METHODS AND COMPOSITIONS FOR VISUALIZATION OF FLUORESCENT PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U. S. C. § 119 (e), this application claims priority to the filing dates of: United States Provisional Patent Application Serial No. 61/170,932 filed April 20, 2009 and United States Provisional Patent Application Serial No. 61/222,052 filed on June 30, 2009; the disclosures of which are herein incorporated by reference.

INTRODUCTION

GFP from hydromedusa Aequorea aequorea (synonym A. victoria), as reported in Johnson et al., J. Cell Comp. Physiol. (1962) 60:85-104, was found as a part of bioluminescent system of the jellyfish where GFP played the role of a secondary emitter transforming blue light from the photoprotein aequorin into green light. cDNA encoding A. victoria GFP (avGFP) was cloned as reported in Prasher et al., Gene (1992) 11 1 :229-33. The amino acid sequence of avGFP is provides herein as SEQ ID NO:01. It was discovered that this gene can be heterologically expressed in practically any organism due to the unique ability of GFP to form the chromophore by itself, as reported in Chalfie et al., Gene (1992) 111 :229-233. This finding has enabled broad applications for the use of GFP in ceil biology as a genetically encoded fluorescent label.

Following the above summarized discovery of GFP, genes encoding fluorescent proteins were then cloned from organisms of different systematic groups including, but not limited to: Hydrozoa, Anthozoa, Arthropoda (Copepoda) and Chordrata (Brachiostoma), e.g., as reported in: Matz et al., Nat. Biotechnol. (1999) 17: 969-973; Chudakov et al., Trends Biotechnol. (2005) 23: 605-613; Shagin et al., MoI. Biol. Evol. (2004) 21 : 841-850; Masudaet al., Gene (2006) 372: 18-25; Deheyn et al., Biol. Bull. (2007) 213: 95-100; and Baumann et al., Biol. Direct. (2008) 3: 28. Currently, the fluorescent protein (FP) family (also referred to in the art as the "GFP family") includes hundreds member proteins. While these proteins may collectively be referred to as members of the "GFP family", where emission maxima may vary widely in terms of wavelength, and therefore not all members of the family have a green emission maximum. FP of the "GFP family" of proteins share a common GFP-like domain with a chromophore group. This domain can be easily identified in the protein amino acid sequence using available software for analysis of domain organization, e.g., by using the Conserved Domain Database (CDD) program available at the website formed by placed "http://www." in front of "ncbi.nlm.nih.gov/Structure/cdd/" and the SMART(a Simple Modular Architecture Research Tool) program available at the website formed by placing "http://smart." in front of "embl-heidelberg.de/".

Fluorescent proteins emit fluorescence upon irradiation with excitation light at an appropriate wavelength. This capacity is conditioned by the interaction of three or more amino acid residues in the chromophore, which chromophore is formed autocatalytically within the fluorescent protein domain. Because the chromophore is formed autocatalytically, no enzymes, cofactors and/or substrates are required for chromophore formation and fluorescence, with the exception of molecular oxygen. Three-dimensional structures have been obtained for several GFP family members. All family members share a common three-dimensional structure called a β-can, which structure includes an 11 -stranded β-barrel enclosing an α- helix (see e.g., Ormo et al., Science (1996) 273: 1392-1395; Wall et al., Nat. Struct. Biol. (2000), 7: 1133-1 138; Yarbrough et al., Proc. Nat'l Acad. Sci. U S A (2001 ) 98: 462-467; Prescott et al., Structure (Camb) (2003) 11 : 275-284;

Petersen et al., J. Biol. Chem. (2003) 278: 44626-44631 ; Wilmann et al., J. Biol Chem (2005), 280: 2401-2404; Remington et al., Biochemistry (2005) 44: 202- 212; and Quillin et al., Biochemistry (2005) 44: 5774-5787). The chromophore is located in the central region of the α-helix and is formed by amino acids corresponding to the Ser65-Tyr66-Gly67 region of avGFP. Corresponding amino acids in fluorescent proteins other than avGFP can be determined using alignment of the amino acid sequence of a protein under examination with avGFP, e.g., as described in Matz et al., Nat. Biotechnol. (1999) 17: 969-973.

It has been demonstrated that the green chromophore in GFP is formed by cyclization of the protein backbone in the Ser65-Tyr66-Gly67 region, followed by dehydrogenation of the Cα-Cβ bond of Tyr66. As a result, a bicyclic structure of 5-(4-hydroxybenzylidene)-3,5-dihydro-4H-imidazol-4-one is formed, in which the six-member aromatic ring of the Tyr66 side chain is linked to an unusual five- member heterocycle, which itself originates from condensation of the carbonyl carbon of Ser65 with the nitrogen of Gly67 (see e.g., Heim et al., Proc Nat'l Acad. Sci USA. (1994) 91 :12501-12504; Ormo et al., Science (1996) 273:1392-1395; and Yang et al., Nat. Biotechnoi. (1996) 14:1246-1251). All of the green proteins apparently possess the avGFP-like chromophore, and the differences in the spectral shapes are explained by modifications of protein's environment (see e.g., Brejc et al., Proc. Nat'l Acad. Sci. USA (1997) 94: 2306-2311 ; Palm et ai., Nat. Struct. Biol. (1997) 4:361-365; and Gurskaya et al., BMC Biochem. (2001) 2:6.

Further chemical modifications of the green chromophore occur in red- shifted GFP-like proteins. In particular, oxidation of a Ca-N bond at position 65 (avGFP numbering) results in an acyiimine group conjugated to a GFP-like core in DsRed (see Gross et al., Proc. Nat'l Acad. Sci USA (2000) 97:11990-11995; Wall et al., Nat. Struct. Biol. (2000) 7:1133-1138; and Yarbrough et al., Proc. Nafl Acad. Sci. USA (2001 ) 98:462-467). The DsRed-like chromophore is formed within many other proteins with red-shifted absorption and fluorescence (See e.g., Pakhomov, A. A. and Martynov, V. I., Chem. Bioi. (2008) 15: 755- 764). In some proteins, the acyiimine moiety of the DsRed chromophore is further attacked by various nucleophiles to form additional types of red-shifted chromophores. For example, the chromophore in the purple chromoprotein asFP595 is formed by hydrolysis of the acyiimine group, resulting in cleavage of the protein backbone and formation of a keto group conjugated to a GFP-like chromophore core (see e.g., Quillinet al., Biochemistry ( 2005) 44: 5774- 5787; and Yampolsky et al., Biochemistry ( 2005) 44: 5788- 5793). In the orange fluorescent proteins mOrange and mKO, nucleophilic addition of Thr65 (in mOrange) or Cys65 (in mKO) side chain groups leads to unusual heterocycles without protein backbone scission (see e.g., Shu et al., Biochemistry ( 2006) 45: 9639- 9647 and Kikuchi et al., Biochemistry ( 2008) 47: 11573- 11580).

The yellow fluorescent protein zFP538 from the button polyp Zoanthus demonstrates unique spectra! characteristics that are intermediate between those of the green and orange-red fluorescent proteins (λmax.ex = 528 nm, and λmax.em = 538 nm) (Mate et al., Nat. Biotechnol. (1999) 17: 969- 973). The chromophore-forming residues in zFP538 are Lys-Tyr-Gly (Yampolsky et al., Biochemistry (2009) 48: 8077-8082). Several amino acid substitutions have been found to strongly affect fluorescence maxima of FPs. These positions crucial for fluorescence of particular color can be found by sequence comparison of fluorescent proteins of different colors. In many cases, one amino acid substitution is required to produce a green fluorescent protein from the red FP (see e.g., Gurskaya et al., BMC Biochemistry (2001 ) 2:6).

Some fluorescent proteins are photoactivatable, photoconvertable or photoswitchable. The fluorescent characteristics of these proteins can be altered by light energy. One of the first observations of photoswitching in FPs was reported in wtGFP and several of its enhanced derivatives (Dickson et al., Nature (1997) 388: 355-358). Photoactivatable, photoconvertable and photoswitchable fluorescent proteins can be switched on at a particular time and location within the cell to track the behavior of a tagged protein. Photoactivatable FPs are capable of being activated from very low ievel to bright fluorescence emission upon illumination with ultraviolet or violet light, whereas photoconvertable FPs can be optically converted from one fluorescence emission bandwidth to another. In contrast, photoswitchable FPs have emission characteristics that can alternatively be turned on or off with specific illumination (see e.g., Day and Davidson, Chem. Soc. Rev. (2009) 38: 2887 - 2921 ). Photoactivation of PA-GFP and photoswitching of PS-CFP2 is believed to occur due to decarboxylation of Glu222 followed by conversion of the chromophore from a neutral to anionic state. Green to red photoconversion for proteins having HYG chromophore (see e.g., Kaede, KikGR, Dendra2, and EosFP) occurs when the FP is illuminated with ultraviolet or violet radiation (405 nm) to induce cleavage between the amide nitrogen and α-carbon atoms in the histidine 62 residue leading to subsequent formation of a conjugated dual imidazole ring system. Photoswitching of Dronpa, mTFP0.7 and KFP1 involves cis-trans photoisomerization induced by alternating radiation between 405 nm and 488 nm (see e.g., Day and Davidson, Chem. Soc. Rev. (2009) 38: 2887 - 2921 ).

Fluorescent proteins currently find extensive use as fluorescent markers in the biomedical sciences, e.g., as discussed in detail by Lippincott-Schwartz and Patterson, Science (2003) 300:87-91 and Stepanenko et al., Curr. Protein Pept. Sci. (2008) 9:338-369). Fluorescent proteins find use in a wide range of applications, including the study of gene expression and protein localization (see e.g., Chalfie et al., Science (1994) 263: 802-805 and Heim et al., in Proc. Nat'l Acad. Sci. (1994) 91 : 12501 -12504); as a tool for visualizing sub-cellular organelles in cells (see e.g., Rizzuto et al., Curr. Biology (1995) 5: 635-642); and for the visualization of protein transport along the secretory pathways (see e.g., Kaether and Gerdes, FEBS Letters (1995) 369: 267-271 ).

For example, fluorescent proteins find use as reporter molecules in cell and molecular biology assays. The assays of interest include, but are not limited to, assays for gene expression, protein localization and co-localization, protein- protein interactions, protein-nucleic acid interactions, nucleic acid-nucleic acid interactions, cell and cell organelle localization and interactions, etc. Fluorescent proteins find use as a biomolecule labels and cell organelle labels in living and fixed cells; as a markers in cell or organelle fusion; as cell or organelle integrity markers; as transfection markers (e.g., as labels for selection of transfected cells containing an expression vector encoding at least one fluorescent protein of the invention); as real-time probes working at near physiological concentrations; etc. For example, fluorescent proteins find use in applications involving the automated screening of arrays of cells expressing fluorescent reporting groups by using microscopic imaging and electronic analysis. Screening can be used for drug discovery and in the field of functional genomics where the subject proteins are used as markers of whole cells to detect changes in multi-cellular reorganization and migration, for example in the formation of multi-cellular tubules (blood vessel formation) by endothelial cells, migration of celis through the Fluoroblok Insert system (Becton Dickinson Co.), wound healing, or neurite outgrowth. Screening can also be employed where the proteins of the present invention are used as markers fused to peptides (such as targeting sequences) or proteins that detect changes in intracellular location as an indicator for cellular activity, for example in signal transduction, such as kinase and transcription factor translocation upon stimuli. Examples include protein kinase C, protein kinase A, transcription factor NFkB, and NFAT; cell cycle proteins, such as cyclin A, cyclin B1 and cyclin E; protease cleavage with subsequent movement of cleaved substrate; phospholipids, with markers for intracellular structures such as the endoplasmic reticulum, Golgi apparatus, mitochondria, peroxisomes, nucleus, nucleoli, plasma membrane, histones, endosomes, lysosomes, or microtubules. Fluorescent proteins are also used in high content screening applications to detect co-localization of other fluorescent fusion proteins with localization markers as indicators of movements of intracellular fluorescent proteins/peptides or as markers alone. Examples of applications involving the automated screening of arrays of cells in which the subject fluorescent proteins find use include U.S. Patent No. 5,989,835; as well as WO 0017624; WO 00/26408; WO 00/17643; and WO 00/03246.

