 2 , R 3 , or R 4 are independently selected from the group consisting of methyl, ethyl, C1- 10 alkyl (linear or branched), alkenes (linear or branched), or wherein R 1 and R 2 or R 3 and R 4 taken together with the nitrogen atom to which they are attached form pyrrolidino or piperidino rings; R 5 is selected from the group consisting of methyl, ethyl, Ci-10 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, an organometallic compound, a polyalkylene glycol moiety, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; and wherein said binding increases the fluorescence intensity of said Formula I, said method comprising the steps of: (a) providing a population of analytes; (b) selecting said analytes that bind said Formula I; (c) contacting said analytes that bind said Formula I; and (d) selecting said analytes that, upon binding said Formula I, increase the fluorescent intensity of the analyte.

13. The method according to claim 12, wherein said analyte is DNA.

14. The method according to claim 12, wherein said analyte is RNA.

15. The method according to claim 12, wherein said analyte is a small molecule.

16. The method according to claim 12, wherein said analyte is a microorganism. 44 WO 2007/086900 PCT/US2006/012942

17. The method according to claim 12, wherein said analyte is a cell.

18. A method of determining whether a nucleic acid of interest interacts with a protein of interest in a cell or an in vitro sample, said method comprising the steps of: (a) expressing in said cell or said sample a fusion nucleic acid comprising said nucleic acid of interest covalently linked to a nucleic acid aptamer which binds a first Formula I R1N R3 N --- I-- -- I - X- IR R RsR4 wherein for Formula I, the NR 1 R 2 and NR3R 4 moieties are in the ortho, meta or para positions; X- is an anionic salt; R 1 , R 2 , R 3 , or R 4 are independently selected from the group consisting of methyl, ethyl, C-10 alkyl (linear or branched), alkenes (linear or branched), or wherein R 1 and R2 or R 3 and R 4 taken together with the nitrogen atom to which they are attached form pyrrolidino or piperidino rings; S R 5 is selected from the group consisting of methyl, ethyl, Cl-o alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, an organometallic compound, a polyalkylene glycol moiety, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; and (b) expressing in said cell or said sample a fusion protein comprising said protein of interest covalently linked to a detectable protein which binds a second Formula I, wherein the emission wavelength of said first Formula I is different from that of said second Formula I, and wherein the emission wavelength of said first Formula I induces the fluorescence of said second Formula I or the emission wavelength of said second Formula I induces the fluorescence of said first Formula I; (c) contacting said cell or said sample with i) said first Formula I and said second Formula I, ii) said first Formula I alone, iii) or said second Formula I alone, whereby said nucleic acid aptamer binds to said first Formula I and increases its fluorescence intensity, and whereby said detectable protein binds to said second Formula I and increases its fluorescence intensity; and 45 WO 2007/086900 PCT/US2006/012942 (d) measuring said fluorescence intensity of said first Formula I in the presence and absence of said second Formula I or measuring said fluorescence intensity of said second Formula I in the presence and absence of said first Formula I, whereby said nucleic acid of interest is determined to interact with said protein of interest if fluorescence resonance energy transfer occurs between said first Formula I and said second Formula I.

19. The method according to Claim 18 further comprising: (a) expressing in said cell or said sample a fusion protein comprising said protein of interest covalently linked to a detectable protein with intrinsic fluorescence, wherein the emission wavelength of said Formula I is different from that of said detectable protein, and wherein the emission wavelength of said Formula I induces the fluorescence of said detectable protein, or wherein the emission wavelength of said detectable protein induces the fluorescence of said Formula I; (b) contacting said cell or said sample with said Formula I, whereby said nucleic acid aptamer binds to said Formula I and increases its fluorescence intensity; and (c) measuring said fluorescence intensity of said Formula I in the presence and absence of said detectable protein or measuring said fluorescence intensity of said detectable protein in the presence and absence of said Formula I, whereby said nucleic acid of interest is determined to interact with said protein of interest if fluorescence resonance energy transfer occurs between said Formula I and said detectable protein.

20. A method for determining the presence or absence of one or more target compounds in a sample, wherein said compound is a fluorescent molecule of a compound of Formula I R N N/R3 1/X- ---- R2 R- R4I wherein for Formula I, the NRIR 2 and NR 3 R 4 moieties are in the ortho, meta or para positions; X" is an anionic salt; 46 WO 2007/086900 PCT/US2006/012942 R 1 , R 2 , R 3 , or R 4 are independently selected from the group consisting of methyl, ethyl, C 1 - 1 0 alkyl (linear or branched), alkenes (linear or branched), or wherein R 1 and R 2 or R 3 and R 4 taken together with the nitrogen atom to which they are attached form pyrrolidino or piperidino rings; R5 is selected from the group consisting of methyl, ethyl, C-10o alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, an organometallic compound, a polyalkylene glycol moiety, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; and the method comprising the steps of: providing a plurality of electrophoretic probes specific for the one or more target compounds, each electrophoretic probe having a target-binding moiety; combining with the sample the plurality of electrophoretic probes such that in the presence of a target compound a complex is formed between each target compound and one or more electrophoretic probes specific therefor; and separating and identifying the compounds to determine the presence or absence of the one or more target compounds.

21. A kit for staining cells, analytes, nucleic acids or microorganisms in a sample, comprising: (a) a staining mixture comprising one or more dyes to form a combined mixture; wherein at least one dye has the Formula I R, N R3 N--- - -- N R2 )C R, R4 wherein for Formula I, the NRR 2 and NR 3 R 4 moieties are in the ortho, meta or para positions; X is an anionic salt; R 1 , R 2 , R 3 , or R 4 are independently selected from the group consisting of methyl, ethyl, CI-o alkyl (linear or branched), alkenes (linear or branched), or wherein R 1 and R 2 or 47 WO 2007/086900 PCT/US2006/012942 R 3 and R 4 taken together with the nitrogen atom to which they are attached form pyrrolidino or piperidino rings; R 5 is selected from the group consisting of methyl, ethyl, C 1 - 1 0 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, an organometallic compound, a polyalkylene glycol moiety, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; and b) instructions for combining said dye or dyes with a sample comprising cells, analytes, nucleic acids and/or microorganisms, said instructions comprising i) combining a sample of cells, analytes, nucleic acids and/or microorganisms with a staining mixture comprising said at least one dye or dyes to form a combined mixture; and ii) incubating the combined mixture for a time sufficient for the dye in the staining mixture to associate with the cells, analytes, nucleic acids or microorganisms in the sample mixture to form stained cells, analytes, nucleic acids or microorganisms complex that gives a detectable optical response upon illumination.

22. The kit of claim 21, wherein said kit is used for cell differentiation.

23. A compound of Formula I RI\ R R N N+R R2 XR R R4 wherein for Formula I, the NR 1 R 2 and NR 3 R 4 moieties are in the ortho, meta or para positions; X- is an anionic salt; R 1 , R 2 , R 3 , or R 4 are independently selected from the group consisting of methyl, ethyl, CI-o 10 alkyl (linear or branched), alkenes (linear or branched), or wherein R, and R 2 or R 3 and R 4 taken together with the nitrogen atom to which they are attached form pyrrolidino or piperidino rings; 48 WO 2007/086900 PCT/US2006/012942 R 5 is selected from the group consisting of methyl, ethyl, C 1 i- 0 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, an organometallic compound, a polyalkylene glycol moiety, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; and said compound is encapsulated or pegylated.

24. The compound of claim 23, wherein said encapsulated or pegylated compound is water soluble.

25. The compound of claim 23, wherein said compound fluoresces in a range from about 400 nm to 700 nm. 49