Fluorescent proteins also find use in high throughput screening assays. For example, destabilized versions of the subject fluorescent proteins with decreased half-lives are used as transcription reporters for drug discovery. For example, a fluorescent protein is fused with a putative proteolytic signal sequence derived from a protein with shorter half-life, such as a PEST sequence from the mouse ornithine decarboxylase gene, a mouse cyclin B1 destruction box or ubiquitin, etc. For a description of destabilized proteins and vectors that can be employed to produce the same, see e.g., U.S. Patent No. 6,130,313. Promoters in signal transduction pathways can be detected using destabilized versions of the subject fluorescent proteins for drug screening such as, for example, AP1 , NFAT, NFkB, Smad, STAT, p53, E2F, Rb, myc, CRE, ER, GR and TRE and the like.

Fluorescent proteins also find use as second messenger detectors by fusing the subject proteins to specific domains such as the PKCgamma Ca binding domain, PKCy DAG binding domain, SH2 domain or SH3 domain, etc. Fluorescent proteins also find use in fluorescence activated cell sorting (FACS) applications. In such applications, the fluorescent protein is used as a label to mark a population of ceils and the resulting labeled population of cells is then sorted with a fluorescent activated cell sorting device, as is known in the art. FACS methods are described in U.S. Patent Nos. 5,968,738 and 5,804,387; the disclosures of which are herein incorporated by reference.

Fluorescent proteins also find use as in vivo labels in transgenic animals. For example, expression of a fluorescent protein can be driven by tissue-specific promoters, where such methods find use in research for gene therapy, such as testing efficiency of transgenic expression, among other applications. One application of fluorescent proteins in transgenic animals that illustrates such applications is found in WO 00/02997.

Fluorescent proteins also find use as biosensors in prokaryotic and eukaryotic cells, such as a Ca ²⁺ ion indicator; a pH indicator; a phosphorylation indicator; or as an indicator of other ions, such as magnesium, sodium, potassium, chloride and halides. Methods of using fluorescent proteins as biosensors also include those described in U.S. Patent Nos. 5,972,638; 5,824,485 and 5,650,135 (as well as the references cited therein) the disclosures of which are herein incorporated by reference. Additional applications in which fluorescent proteins find use include the use of fluorescent proteins as markers following injection of purified recombinant proteins into cells or animals and in calibration for quantitative measurements; as markers or reporters in oxygen biosensor devices for monitoring cell viability; as markers or labels for animals, pets, toys, food, and the like. To enhance the use of fluorescent proteins in many applications, the wild- type fluorescent proteins are subjected to mutagenesis to achieve one or more desirable features, such as to increase fluorescence intensity and maturation rate at 37°C, to modify excitation and emission spectra, and/or to reduce aggregation and oligomerization capacity, etc. Moreover, codon usage has been optimized for high expression in a desired heterological system, for example in mammalian cells (Haas, et a!., Current Biology (1996), 6: 315-324; Yang, et al., Nucleic Acids Research (1996), 24: 4592-4593).

One characteristic of fluorescent proteins that impacts their use in a variety of applications is "photostability". Photochemical destruction of the chromophore is assumed to be the main reason for fluorescence interruption, although it has been shown that fluorescence can be recovered by the decay of exceptionally long-living dark states (see e.g., Jung et al., Bioimaging (1998) 6: 54-61 ). Photobleaching therefore subsumes these processes, and the molecular quality "photostability" characterizes the ability of different FPs to withstand these processes. Photostability of a fluorescent protein mainly depends on the chromophore structure, protein environment of the chromophore, and presence of molecular oxygen (see e.g., Jung et al., Bioimaging (1998) 6: 54-61 ; and Jung et al., Biophys. J. (2005) 88:1932-1947). Enhancement of photostability of fluorescent proteins is desirable because such enhance affords the ability to increase the time of FP visualization and increase the number of applications in which FPs find use.

SUMMARY

Cell visualization mediums formulated to modify the photostability of a fluorescent protein associated with a cell when that cell is present in the medium are provided. The visualization mediums are based on the inventors' surprising discovery that the composition of a cell medium used during cell visualization can influence photostabiiity and photobleaching of fluorescent proteins (FPs) that are associated with, e.g., expressed in or injected into, host-cells. Also provided are methods of using the cell visualization mediums, e.g., in visualization methods, as well as kits for use in the practicing embodiments of the invention. As reviewed in greater detail below, aspects of the invention include visualization media which have a concentration of general organic oxidants that results in an increase in the photostability of a fluorescent protein. In yet other embodiments, visualization media are provided in which a change in the concentration of general organic oxidants in the cell medium results in a decrease in the photostability of a fluorescent protein.

In certain embodiments, the riboflavin concentration of the visualization media is decreased in the visualization medium (e.g., as seen in embodiments of visualization medium VM1), as compared to a control, such as DMEM. In some embodiments, the riboflavin concentration is 0.01 mg/L or less, such as 0.005 mg/L or less. !n certain embodiments, the visualization medium does not include riboflavin.

In some embodiments, the concentration of vitamin B6 concentration is also decreased in the visualization medium (e.g., as seen in certain embodiment of VM 1 ) as compared to a control, such as DMEM. In some instances, the vitamin B6 (e.g., any form thereof, including pyhdoxine, pyridoxamine, and pyridoxal) concentration is 0.01 mg/L or less, such as 0.005 mg/L or iess. In some instances, the visualization medium does not include vitamin B6.

In some instances, nicotinamide is present in the visualization medium in a concentration ranging from 0 to 40 mg/L. For example, the concentration of nicotinamide may be 0.01 mg/L or more, such as 1 mg/L or more, including 2 mg/L (for example 3 mg/L; 4 mg/L) or more. In some instances, the concentration of nicotinamide is 4 mg/L or more and can be up to 40 mg/L or more. In some embodiments, the nicotinamide is absent in the visualization medium. Visualization media of the invention find use with a variety of different fluorescent proteins. Fluorescent proteins for which photostability is increased when visualization is performed in visualization media of the invention with decreased concentration of organic oxidants (e.g., riboflavin and\or vitamin B6), such as VM1 , include, but are not limited to, green fluorescent proteins, e.g., Aequorea victoria GFP and mutants thereof, green fluorescent mutants of Aequorea coerulescens GFP-like protein, Aequorea macrodactyta GFP and mutants thereof, Copepoda green fluorescent proteins and mutants thereof, Anthozoa green fluorescent proteins and mutants thereof, Brachiostoma green fluorescent proteins and mutants thereof (for example Aequorea victoria GFP (SEQ ID NO:01), EGFP (SEQ ID NO:02), AcGFPI (SEQ ID NO: 05), mTagGFP (also known as TagGFP2, SEQ ID NO:06); and photoactivatable and photo switch able fluorescent proteins, for example PA-GFP (SEQ ID NO: 15), PS- CFP, PS-CFP2 (SEQ ID NO:04), Dendra, Dendra2 (SEQ ID NO:08) and homologs of these proteins. In certain embodiments, the fluorescent proteins exhibit green fluorescence. In certain embodiments, the fluorescent proteins comprise a Tyr amino acid residue at the position corresponding to Tyr66 of avGFP and have avGFP-like chromophore.

Examples of fluorescent proteins in which photostability is decreased when visualization is performed in the visualization media that have decreased concentrations of organic oxidants (e.g., riboflavin and\or vitamin B6) include, but not limited to, TagBFP (also known as mTagBFP, SEQ ID NO: 12) and related proteins. These proteins are characterized by an unusual type of chromophore, N-[(5-hydroxy-1H-imidazole-2-yl)methylidene]acetamide, which contains the N- acylimine but does not have the Ca-Cb double bond in the Tyr64 side chain and are referred to herein as comprising a TagBFP-type chromophore. The chemical pathway for the formation of the DsRed-like chromophore, such as that observed in TagRFP via the TagBFP-like intermediate has been proposed (Subach et al., Chemistry & Biology, 2010, doi: 10.1016 /j.chembiol.2010.03.005, in press).

In other embodiments, the riboflavin concentration of the visualization media is increased relative to a control (see e.g., visualization medium VM2). In certain of these embodiments, riboflavin concentration ranges from 2-40 mg/L (such as example 4 mg/L or more; 6 mg/L or more; 10 mg/L or more).

In certain embodiments, the visualization medium comprises vitamin B6 (any form) in a concentration ranging from 2 to 40 mg/L (such as 4 mg/L or more; 6 mg/L or more; 10 mg/L or more), e.g., as found in certain embodiments of VM2. In some embodiments, nicotinamide is present in the visualization medium in concentration that ranges from 0 to 4 mg/L. For example, the concentration of nicotinamide may be 0.01 mg/L or more, such as 1 mg/L or more, and including 2 mg/L or more (for example 3 mg/L; 4 mg/L). In some embodiments, the nicotinamide is absent in the visualization medium. Such embodiments include certain instances of VM2, described in greater detail below. Examples of fluorescent proteins in which photostability is decreased in media that include enhanced amounts of riboflavine, e.g., embodiments of medium VM2, include, but are not limited to: green fluorescent proteins, for example Aequorea victoria GFP and mutants thereof, green fluorescent mutants of Aequorea coerulβscens GFP-like protein, Aequorea macrodactyla GFP and mutants thereof, Copepoda green fluorescent proteins and mutants thereof, Anthozoa green fluorescent proteins and mutants thereof, Brachiostoma green fluorescent proteins and mutants thereof (for example Aequorea victoria GFP, EGFP, AcGFPI , TagGFP2); and photoactivatable and photoswitchable fluorescent proteins, for example PA-GFP, PS-CFP, PS-CFP2, Dendra, Dendra2 and homologs of these proteins. In certain embodiments, the fluorescent proteins exhibit green fluorescence. In certain embodiments, the fluorescent proteins comprise Tyr amino acid residue at the position corresponding to Tyr66 of avGFP and has avGFP-ϋke chromophore.

Examples of proteins for which photostability is increased when visualization is performed in visualization media having increased concenrations of riboflavin, e.g., embodiments of VM2, include, but are not limited to, TagBFP and related proteins, e.g., proteins that include a TagBFP type chromophore, as reviewed in greater detail above.

In some embodiments, the visualization media having increased concentrations of organic oxidants (e.g., riboflavin and\or vitamin B6) are used to enhance photo-conversion of a fluorescent protein to another spectrally detectable form. Examples of such photo-conversion events include, but are not limited to, green-to-red photoconversions of Aequorea victoria GFP, EGFP, AcGFPI , TagGFP2, Dendra2, and other green fluorescent proteins. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 (parts 1-3) shows multiple sequence alignments of several fluorescent proteins. Conservative amino acids are marked by black. GFP-like domain start and finish are specified at the top by square brackets ("["and "]") Chromophore is marked by pluses ("+++"). Figure 1 part 1 shows multiple sequence alignments of FP sequences that correspond to amino acid residues from 1 to 102 of avGFP; figure 1 part 2 shows multiple sequence alignments of FP sequences that correspond to amino acid residues from 103 to 234 of avGFP; figure 1 part 3 shows multiple sequence alignments of FP sequences that correspond to amino acid residues from 235 to 238 of avGFP.

Figure 2 illustrates chemical structures of riboflavin (vitamin B2), D-Ca pantothenate (vitamin B5), choline chloride (vitamin B4), folic acid (vitamin B9), niacinamide (vitamin B3), vitamin B6 (pyridoxal and pyridoxine), and thiamine HCI (vitamin B1 ). Figure 3 illustrates influence of cell medium on fluorescent protein photostability. The graph shows normalized bleaching curves for EGFP in live HEK293T cells maintained in DMEM (squares), DMEM-RF (circles), or DMEM-V (triangles). Standard deviations (n = 20 cells) are shown. The EGFP-N 1 plasmid was used for transfection. Figure 4 illustrates influence of cell medium on EGFP-tubulin fusion protein photostability. The graph shows normalized bleaching curves for EGFP-tubulin in live HEK293T cells maintained in DMEM (squares), DMEM-RF (circles), or DMEM-V (triangles). Standard deviations (n = 15-20 cells) are shown. Figure 5 illustrates influence of cell medium on photostability of mitochondria targeted EGFP. The graph shows normalized bleaching curves of the fluorescent protein in live HEK293T cells maintained in DMEM (squares), DMEM-Rf (circles), or DMEM-V (triangles). Standard deviations (n = 15-20 cells) are shown.

Figure 6 illustrates the influence of cell medium on the photostability of AcGFPI . The graph shows normalized bleaching curves of the fluorescent protein in live HEK293T cells maintained in DMEM (squares), DMEM-Rf (circles), or DMEM-V (triangles). Standard deviations (n = 15-20 cells) are shown.

Figure 7 illustrates influence of cell medium on photostability of TagGFP2. The graph shows normalized bleaching curves of the fluorescent protein in live HEK293T cells maintained in DMEM (squares), DMEM-Rf (circles), or DMEM-V (triangles). Standard deviations (n = 15-20 cells) are shown.

Figure 8 illustrates signal intensity of normalized green fluorescence bleaching curves for Phoenix eco-produced EGFP. Each curve depicts bleaching with different medium (pointed above the curve). EGFP-N 1 plasmid had been used for transfection.

Figure 9 illustrates the influence of folic acid on the photostability of EGFP. The graph shows normalized bleaching curves of the fluorescent protein in live HEK293T cells maintained in DMEM-V (black squares) or in DMEM-V supplemented with folic acid (4mg/i_) (black circles). Standard deviations (n = 15- 20 cells) are shown.

Figure 10 illustrates the influence of choline on the photostability of EGFP. The graph shows the normalized bleaching curves of the fluorescent protein in live HEK293T cells maintained in DMEM-V (black squares) or in DMEM-V supplemented with choline chloride (4 mg/L) (black circles). Standard deviations (n = 15-20 cells) are shown.

Figure 11 illustrates influence of riboflavin on the photostability of EGFP. The graph shows normalized bleaching curves of the fluorescent protein in live HEK293T ceils maintained in DMEM-V (black squares) or in DMEM-V supplemented with riboflavin (0.4 mg/L) (black circles). Standard deviations (n = 15-20 cells) are shown.

Figure 12 illustrates the influence of pyridoxal on the photostability of EGFP. The graph shows normalized bleaching curves of the fluorescent protein in live HEK293T cells maintained in DMEM-V (black squares) or in DMEM-V supplemented with pyridoxal (4 mg/L) (black circles). Standard deviations (n = 15-20 cells) are shown. Figure 13 illustrates the influence of nicotinamide on the photostability of EGFP. The graph shows normalized bleaching curves of the fluorescent protein in live HEK293T cells maintained in DMEM-V (black squares) or in DMEM-V supplemented with nicotinamide (4 mg/L) (black circles). Standard deviations (n = 15-20 cells) are shown.

Figure 14 shows the fluorescent microscopy of cytoskeletal proteins of live HeLa cells after five days cultivation in DMEM-V. Figure 14, left column shows TagGFP2-tagged α-tubulin fusion expressed in HeLa cells; Figure 14, right column shows mKate2-tagged β-actin fusion expressed in HeLa cells. Figure 15 shows Scratch wound healing assay on REF52 cells in different media. Cells are shown just after scratching (upper panels) and 10 h later (bottom panels) maintained in DMEM (left panels) or DMEM-V (right panels).

DEFINITIONS In accordance with various embodiments of the invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and Il (D.N. Glover ed. 1985); Oligonucleotide Synthesis" (MJ. Gait ed. 1984); "Nucleic Acid Hybridization" (B.D. Hames & SJ. Higgins eds. (1985)); "Transcription and Translation" (B.D. Hames & SJ. Higgins eds. (1984)); "Animal Cell Culture" (R.I. Freshney, ed. (1986)); "Immobilized Cells and Enzymes" (iRL Press, (1986)); B. Perbal, "A Practical Guide To Molecular Cloning" (1984). A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. A DNA "coding sequence" is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3 ¹ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. A polyadenylation signal and transcription termination sequence may be located 3' to the coding sequence.

"DNA regulatory sequences", as used herein, are transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for and/or regulate expression of a coding sequence in a host cell.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. A promoter sequence may be bounded at its 3' terminus by the transcription initiation site and extend upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including inducible promoters, may be used to drive the various vectors of the present invention.

A cell has been "transformed", "transfected" or "transduced" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal (extrachromosomal) element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, heterologous DNA includes coding sequence in a construct where portions of genes from two different sources have been brought together so as to produce a fusion protein product. Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

As used herein, the term "reporter gene" refers to a coding sequence attached to heterologous promoter or enhancer elements and whose product may be assayed easily and quantifiably when the construct is introduced into tissues or cells.

The amino acids described herein may be in the "L" isomeric form. The amino acid sequences are given in three-letter or one-letter code (A: alanine; C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan; Y: tyrosine; X: any residue). NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J Biol. Chem., 243 (1969), 3552-59 is used. As used herein the term "isolated" is meant to describe a nucleic acid, a polypeptide, an antibody, or a host cell that is in an environment different from that in which the polynucleotide, the polypeptide, the antibody, or the host cell naturally occurs.

As used herein the term "fluorescent protein" or "fluoroprotein" means a protein of GFP family that is fluorescent; e.g., it may exhibit low, medium or intense fluorescence upon irradiation with light of the appropriate excitation wavelength. The fluorescent characteristic of these proteins is one that arises from the interaction of three or more amino acid residues of the protein, and not from a single amino acid residue. As such, the fluorescent proteins of the present invention do not include proteins that exhibit fluorescence only from residues that act by themselves as intrinsic fluors, i.e., tryptophan, tyrosine and phenylalanine.

As used herein the term "fragment" is meant to comprise e. g. an alternatively spliced, or truncated, or otherwise cleaved nucleic acid molecule or protein.

As used herein the term "mutant" refers to protein disclosed in the present invention, in which one or more amino acids are added and/or substituted and/or deleted and/or inserted at the N-terminus, and/or the C-terminus, and/or within the native amino acid sequences of the proteins of the present invention. As used herein the term "mutant" refers to nucleic acid molecule that encodes a mutant protein. Moreover, the term "mutant" refers to any shorter or longer version of the protein or nucleic acid herein.

As used herein, "homologue or homology" is a term used in the art to describe the relatedness of a nucleotide or peptide sequence to another nucleotide or peptide sequence, which is determined by the degree of identity and/or similarity between said sequences compared. As used herein, "fluorescent property" or "spectral property" refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or emission spectrum, the excitation wavelength maximum and emission wavelength maximum, the ratio of excitation amplitudes at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime, or the fluorescence anisotropy. A measurable difference in any one of these properties of reference protein in different conditions is useful. A measurable difference can be determined as the amount of any quantitative fluorescent property, e.g., the amount of fluorescence at a particular wavelength, or the integral of fluorescence over the emission spectrum. The terms "cell culture medium" and "culture medium" refer to the aqueous environment in which eukaryotic cells are grown in culture. The medium comprises the physiochemical, nutritional, and hormonal environment. Traditionally the medium has been formulated by the addition of nutritional and growth factors necessary for cell growth or survival. The terms "ceil visualization medium" and "visualization medium" refer to the aqueous environment in which eukaryotic cells are visualized using common methods to test fluorescence in cells, e.g., fluorescence microscopy, spectrophotometry, spectrofluorometry, fluorescence-activated flow cytometry. In some embodiments, cell visualization medium is used in fluorescence microscopy.

The term "oxidant" refers to a molecule that can accept one or more electrons from another reagent in aredox chemical reaction. For example, the term "oxidant" refers to riboflavin (vitamin B2) and vitamin B6.

"Host cell" refers to a cell into which has been introduced (e.g., transformed or transfected) an exogenous polynucleotide sequence, i.e. a heterologogus nucleic acid molecule. Host cells are typically eukaryotic cells such as yeast, insect, amphibian, green plant, or mammalian cells and may be prokaryotic cells such as bacteria, e.g., Escherichia coii, where the relevant exogenous genes exist.

DETAILED DESCRIPTION

Cell visualization mediums formulated to modify the photostability of a fluorescent protein associated with a cell when present in the medium are provided. Also provided are methods of using the cell visualization mediums, e.g., in visualization methods, as well as kits for use in the practicing embodiments of the invention. Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

In further describing various aspects of the invention, the visualization media will be described first in greater detail, followed by a review of various embodiments of methods of using the media, as well as a review of examples of kit embodiments of the invention. CELL VISUALIZATION MEDIA

As summarized above, aspects of the invention include cell visualization media. Cell visualization media of the invention are cell culture media that are formutated to modify the photostability of a fluorescent protein as compared to a reference medium (e.g., a standard cell culture medium, such as Dulbecco's Modified Eagle's Medium (DMEM)).Specifically, the cell visualization media according to certain embodiments of the invention change the photostability of a fluorescent protein when associated with a cell (e.g., when inside of a cell) as compared to a suitable control, i.e., the same cell that includes the same fluorescent protein present in a standard cell culture medium, e.g., DMEM. As used herein, "photostability" is stability of protein fluorescence upon light irradiation under excitation wavelength. As used herein, "photobleaching" means the photochemical destruction of a fluorophore (chromophore) of fluorescent protein. The photostability rate is defined as a relative photobleaching speed. Photostability and photobleaching are characterized by fluorescence intensity decrease in course of irradiation by light of excitation wavelength and certain intensity.

Photobleaching and photostability may be measured using any convenient protocol, where protocols of interest include those described in: Chiu et al., J. Neurosci. Methods (2001 ) 105: 55-63); Wiedenmann et al., Proc. Nat'l Acad. Sci USA. (2002) 99:11646-11651 ; Rettig et al., Springer- Verlag (1999) p.206-207; Shaner et al., Nat. Biotechnol. (2004), 22:1567-1572; and Nat. Methods. (2005) 2:905-909). For example, photostability comparison between different FPs and/or the same FPs under various conditions can be performed using bleaching curves. These curves allow one to extract photobleaching half-time (i.e., irradiation duration needed to halve fluorescence intensity relative to the initial level) that is a measure of photostability reasonable for practical usage. Comparing photobleaching half-times calculated from bleaching curves normalized on the initial signal values allows one to compare the photostability. Longer photobleaching half-time means higher photostability. Photostability of FPs expressed in host-cells can be measured under various conditions (e.g., composition of the visualization medium) using a fluorescent microscope (such as inverted microscopes DMIRE2 and DMI 6000 B (Leica), 1X81 (Olympus), Axiovert 200M (Zeiss), Eclipse Ti (Nikon), and others) equipped with an appropriate excitation light source, such as arc mercury lamp, HXP short arc lamp, xenon-arc lamp, LEDs (Light Emitting Diodes), or a laser beam. Fluorescent intensity of a cell may be measured before and after bleaching by extensive irradiation with excitation light. After several rounds of sequential detection-bleaching (usually after 10 - 300 rounds, more usually after 20-200 rounds, and preferable after 30-100 rounds) bleaching curves may be prepared using data obtained from a detecting channel of the microscope and appropriate software. For comparison between different conditions or fluorescent proteins used, the curves are normalized to initial fluorescent signal.

In one embodiment, a Leica AF6000 LX imaging system based on a DMI 6000 B inverted microscope is used for photostability measurements of fluorescent proteins in host-cells with 63χ immersion oil objective (effective magnification ratio 630χ). A GFP filter cube can be used for green fluorescent proteins and TX2 filter cube can be used for red fluorescent proteins. The following procedure may be employed: a selected field of view (containing several fluorescent cells) is irradiated with appropriate filter set in series of detecting (e.g., intensity 1 , gain 1, exposure length 10-100 ms) and bleaching (e.g., intensity 5, gain 1 , exposure length 5 s) light. After 30-100 frames of detection/bleaching, one can draw the bleaching curves using data from detecting channel and appropriate software. Bleaching curves are normalized and compared.

The detection light allows one to measure the fluorescence signal value. It is a relatively low intensity light of fluorescence excitation wavelength. An intensity of detecting light may be calibrated with the output signal value in such a way as to fit this value into the dynamic range of CCD detector used. In each particular case, the detecting light intensity depends on the initial fluorescence brightness, microscope, detector and excitation light source type. Also detecting light does not considerably bleach the fluorescence (or its bleaching ability is as low as possible).

As used herein, "bleaching light" means a relatively high intensity (for example 1 W/cm ² ) light of fluorescence excitation wavelength. Its intensity may be chosen as a maximum available with a particular microscope. Output signal usually doesn't matter and is not acquired for the further calculations. Bleaching light should provide the effective bleaching of fluorescence (bleaching is controlled by the detecting channel data respectively).

In some cases, one can use bleaching light for both fluorescent protein bleaching and fluorescence signal measurement. "Normalization" means data reduction to unified, convenient for the further comparison form. Technically, to normalize bleaching curves acquired with the identical detection mode one may divide all the fluorescence signal values by the initial signal value.

Turning back to visualization media of the invention, the nature of the change in photostability that is achieved with visualization media of the invention may vary. In some instances, the photostability of a given fluorescent protein is enhanced by the visualization medium that is employed, as compare to a control. The magnitude of enhancement in stability may, in certain instances, be 1.3 fold or more, such as 2.0 fold or more, including 3.0 fold or more, 5.0 fold or more, and 9.0 fold or more. In some instances, the photostability of a given fluorescent protein is decreased by the visualization medium that is employed, as compared to a control. The magnitude of decrease in stability may, in certain instances, be 1.3 fold or more, such as 2.0 fold or more, including 3.0 fold or more and 5.0 fold or more. Visualization media according to aspects of the invention are culture media that have been modified to provide the desired impact on photostability, e.g., increase or decrease in photostability. The term "culture medium" is used according to its art accepted meaning to refer to aqueous compositions that generally include essential amino acids, salts, vitamins, trace metals, sugars, lipids and nucleosides. Cell culture media are compositions that supply the components to a cell necessary to meet the nutritional needs required to the cell in a controlled, artificial and in vitro environment. Nutrient formulations, pH, and osmolarity may vary in accordance with parameters such as ceil type, cell density, and the culture system employed. Many cell culture medium formulations are documented in the literature and a number of media are commercially available. In general, standard ceil culture media include riboflavin in amounts ranging from 0.1 to 1.0, and vitamin B6 in amounts ranging from 0.05 (e.g., 0.062) to 4.0.

Visualization media of embodiments of the invention may be variants of a variety of different culture media, including well defined and commercially available culture media. Cell culture medium of which the subject media are variants (i.e., may differ from in terms of one or more components, such as riboflavin and/or vitamin B6) may be any cell culture medium which adequately addresses the nutritional needs of cells being cultured therein. Examples of cell culture media of interest include, but are not limited to: Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12, RPM1 1640, Iscove's, McCoy's and other media formulations readily apparent to those skilled in the art, including media found in Methods For Preparation of Media, Supplements and Substrate For Serum-Free Animal CeIi Culture Alan R. Liss, New York (1984) and Cell &Tissue Culture: Laboratory Procedures, John Wiley & Sons Ltd., Chichester, England 1996, both of which are incorporated by reference herein in their entirety. In some instances, the cell culture media may be supplemented, e.g., with any components necessary to support the desired cell or tissue culture. Additionally serum, such as bovine serum, which is a complex solution of albumins, globulins, growth promoters and growth inhibitors may be added if desired. Hormone addition into the medium may or may not be desired.

As indicated above, visualization media according to embodiments of the invention may be variants of a cell culture medium. In certain embodiments, a given visualization medium of the invention comprises all of the components of a reference culture medium in the same concentration, except for general organic oxidants, e.g., riboflavin (vitamin B2), and optionally vitamin B6. In some embodiments, other vitamins including D-Ca pantothenate (vitamin B5), choline chloride (vitamin B4), folic acid (vitamin B9), niacinamide (vitamin B3), and thiamine HC! (vitamin B1 ) are also varied in concentration in the visualization medium, e.g., in comparison to a reference cell culture medium. Figure 2 provides chemical structures of these well-known components. As such, visualization media according to certain embodiments of the invention are aqueous compositions that include: essential amino acids; salts; trace metals; sugars; lipids and nucleosides.

Essential amino acids of interest include: L-arginine, L-cystine (or L- cysteine), L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L- phenylalanine, L-threonine, L-triptophan, L-tyrosine, L-valine and the like. Amino acids of interest may also include: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-glutamine, L-hydroxyproline, L-proline, L-serine, and the like. When present, the amount of individual essential amino acid in the visualization medium may range from 1 mg/L to 550 mg/L, such as 2 to 300 mg/L and including 5 to 150 mg/L.

Inorganic salts of interest include: Ca(NO ₃ ) ₂ *4H ₂ O; CaCI ₂ ; CaCI ₂ ^* 2H ₂ O, CaNO ₃ MH ₂ O; KCI; KH ₂ PO ₄ , KNO ₃ , MgCI ₂ ; MgCI ₂ ^* 6H ₂ O, MgSO ₄ ; MgSO ₄ VH ₂ O; NaHCO ₃ ; Na ₂ H ₂ PO ₄ VH ₂ O, Na ₂ HPO ₄ ; NaCI; NaH ₂ PO ₄ ; NaHCO ₃ and the like. When present, the amount of individual salt in the visualization medium may range from 8,000 to 1 mg/L depending on salt. For example, NaCI amount may range from 8000 mg/L to 800 mg/L, such as 6400 mg/L, 6800 mg/L, 6000 mg/L, 7599 mg/L. The amount of KCI, when present, may range from 500 mg/L to 150 mg/L, such as 400 mg/L and 224 mg/L. The amount of CaCI ₂ , when present, may range from 270 mg/L to 30 mg/L, such as 200 mg/L and 33.2 mg/L. The amount of MgSO ₄ *7H ₂ O, when present, may range from 400 mg/L to 90 mg/L, such as 100 mg/L and 200 mg/L. The amount of MgSO ₄ (anhydrous), when present, may range from 110 mg/L to 30 mg/L. The amount of NaHCO ₃ , when present, may range from 3700 mg/L to 350 mg/L, such as 2240 mg/L, 2200 mg/L, 1176 mg/L, and 1 125 mg/L. The amount of NaH ₂ PO ₄ , Na ₂ H ₂ PO ₄ VH ₂ O, Na ₂ HPO ₄ , and/or NaH ₂ PO ₄ , when present, may range from 47 mg/L to 1512 mg/L, such as 60 to 600 mg/L and including 78 to 140 mg/L. KNO3, when present, may range in concentration from 0.02 mg/L to 0.006 mg/L. The amount of MgCb, when present, may range from 60 mg/L to 15 mg/L.

Inorganic metal salts, commonly called trace metals or trace elements, may include: CuSO ₄ ; FeSO ₄ ; Fe(NO ₃ ), ZnSO ₄ ; Na ₂ SeO ₃ , NH ₄ VO ₃ , MoO ₃ , MnSO ₄ and the like, and may be present in low concentrations range from 1 mg/L to

0.001 mg/L, such as 0.9 mg/L to 0.002 mg/L and including 0.8 mg/L to 0.005 mg/L.

Sugars of interest include: glucose, galactose, and the like. When present, the collective amount of sugars in the visualization medium may range from 500 mg/L to 10,000 mg/L, such as 1 ,000 mg/L to 9,000 mg/L and including 1 ,800 mg/L to 5,000 mg/L. Lipids of interest include: lipoic acid, linoleic acid and the like. When present, the collective amount of lipids in the visualization medium may range from 0.08 mg/L to 30 mg/L, such as 0.085 to1 mg/L. Nucleosides of interest include: adenosine, thymidine, hypoxanthine and the like. When present, the collective amount of nucleosides in the visualization medium may range from

0.1 mg/L to 10 mg/l, such as 0.2 mg/L to 6 mg/L and including 0.3 mg/L to 4.1 mg/L.

Other components of interest include, but are not limited to: phenol red, sodium pyruvate, L-inositol. When present, the amount of phenol red may range from 5 to 20 mg/L (for example 12, 15, 17 mg/L). When present, the amount of sodium pyruvate may range from 600 mg/L to 100 mg/L, such as from 550 mg/L to 110 mg/L.

Inositol (or cyclohexane-1 ,2,3,4,5,6-hexol) is a chemical compound with formula C ₆ Hi ₂ OeOr (-CHOH-) ₆ . It exists in nine possible stereoisomers, of which the most prominent form, widely occurring in nature, is c/s-1 ,2,3,5-trans-4,6- cyclohexanehexol, or myo-inositol. Inositol is a carbohydrate, though not a classical sugar. When present, amount of inositol in the visualization medium may range from 0.01 mg/L to 50 mg/L, such as 0.1 mg/L to 35 mg/L and including

1 mg/L to 20 mg/L. In certain embodiments, the concentration of at least riboflavin is altered in the visualization medium as compared to a control cell culture medium. Riboflavin, also known as vitamin B2, is the central component of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), and is therefore required by all flavoproteins. The molecular formula is Ci ₇ H ₂ oN ₄ θ6 and ribovlavin has a Molar mass of 376.36. FMN and FAD function as coenzymes for a wide variety of oxidative enzymes. Flavins can act as oxidizing agents because of their ability to accept a pair of hydrogen atoms. !n one embodiment, the concentration of riboflavin is decreased in the visualization medium (e.g., visualization medium VM1 as described in greater detail below), as compared to a control. In some instances, the visualization medium comprises substantially no or no riboflavin. In these instances, the concentration of riboflavin may be 0.01 mg/L or less, such as 0.001 mg/L or less. In others embodiment, the visualization medium does not include any riboflavin.

In some embodiments, the concentration of vitamin B6 is also altered in the visualization medium, e.g., as compared to a control. The term "Vitamin B6" refers to all natural forms of the vitamin, e.g., pyridoxal, pyridoxamine and pyridoxine (also called "pyridoxol"). All of these forms are converted in cells into a single biologically active form, pyridoxal 5-phosphate. There are generally three pyridoxal-phosphate (PLP) reactions: transamination, alpha elimnation, and beta- elimination. The molecular formula of this compound is C ₈ H ₉ NO ₃ and has a molar mass 167.16. In certain embodiments, the visualization medium may include substantially no or no vitamin B6 (e.g., pyridoxal HCI). In these instances, the concentration of vitamin B6 may be 0.01 mg/L or less, such as 0.001 mg/L or less. In another embodiment, the visualization medium does not include any vitamin B6. In one embodiment, the visualization medium does not include any riboflavin or vitamin B6.

In other embodiments, the concentration of riboflavin is increased in the visualization medium (see e.g., visualization medium VM2) as compared to control, in these embodiments, the concentration of riboflavin may range from 2 to 40 mg/l, such as from 3 to 10 mg/l, e.g., from 4-5 mg/L. In some embodiments, the concentration of vitamin B6 is also increased in the visualization medium as compared to a control (see e.g., VM2). In these embodiments, the concentration of vitamin B6 may range from 2 to 40 mg/!, such as from 3 to 10 mg/l, e.g., 4-5 mg/L.

In some instances, the visualization medium includes a modified amount of D-Ca pantothenate as compared to a control. D-Ca pantothenate or pantothenic acid, also called vitamin B5, is needed to form coenzyme-A (CoA), and is critical in the metabolism and synthesis of carbohydrates, proteins, and fats. In chemical structure, it is the amide between D-pantoate and beta-alanine. It is commonly found as its alcohol analog, the provitamin panthenol, and as calcium pantothenate. Its molecular formula is C ₉ H ₁₇ NO ₅ and it has a molar mass 219.235. in some embodiments, the concentration of D-Ca pantothenate is decreased in the visualization medium as compared to a control. In these embodiments, the concentration of D-Ca pantothenate may be 4 mg/L or less, such as 1 mg/L or less, including 0.01 mg/L or less. In some instances, the visualization medium may be free from D-Ca pantothenate. In other embodiments, the concentration D-Ca pantothenate is not altered in the visualization medium as compared with the control cell culture medium.

In some instances, the visualization medium includes a modified amount of choline chloride as compared to a control. Choline chloride is an organic compound, classified as a water-soluble essential nutrient and usually grouped within the Vitamin B complex. This natural amine is found in the lipids that make up cell membranes and in the neurotransmitter acetylcholine. Choline and its metabolites are needed for three main physiological purposes: structural integrity and signaling roles for cell membranes, cholinergic neurotransmission (acetylcholine synthesis), and as a major source for methyl groups via its metabolite, trimethylglycine (betaine) that participates in the S- adenosylmethionine synthesis pathways. The molecular formula of choline is C ₅ Hi ₄ NO ⁺ and it has a molar mass 104.17. In some embodiments, the concentration of choline chloride s decreased in the visualization medium as compared to a control. In these embodiments, the concentration of choline may be mg/L or less, such as 1 mg/L or less, including 0.01 mg/L or less. In some instances, the visualization medium may be free from choine. In other embodiments, the concentration choline is not altered in the visualization medium as compared with the control cell culture medium.

In some instances, the visualization medium includes a modified amount of folic acid as compared to a control. Folic acid (also known as Vitamin B9 or Folacin) and Folate (the naturally occurring form) are forms of the water-soluble Vitamin B9. Vitamin B9 (Folic acid and Folate inclusive) is essential to numerous bodily functions ranging from nucleotide synthesis to the remethylation of homocysteine. This compound has a molecular formula C ₁ 9H ₁ 9N7O6 and a molar mass of 441.4. In some embodiments, the concentration of folic acid is decreased in the visualization medium as compared to a control. !n these embodiments, the concentration of folic acid may be 4 mg/L or less, such as 1 mg/L or less, including 0.01 mg/L or less. In some instances, the visualization medium may be free from folic acid. In other embodiments, the concentration folic acid is not altered in the visualization medium as compared with the control cell culture medium.

In some instances, the visualization medium includes a modified amount of niacinamide compared to a control. Niacinamide, also known as nicotinamide and nicotinic acid amide, is the amide of nicotinic acid (vitamin B3). Nicotinamide is a water-soluble vitamin and is part of the vitamin B group. In cells, niacin is incorporated into nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), although the pathways for nicotinamide and nicotinic acid are very similar. NAD+ and NADP+ are coenzymes in a wide variety of enzymatic oxidation-reduction reactions. This compound has a molecular formula CeHeN ₂ O and a molar mass of 122.12.

Thiamine, or thiamin, sometimes called aneurin, is a water-soluble vitamin of the B complex (vitamin B1 ), whose phosphate derivatives are involved in many cellular processes. The best characterized form is thiamine diphosphate (ThDP), a coenzyme in the catabolism of sugars and amino acids. The molecular formula of this compound is Ci ₂ Hi ₇ N ₄ OS ⁺ and this compound has a molar mass of 265.35. In some embodiments, the concentration of niacinamide in the visualization medium is not changed as compared to the cell culture medium and, in some instances, ranges between 0.001 mg/L to 40 mg/L, such as between 0.01 mg/L to 5 mg/L, including 1 mg/L, 2 mg/L, 3 mg/L; and 4 mg/L. In some embodiments, the concentration of nicotinamide is increased in the visualization medium as compared to the cell culture medium and is 4 mg/L or greater, and can be up to 40 mg/L or greater (e.g., 4-5 mg/L, 10-15 mg/L, 20-25 mg/L, 30-35 mg/L, 39-40 mg/L). In some embodiments, the nicotinamide is absent in the visualization medium VM 1.

In some embodiments, the concentration of thiamine is decreased in the visualization medium as compared to a control. In these embodiments, the concentration of thiamine HCI may be 4 mg/L or less, such as 1 mg/L or less, including 0.01 mg/L or less. In some instances, the visualization medium may be free from thiamine. In other embodiments, the concentration thiamine HCI is not altered in the visualization medium as compared with the control cell culture medium.

Specific visualization media of interest are provided in Tables 1 to 3, below.

Table 1. Visualization media prepared on the basis of DMEM.

VM1-1 (DMEM-V) and VM1-2 (DMEM-Rf) are the media with decreased concentrations of organic oxidants and VM2-1 is the medium with increased riboflavin concentration.

Table 2. Visualization media prepared on the basis of Ham's F12.

VM 1-3 and VM 1-4 are the media with decreased concentrations of organic oxidants and VM2-2 is the medium with increased riboflavin concentration

Table 3.

Visualization media prepared on the basis of RPM! 1640.

VM1-5 and VM1-6 are the media with decreased concentrations of organic oxidants and VM2-3 is the medium with increased riboflavin concentration

METHODS

Visualization media, e.g., as described above, find use in a variety of different applications. In methods of using the visualization media, a host cell having a fluorescent protein associated therewith, e.g., expressed in the cell, is combined with a visualization medium in accordance with the invention. The cell, which in some instances is present in a population of multiple cells, may be combined with a suitable amount of medium in any convenient containment vessel. The amount of medium that is combined with the ce!l(s) is generally sufficient to immerse the cell(s) in the medium. In some instances, the cell(s) which are combined with the visualization medium have been previously cultured in a standard cell culture medium. Standard culture mediums in which the cell(s) may have been cultured prior to practice of the methods of the invention include, but are not limited to: Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640, Iscove's, McCoy's and other media formulations readily apparent to those skilled in the art, including media found in Methods For Preparation of Media, Supplements and Substrate For Serum-Free Animal Cell Culture Alan R. Liss, New York (1984) and Cell &Tissue Culture: Laboratory Procedures, John Wiley & Sons Ltd., Chichester, England 1996, both of which are incorporated by reference herein in their entirety. In some instances, the cell culture media may be supplemented, e.g., with any components necessary to support the desired cell or tissue culture. Additionally serum, such as bovine serum, which is a complex solution of albumins, globulins, growth promoters and growth inhibitors may be added if desired. Hormone addition into the medium may or may not be desired.

The cell or cells comprising the fluorescent protein may be combined with the visualization medium using any convenient protocol. In some instances, a washing and incubation protocol is employed. For example, in one protocol, the cell culture medium may be replaced for visualization medium before visualization experiment. The replacement may be performed under sterile conditions. For example, culture medium replacement can be performed as follows: (1 ) the culture medium is taken away from host-cells; (2) a sufficient volume of the visualization medium is carefully added to the cell monolayer to wash the cells; (3) the washing aliquot of the visualization medium is taken away and the visualization medium is carefully added; and (4) the cells are incubated in the visualization medium for a sufficient stabilization period of time at a suitable temperature, e.g., 30 min at 37°C in a Cθ ₂ -incubator. The resultant composition comprises a cell having associated therewith a fluorescent protein, where the cell is present in a visualization medium of the invention. Following combination and any desired stabilization period, the cell may be imaged using any protocol suitable for the particular application being performed. In imaging the cell, the cell will be irradiated with light of an appropriate excitation wavelength, where this excitation wavelength may range in some instances from 300 nm to 510 nm, such as 350 nm to 500 nm and including 380 nm to 490 nm. Emitted light from the cells will then be detected, where the wavelength of the emitted light may have a maximum that varies depending on the particular fluorescent protein being imaged, in some instances, this maximum has a wavelength ranging from 420 nm to 550 nm, such as 440 nm to 530 nm and including 450 nm to 510 nm.

As reviewed above, in some instances the photostability of a fluorescent protein is enhanced by using the visualization media of the invention. In such embodiments, the photobleaching half-time of a given fluorescent protein may be enhanced by 1.3 fold or more, such as 2.0 fold or more and including 3.0 fold or more. Because of this enhanced photostability, the fluorescent protein may be imaged for an extended period of time as compared with a control sample, where this extended period of time may be 50 seconds or longer, such as 100 seconds or longer, including 150 seconds or longer, e.g., 200 seconds or longer, 250 seconds or longer under bleaching light with 1 W/cm ² intensity. Because of this enhanced photostability, the photobleaching half-time of a fluorescent protein under exposure to bleaching light with 1 W/cm ² intensity may be 40 seconds or longer, such as 70 seconds or longer, including 100 seconds or longer, e.g., 200 seconds or longer, 250 seconds or longer, 300 seconds or longer.

Accordingly, embodiments of the invention include irradiating and imaging a fluorescent protein in a continuous manner for an extended period of time with increased photobieaching hilf-time, where this extended period of time may be 50 seconds or longer, such as 100 seconds or longer, including 150 seconds or longer, e.g., 200 seconds or longer, 250 seconds or longer under bleaching light with 1 W/cm ² intensity.

Following imaging, where desired the cell(s) may be placed back into a cell culture medium for further maintenance and/or manipulation. Any convenient transfer protocol may be employed. Visualization media of the invention may be used with a variety of different fluorescent proteins and host cells, e.g., where examples of each are reviewed in more detail below.

Fluorescent proteins

The visualization media of the invention may be used to modulate the photostability of a variety of different fluorescent proteins. The effect of the visualization medium on a given fluorescent protein's photostability and photobleaching can be tested by the analysis of the bleaching curves, e.g., as described in the above.

As reviewed above, fluorescent proteins possess detectable fluorescence and are characterized by GFP-like domain that is homologues to the Green Fluorescent Protein (avGFP) from Aequorea victoria. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. (1990), J. MoI. Biol. 215:403-10 (using default settings, i.e. parameters w=4 and T= 17). GFP-like domain can be identified in the protein amino acid sequence using available software for analysis of domain organization, for example using Conserved Domain Database (CDD) (available at the website formed by placing "http://www." in front of "ncbi.nlm.nih.gov/Structure/cdd/") and SMART (a Simple Modular Architecture Research Tool, available at the website formed by placing "http://smart." in front of "embl-heidelberg.de/").

In certain embodiments, the GFP-like domain starts from the amino acid residue corresponding to the position 6 of avGFP that can be revealed using sequence alignment of a fluorescent protein under examination and avGFP (SEQ ID No: 1 ). GFP-like domain may be 200 amino acids in length or longer, such as 215 amino acids long in length or longer, and including 220 amino acids in length or longer (e.g., 220; 221 ; 222; 223; 224; 225; 230; 231 ; 232; 233 amino acids in length or longer). In some embodiments, the GFP-like domain ends at the amino acid residue corresponding to the position 229 of avGFP. In certain embodiments, a fluorescent protein whose photostability is modified by the media of the present invention has a sequence identity to a corresponding homolog on the amino acid levels of at least about 10%, and, preferably about 20%, 25%, 30%, 35%, 40%, 45%, 55%, 60%, 65%, 70%, or higher, including about 75%, 80%, 85%, 90% and 95% or higher. A reference sequence will usually be at least 150 amino acids long, usually at least 200 amino acids long, more usually at least about 215 amino acids long (e.g. 218; 225; 229; 230; 231 ; 232; 233 amino acids long), and may extend to the complete protein sequence that is being compared. Sequence similarity is calculated based on a reference sequence. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al., J. MoI. Biol., 215, pp. 403-10 (1990) (for example, using default settings, i.e., parameters w=4 and T=17).

Fluorescent proteins of interest are naturally occurring fluorescent proteins or mutants of naturally occurring fluorescent or chromoproteins, e.g., of the GFP family. Fluorescent proteins of interest also include any fusions of the fluorescent proteins with any other peptide(s) and\or protein(s). The methods of preparation of such fusions are recombinant technologies well-known in the art on the nucleic acid level. Examples of fusion partners include a degradation sequence, a sequence of sub-cellular localization (e.g., nuclear localization signal, peroximal targeting signal, Golgi apparatus targeting sequence, mitochondrial targeting sequence, etc.), a signal peptide, or any protein or polypeptide of interest. Fusion proteins may comprise for example, a fluorescent protein and a second polypeptide ("the fusion partner") fused in-frame at the N-terminus and/or C- terminus of the fluorescent protein. Fusion partners include, but are not limited to, polypeptides that can bind antibodies specific to the fusion partner (e.g., epitope tags), antibodies or binding fragments thereof, polypeptides that provide a catalytic function or induce a cellular response, ligands or receptors, cytoskeletal and adhesion proteins, gap junction proteins, nuclear proteins, signal transduction proteins, vesicular transport proteins and the like. In such fusion proteins, the fusion partner is generally not naturally associated with the fluorescent protein portion of the fusion protein. A great number of experiments shown that fusion proteins in which fusion partner is fused to the C-terminus or N- terminus of a fluorescent protein exhibit substantially similar fluorescent properties as the fluorescent protein alone (see e.g., Niedenthal et al., Yeast (1996) 12: 773 - 786; Day and Davidson, Chem. Soc. Rev. (2009) 38: 2887; Lippincott-Schwartz and Patterson, Science (2003) 300:87-91 ; and Stepanenko et al., Curr. Protein Pept. Sci. (2008):338-369).

In certain embodiments, the fluorescent proteins of interest comprise a avGFP-like chromophore formed by Ser65-Tyr66-Gly67 region (numbering of avGFP; corresponding amino acids in other proteins can be identified by alignment of a protein with avGFP) within a β-can.

In certain embodiments, the fluorescent proteins of interest exhibit green fluorescence, e.g., with emission maximum between 470 nm and 520 nm, such as between 475 nm and 520 nm, and including between 477 nm and 515 nm, e.g., between 480 nm and 510 nm (such as 480 nm, 481 nm, 482 nm, 483 nm, 484 nm, 485 nm, 486 nm, 487 nm, 488 nm, 489 nm, 490 nm, 491 nm, 492nm, 493 nm, 494 nm, 495 nm, 496 nm, 497 nm, 498 nm, 499 nm, 500, nm, 501 nm, 502 nm, 503 nm, 504 nm, 505 nm, 506 nm, 507 nm, 508 nm, 509 nm, 510 nm). In certain embodiments, the fluorescent proteins of interest exhibit blue or cyan fluorescence, e.g., with emission maximum between 400 nm and 470 nm, such as between 440 nm and 460 nm, e.g., 457 nm.

In certain embodiments, the fluorescent proteins of interest exhibit yellow or red fluorescence, e.g., with emission maximum between 520 nm and 750 nm, such as between 523 nm and 700 nm, including between 550 nm and 650 nm. In some embodiments, the fluorescent proteins of interest are photoactivatable, photoconvertable or photoswitchable. These fluorescent proteins change their spectral properties upon light irradiation of a specific wavelength. For example, the observed conversion may be reversible or irreversible from a non-fluorescent form to a bright fluorescent form (e.g., PA- GFP), photoconversion from the blue fluorescent form to the green fluorescent form (e.g. PS-CFP, PS-CFP2), from a green fluorescent form to a red fluorescent form (e.g., Dendra, Dendra2, Kaede, EosFP,) etc. In certain embodiments, the proteins of interest are bright, where by "bright" is meant that the fluorescence of these proteins can be detected by common methods (e.g., visual screening, spectrophotometry, spectrofluorometry, fluorescent microscopy, by FACS machines, etc.) Fluorescence brightness of particular fluorescent proteins is determined by its quantum yield multiplied by maximal extinction coefficient.

In some instances, the proteins of interest have an absorbance maximum ranging from about 300 nm to 700 nm; a maximum extinction coefficient ranging from about 25,000 to 150,000 M-1cm-1. The proteins of interest may include GFP-like domain that ranges in length from about 150 to 300 amino acids, such as from 200 to 300 amino acid residues.

Specific fluorescent proteins of interest include fluorescent proteins whose photostability depends on organic oxidant concentration in a visualization medium, for example Aequorea victoria GFP (SEQ ID NO; 1 ) and related proteins and mutants thereof (e.g., EGFP (SEQ ID NO:02), PA-GFP (SEQ ID NO:15), VisGreen (SEQ ID NO:03), Emerald, Superfolder avGFP, T-Sapphire, etc), fluorescent mutants of the Aequorea coerulscens non-fluorescent protein (e.g., AcGFPI (SEQ ID NO:05), PS-CFP, PS-CFP2 (SEQ ID NO:04)) and related proteins, Aequorea macrodactila GFP and related proteins and mutants thereof (e.g., mTagGFP, also known as TagGFP2 (SEQ ID NO:06)), sea anemone Entacmaea quadricolor fluorescent protein eqFP578 and related proteins and mutants thereof (e.g., TagBFP (SEQ ID NO:12) and TagRFP), Copepoda green fluorescent proteins and related proteins and mutants thereof (e.g., TurboGFP (SEQ ID NO: 14), CopGFP), Anthozoa fluorescent proteins and related proteins and mutants thereof (e.g., DsRed, DsRed2 (SEQ ID NO:11 ), mCherry, mRasberry, Azami (SEQ ID NO:07) Green, mWasabi (SEQ ID NO:09), ZsGreen (SEQ ID NO:13), Dendra2 (SEQ ID NO:08), Dronpa (SEQ ID NO:10), EosFP, Kaede, Renilla GFP), etc. Homologs of the above noted fluorescent proteins are also of interest. The amino acid sequences of certain fluorescent proteins of interest are provided in FIG. 1. As used herein "related protein" is a protein that comprises an amino acid sequence that has 50% or more, such as 55% or more and including 60% or more amino acid sequence identity to the sequence of the referred protein as determined using, for example, MegAlign, DNAstar clustal algorithm as described in D. G. Higgins and P.M. Sharp, "Fast and Sensitive multiple Sequence

Alignments on a Microcomputer," CABIOS, 5 pp. 151-3 (1989) (using parameters ktuple 1 , gap penalty 3, window 5 and diagonals saved 5). Related proteins of interest may have much higher sequence identity, e.g., 70%, 75%, 80%, 85%, 90% (e.g., 92%, 93%, 94%) or higher, e.g., 95%, 96%, 97%, 98%, 99%, 99.5%, particularly for the amino acid sequence that provides the functional regions of the protein, e.g. GFP-like domain. The length of the GFP-like domain can be identified using sequence alignment of the reference protein with the avGFP as shown in Fig.1. In some embodiments, sequence identity is identified over a complete protein sequence. Mutations that may be present include single amino acid changes, deletions or insertions of one or more amino acids, N-terminal truncations or extensions, C-terminal truncations or extensions and the like. Mutants can be generated on a template nucleic acid by modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid. The modifications, additions or deletions can be introduced by any method known in the art (see for example Gustin et al., Biotechniques (1993) 14: 22; Barany, Gene (1985) 37: 111-123; and Coiicelli et al., MoI. Gen. Genet. (1985) 199:537-539, Sambrook et al., Molecular Cloning: A Laboratory Manual, (1989), CSH Press, pp. 15.3-15.108) including error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-directed mutagenesis, random mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a combination thereof. The modifications, additions or deletions may be also introduced by a method comprising recombination, recursive sequence recombination, phosphothioate- modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction- purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof. In some embodiments, fluorescent proteins encoded by mutant or derived nucleic acids have the same fluorescent or biochemical properties as the wild type fluorescent protein. In other embodiments, mutant or derived nucleic acids encode fluorescent proteins with altered properties.

The fluorescent proteins of interest may be expressed in host-cells. For certain applications, DNA comprising a fluorescent protein coding sequence (alone or in fusion construct) is linked with a suitable DNA regulatory sequence(s) and is introduced into host-cells as described below. To express fluorescent protein in host cells, expression cassettes or systems used, inter alia, for the production of the fluorescent proteins are used. The expression cassette may exist as an extra-chromosomal element or may be integrated into the genome of the cell as a result of introduction of said expression cassette into the cell. For expression, the gene product encoded by a nucleic acid is expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian, or mammalian systems. In the expression cassette or expression vector, a subject nucleic acid is operably linked to a regulatory sequence(s) that can include promoters, enhancers, terminators, operators, repressors and inducers. Methods for preparing expression cassettes or systems capable of expressing the desired product are known for a person skilled in the art. The term "operably linked" as used herein refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that the reading frame is maintained and a functional protein is produced. The terms "operatively linked" or "operably linked" or the like, when used to describe chimeric proteins, refer to polypeptide sequences that are placed in a physical and functional relationship to each other. In one embodiment, the functions of the polypeptide components of the chimeric molecule are unchanged compared to the functional activities of the parts in isolation. For example, a fluorescent protein of the present invention can be fused to a fusion partner of interest. In this case, the fusion molecule retains its fluorescence, and the polypeptide of interest retains its original biological activity. In some embodiments of the present invention, the activities of either the fluorescent protein or the protein of interest can be reduced relative to their activities in isolation. Such fusions can also find use with the present invention.

Exempliary nucleic acids comprising coding sequence of fluorescent proteins of interest are shown in SEQ ID NOs: 16-27.

Cell lines, which stably express the fluorescent proteins of present invention, can be selected by the methods known in the art (e.g., the co- transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells that contain the gene integrated into a genome). The above-described expression systems may be used in prokaryotic or eukaryotic hosts. Host-cells such as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, e.g., COS 7 cells, HEK 293, CHO, Xenopus oocytes, etc., may be used for production of the protein.

Host cells

The fluorescent proteins of interest are associated with, e.g., present in, host-cells. Suitable host-cells are any eukaryotic cells including primary culture or cell lines. Host-cells such as insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, e.g., COS 7 cells, HEK 293, CHO, Xenopus oocytes, etc., may be used for expression of fluorescent protein. In certain embodiment, host cells comprise at least one fluorescent protein. The isolated nucleic acid encoding florescent protein can be introduced into the host cell by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the nucleic acid molecules (i.e. DNA) into host-cells are widely known and provided in references such as Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3nd Ed., (2001 ) Cold Spring Harbor Press, Cold Spring Harbor, NY).

In one embodiment, the host cells can be a fungus, for example yeast. Yeast is widely used as a vehicle for heterologous gene expression (for example see Goodey et al Yeast biotechnology, D R Berry et ai, eds, (1987) Allen and Unwin, London, pp 401 -429) and by King et a! Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, Blackie, Glasgow (1989) pp 107- 133). Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.

Host cells of interest also include mammalian cells. Host cells (primary culture) can be derived from transgenic animals obtained by transgenic techniques well known in the art and provided in references such as Pinkert, Transgenic Animal Technology: a Laboratory Handbook, 2nd edition (2203) San Diego: Academic Press; Gersenstein and Vintersten, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed, (2002) Nagy A. (Ed), Cold Spring Harbor Laboratory; Blau et al., Laboratory Animal Medicine, 2nd Ed., (2002) Fox J. G., Anderson L. C, Loew F. M., Quimby F.W. (Eds), American Medical Association, American Psychological Association; Gene Targeting: A Practical Approach by Alexandra L. Joyner (Ed.) Oxford University Press; 2nd edition (2000). For example, transgenic animals can be obtained through homologous recombination, where the endogenous locus is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like.

The nucleic acid can be introduced into the cell, directly or indirectly, by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus or with a recombinant viral vector and the like. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant nucleic acid molecule. This nucleic acid molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.

DNA constructs for homologous recombination will comprise at least a nucleic acid encoding fluorescent protein, wherein the gene has the desired genetic modification(s), and includes regions of homology to the target locus. DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection may be included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al., Meth. Enzymol. (1990) 185:527-537. For embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, such as a mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LiF). Tansformed ES or embryonic ceils may be used to produce transgenic animals using the appropriate technique described in the art.

The transgenic animals may be any non-human animals including non- human mammal (e.g., mouse, rat), a bird or an amphibian, etc., and used in functional studies, drug screening and the like. Representative examples of the use of transgenic animals include those described infra. Plant cells also may be produced. Methods of preparing transgenic plant cells and plants are described in U.S. Patent Nos. 5,767,367; 5,750,870; 5,739,409; 5,689,049; 5,689,045; 5,674,731 ; 5,656,466; 5,633,155; 5,629,470; 5,595,896; 5,576,198; 5,538,879; 5,484,956; the disclosures of which are herein incorporated by reference. Methods of producing transgenic plants also are reviewed in Plant Biochemistry and Molecular Biology (eds. Lea and Leegood, John Wiley & Sons) (1993) pp. 275-295 and in Plant Biotechnology and Transgenic Plants (eds. Oksman-Caldentey and Barz), (2002) 719 p.

For example, embryogenic explants comprising somatic cells may be used for preparation of the transgenic host. Following cell or tissue harvesting, exogenous DNA of interest is introduced into the plant cells, where a variety of different techniques is available for such introduction. With isolated protoplasts, the opportunity arises for introduction via DNA-mediated gene transfer protocols, including incubation of the protoplasts with naked DNA, such as plasmids comprising the exogenous coding sequence of interest in the presence of polyvalent cations (for example, PEG or PLO); or electroporation of the protoplasts in the presence of naked DNA comprising the exogenous sequence of interest. Protoplasts that have successfully taken up the exogenous DNA are then selected, grown into a callus, and ultimately into a transgenic plant through contact with the appropriate amounts and ratios of stimulatory factors, such as auxins and cytokinins.

Other suitable methods for producing plants may be used such as "gene- gun" approach or Agrobacterium-mediated transformation available for those skilled in the art.

UTILITY

The media and methods of the invention, e.g., as described above, find use in a variety of different applications. For example, the media described above are useful for short-term fluorescent cell culturing following with cell vizualization, wherein short-term visualization is up to 10 hours, such as 7 hours or less, including 5 hours or less. For the lengthy experiments, media compositions which include supplementary components, such as glutamine, antibiotics (e.g., penicillin, streptomycin), and fetal serum with suitable concentration. For example, 600 mg/l glutamine, 50000 units/I penicillin, 50 mg/l streptomycin, 2- 10% fetal serum may be employed. The methods of the present invention find use in applications wherein fluorescent proteins are expressed in host-cells and visualized, for example when fluorescent proteins are used for visualization of cells, cellular organelles and protein localization and interaction. Visualization medium of the present invention find use when the photostability of a fluorescent protein in host cells should be altered in some manner, e.g., enhanced or decreased. The uses described herein are merely exemplary and are in no way meant to limit the use of the proteins of the present invention to those described. For example, visualization medium of the present invention find use in time-lapse imaging, i.e., imaging of the same region of interest during some time interval. In this case high photostability of FPs is especially important to ensure generation of large series of images (typically more than 10 images) where (i) fluorescence is clearly detectable above background and (ii) preferably, fluorescence intensity remains constant from image to image. Time-lapse imaging is applied to detect changes of the fluorescent signal in time and space (e.g., for tracking of intracellular distribution of labeled proteins inside living cells; tracking cell movements and division in culture or in organism; etc.).

Also, visualization medium of the present invention find use in super- resolution imaging based on so called Photo-Activation Localization Microscopy (PALM) and related techniques as described in Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott- Schwartz J, Hess HF. Imaging intracellular fluorescent proteins at nanometer resolution. Science. 2006, 313(5793):1642-5; Shroff H, Galbraith CG, Galbraith JA, White H, Gillette J, Olenych S, Davidson MW, Betzig E. Dual-color super- resolution imaging of genetically expressed probes within individual adhesion complexes. (See e.g., Proc. Nat'l Acad. Sci U S A. (2007) 104:20308-13; Bates et al., Curr. Opin. Chem. Biol. (2008) 12:505-14. To achieve ultra-high resolution in super-resolution imaging techniques, it is crucial to collect from a photoconverted fluorescent protein molecule (such as PA-GFP and PS-CFP2) as many photons as possible before its photobleaching. Thus, enhanced photostability of fluorescent proteins can result in better spatial resolution achieved. Also, visualization medium with increased concentration of organic oxidants (e.g., riboflavin) of the present invention find use in super-resolution imaging to achieve efficient photoconversion of fluorescent proteins (otherwise commonly used as simple, non-photoconvertible fluorescent proteins). Examples of such photoconversion include, but not limited by, green-to-red photoconversion of Aequorea victoria GFP, EGFP, AcGFPI , mTagGFP, and other green fluorescent proteins.

In addition, photostability-enhancing visualization medium of the present invention find use in Fluorescence Correlation Spectroscopy (FCS) and

Fluorescence Cross-Correlation Spectroscopy (FCCS) (see Haustein E, Schwille P. Fluorescence correlation spectroscopy: novel variations of an established technique. Annu Rev Biophys Biomol Struct. 2007; 36:151-69).

Visualization medium of the present invention with increased concentration of organic oxidants (e.g., riboflavin and/or vitamin B6) find use in applications where low photostability of fluorescent proteins is advantageous. Examples of such applications include, but not limited by, estimation of protein mobility using Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP) and related techniques (see Lippincott-Schwartz, J., Snappl, E., Kenworthy, A. (2001 ). "Studying protein dynamics in living cells". Nature Reviews Molecular Cell Biology 2: 444-456); probing protein-protein interactions by estimation of Fluorescence Resonance Energy Transfer (FRET) between two fluorescent proteins using acceptor photobleaching approach (see Kenworthy AK. Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods. 2001 , 24(3):289-96); regional optical labeling; and others. As such, the methods of the present invention find use in applications where fast photobleaching and/or photoconversion of a reporter fluorescent protein is required, for example in the method of fluorescence recovery after photobleaching (FRAP, Reits and Neefjes, Nat. Cell Biol. 3 (2001 ), pp. E145-E147; ϋppincott-Schwartzet al., Nat. Cell Biol. (2003) pp. S7-S14; Lippincott-Schwartzet al., Nat. Rev. MoI. Cell Biol. 2 (2001 ), pp. 444^56), fluorescence Loss in Photobleaching (FLIP, Hanson and Kohler, 2001 , Journal of Experimental Botany, Vol. 52, No. 356, pp. 529-539).

KITS

Aspects of the invention also include kits. Kits include at least a visualization medium, e.g., as described above. Kits may further include one or more vectors, e.g., comprising a coding sequence of a fluorescent protein, etc., Kits may further include one or more host cells. Components of the kits, as desired, may be present in a suitable storage medium, e.g., buffered solution, typically in a suitable container. Components of the kits may be packaged in separate containers, or compatible components may be present in the same container, e.g., as desired. in addition to the above components, the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

The following examples are offered by way of illustration and not by way of limitation. Specifically, the following examples are of specific embodiments for carrying out the present invention. The examples are for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. EXPERIMENTAL

I. Example 1 Measurement of photostability of fluorescent proteins in mammalian cells.

Commercially available expression vectors pTagGFP2-N, pTagBFP-N, pPS-CFP2-N, pTagRFP-vinculin (Evrogen) and pEGFP-actin, pEGFP-tubulin, pEGFP-mito, pAcGFP1-N1 , pEGFP-N1 and pEGFP-C1 (Clontech) were used for fusion generation and transfection of cells. To generate the pEGFP-vimentin expression vector, EGFP coding sequence was cloned commercially available into pmKate2-vimentin vector (Evrogen) in place of mKate2. To generate the pPA-GFP expression vector, a nucleic acid comprising PA-GFP coding sequence was synthetically produced and cloned into the pEGFP-N1 vector instead of EGFP.

Human embryonic kidney 293 (HEK293T) cells were transfected with vectors encoding fluorescent proteins using the FuGene® transfection reagent (Roche) and grown in DMEM culture medium (Invitrogen) supplemented with 600 mg/L L-glutamine, 50,000 units per L of penicillin, 50 mg/L streptomycin and 10% fetal bovine serum (Sigma) at 37°C in a 5% CO2 atmosphere. Live cells were imaged 24-48 h after transfection at room temperature. The cell culture medium was replaced with visualization media before imaging experiments. The replacement was performed under sterile conditions. The cells were incubated in the visualization medium for 20-30 min at 37 ⁰ C in a CCvincubator and imaged using a fluorescence microscope.

For fluorescence microscopy, a Leica AF6000 LX imaging system based on a DMI 6000 B inverted microscope equipped with an HCX PL APO Ibd. BL 63x 1.4NA oil objective, a Photometries CoolSNAP HQ CCD camera, and a 120W HXP short arc lamp (Osram) as a light source was used. The photoactivatable fluorescent proteins PA-GFP and PS-CFP2 were pre-irradiated with a BFP filter cube (excitation BP360/40, emission BP470/40) to induce their photoconversion into the green fluorescent state. The selected field of view (containing several fluorescent cells) was irradiated with a GFP filter cube (excitation BP470/40, emission BP525/50) in a series of detecting (light intensity 50 mW/cm ² , exposure 10-100 ms) and bleaching (light intensity 1 W/cm ² , exposure 5 s) light. The bleaching curves for different cells were drawn using data from the detection channel and normalized by dividing all the fluorescence values by the initial signal value. A time to 50% fluorescence decrease (bleaching half-time) was used as a quantitative characteristic to compare the photostability of a fluorescent protein in different media.

First, EGFP expressed in human embryonic kidney (HEK293T) cells was tested in phosphate-buffered saline (PBS) as a medium for cell imaging. Commonly used for cell growth and imaging, Dulbecco's Modified Eagle's

Medium (DMEM) without phenol red was used as a control (standard conditions). Impressively, a dramatic (about 5-fold) enhancement of EGFP photostability in PBS compared to DMEM was observed. However, PBS is not suitable for long- term cell maintenance. To find a minimally depleted medium that was optimal for imaging, modified DMEM media lacking either riboflavin (DMEM-Rf) or all vitamins (DMEM-V) (Table 4) were tested. It was found that the absence of riboflavin was sufficient to increase EGFP photostability 5.1-fold, up to the level observed in PBS (Fig. 3, Table 5). Depletion of other vitamins resulted in a further improvement of photostability (9.3-fold compared to DMEM). Notably, the cell-to- cell variability of bleaching rates was much higher in DMEM compared to the depleted media tested (Fig. 3).

Further, EGFP fused to the cytoskeletal proteins β-actin and α-tubulin were tested and drastic improvements were observed in their photostabilities in DMEM-Rf and DMEM-V. At the same time, photostability of mitochondrially localized EGFP demonstrated a low dependence on medium composition, and was essentially higher compared to that of cytosolic EGFP in DMEM (Table 5, Figs 4, 5).

Considerable enhancement of the photostabilities was observed for commercially available green fluorescent proteins AcGFP (Clontech) and TagGFP2 (Evrogeπ) (Table 5, Figs 6, 7). In addition, a strong increase in the photostabilities of the photoactivatable fluorescent proteins PA-GFP and PS-CFP (Evrogen) in their activated (green fluorescent) state in DMEM-Rf and especially in DMEM-V (Table 5) was observed. No effect or low increase of photostability was obtained for cells expressing red fluorescent proteins DsRed2, TagRFP, and TagRFP-actin.

Table 4. Compositions of media for cell maintenance. and fluorescence microscopy.

Table 5. Medium-dependent changes in the photostability of fluorescent proteins expressed in HEK293T cells.

Fluorescent Photostability increase, fold ^a protein DMEM-Rf DMEM-V

EGFP 5.1 9.3

EGFP-actin 3.5 5.9

EGFP- 4.4 6.4 tubulin

EGFP-mito 1.4 1.4

AcGFPI 1.7 2.1

TagGFP2 2.0 3.3

PA-GFP 1.3 4.5

(activated)

PS-CFP2 3.0 4.3

(activated) a Increases of photobleaching half-time compared to DMEM. Bleaching curves were measured under identical conditions except for the cell media. Activated PA-GFP and PS-CFP2 are the green fluorescent forms of these proteins generated under violet (405 nm) light irradiation.

In addition, a stably transfected cell line expressing EGFP was produced by EGFP lentivirus-mediated gene transduction. Firstly, a packaging cell line (HEK293T) was co-transfected with special vectors for lentivirus production. There were 3 commercially available vectors from Addgene:

• modified pRRLSIN.cPPT.PGK-GFP.WPRE (whose PGK promoter was replaced with EF-1 promoter using standard methods),

• pMD2.G (coding viral envelope proteins) and • pCMVR8.2 (coding viral GAG/POL genes).

Co-transfection was performed using FuGene® transfection reagent (as described above) with 2.5:2:0.6 proportion of pRRLSIN.cPPT.PGK-GFP.WPRE: pCMVR8.2: pMD2.G DNA respectively. Transfected cells were grown for 48h. Then cultural medium aliquot was replaced with the fresh one. After viral particle maturation (typically on the third day of cultivation) which is accompanied by mass cell destruction, the liquid phase (supernatant) was collected, centrifuged (to clear off cell debris) and filtered with 0.45 mkm Millipore filter. The obtained suspension in turn was used to infect HEK293 cells. After the infection, several rounds of selection were performed. This selection was based on sub-cultivation of single cells showing high EGFP expression level. A strong increase in the EGFP photostability in the stably of the transfected cells in DMEM-Rf (5.6 fold) and especially in DMEM-V (7.0 fold) was observed similar to that of EGFP in transiently transfected cells.

Then, EGFP was expressed in Phoenix Eco and 3T3 cells. Cell transfection and cultivation were performed as described above in DMEM media. Within 48-72 hours after transfection, the cells were visualized as described above in DMEM and DMEM-V media. Exemplary bleaching curves are shown in Fig. 8. In all cases the greatest increase of EGFP photostability occurred in DMEM-V medium. Phoenix Eco cells expressed EGFP demonstrated the same range of photostability increase in DMEM-V media as HEK293T-expressed EGFP, while photostability increase in this medium was less for 3T3 cells (about 2 times).

II. Example 2 - Cell vitality in the visualization medium

An influence of riboflavin starvation on eukaryotic cell physiology was previously described in literature (See e.g., J. Nutr. Biochem. (2005) 16: 617- 624.). Significant changes usually take place after 5-7 days cultivation with poor in riboflavin medium. We checked the influence of the media developed for the FP photostability enhancement on HEK293T (available from ATCC) cells vital functions. Cells originated from the same passage have been cultivated in

DMEM-V, DMEM-Rf, and DMEM media for a week on different substrates (30 mm thin-walled glass plates and 60 mm plastic plates). All media were supplemented with 600 mg/l glutamine, 50000 units/I penicillin, 50 mg/l streptomycin, 10% fetal serum. During the cultivation, all the cells were sub- cultured several times. State and morphology monitoring was performed with the light microscopy (with Nikon Eclipse TS100 microscope). Cells were observed to keep their normal morphology, division and fastening abilities, and also their transfection ability for the whole observation period. It was also observed that no specific physiological responses concerned with the medium composition in the first week of monitoring, while in the second week cells incubated in DMEM-V medium showed depressed viability and pathologic morphology changes. HeLa cells transfected with fluorescent protein-tagged α-tubulin or β-actin had a normal cytoskeleton after 5-day culture in DMEM-V (Fig. 14). Moreover, a standard scratch wound healing assay revealed no effect of growth in DMEM-V medium (5-day cultivation) on migration of rat embryonic fibroblasts (Fig. 15).

IEI. Example 3

The commercially available expression vector pDendra2-N (Evrogen) was used for dendra2 photoswitchable protein expression. Dendra2 is capable of irreversible photoconversion from the green to the red fluorescent form. Initially, the Dendra2 protein emits green light (emission maximum at 507 nm) under 490 nm excitation irradiation. Strong irradiation with 488nm laser (or lamp) causes photoconvertion into the stable red form. pDendra2-N vector was transfected into HeLa cells (from ATCC) using the iipofection method as described in the Example 1 , above. Cells were cultivated in the DMEM medium also comprising 600 mg/l glutamine, 50000 units/I penicillin, 50 mg/l streptomycin, 10% fetal serum. Cell visualization was performed in 24-72 hours after transfection. Imaging and Dendra2 photoconversion were made with Leica SP2 laser confocal microscope and 63 ^χ immersion oil objective. Fields of view containing one or more entire fluorescent cells were irradiated in series manner (xyt-mode, 512*512 format, beam exp. 3, pinhole value 114,69 mkm, 8χ zoom, between frames with default settings) sequentially with green detecting (3% Ar-laser 488 nm, DD 488/543, gainPM 650V), red detecting (100% HeNe-laser 543 nm, DD 488/543, gainPM 750V) and green activating light (100% Ar-laser 488 nm, DD 488/543, gainPM 300V). Usually 5 frames series were used for full green-to-red photoswitch. Each confocal 30 mm dish was observed firstly with DMEM medium and then with VM2-1 comprising 40 mg/l of riboflavin. Red channel data allowed one to draw the red signal formation curves for each particular case. These curves obtained from DMEM and VM 2-1 experiments were then compared among themselves. Comparison among DMEM and VM2-2 results showed a measurable difference in photoconversion efficiency. At the average (calculated from 20 series) dendra2 underwent green-to-red photoswitch 1.7-times better with VM 2-1 medium than with DMEM. Accordingly, embodiments of the invention include methods in which the photocoversion of a protein is enhanced by 1.2 fold or more, such as 1.4 fold or more, including 1.5 fold or more, e.g., 1.75 fold or more.

IV. Example 4

Whether the supplementary medium compounds needed for the norma! cell growth and long (typically longer than 5 hours) visualization experiments influence the photostability of various FPs was assessed. HEK293T cells (from ATCC) were transfected with pEGFP-N1 (Clontech) and pTagGFP2-N (Evrogen) plasmids as described in the Example 1 , above. Cells were visualized with Leica AF6000LX fluorescent microscope to measure the photostability of FPs. Bleaching curves for DMEM, VM 1-1 and VM 1-2 media were obtained for at least 10 fields of view with and without all appropriate supplements (600 mg/L L- glutamine, 50000 units per L of penicillin, 50 mg/L streptomycin and 10% fetal bovine serum). These experiments demonstrated no influence of the aforementioned supplementary components on the photostability of various FPs.

V. Example 5

The photostability of EGFP-tubulin fusion transiently expressed in 3T3 cells was evaluated using laser scanning confocal microscope Leica TCS SP2 equipped with HCX PL APO Ibd.BL 63x 1.4NA oil objective and 125 mW Argon laser. Green fluorescent signal was acquired at excitation 488 nm laser line and detected at 500-530 nm wavelength range. Scanning was performed using 200 MHz line frequency, 1024x1024 format, beam exp. 3, at zoom 2. It was found that the photostability of EGFP-tubulin green signal increases about 2.5-3-fold in DMEM-V compared to that in DMEM. V!. Example 6

pTagBFP-N vector (Evrogen) was transiently transfected in HEK293T cells and the photostability of this fluorescent protein was evaluated using Leica AF6000 LX imaging system as described in the Example 1 , above. In contrast to other fluorescent proteins studied, the photostability of TagBFP was found to decrease dramatically (about 3-5-fold) in DMEM-Rf and DMEM-V media as compared to DMEM. Notably, appearance of red fluorescent signal was observed upon TagBFP bleaching. The results indicate that TagBFP becomes an efficient blue-to-red photoconvertible fluorescent protein in the vitamin-depleted media.

VM. Example 7 Influence of different vitamins on photostability of EGFP

pEGFP-N vector (Clontech) was transiently transfected in HEK293T cells and the photostability of this fluorescent protein was evaluated using Leica AF6000 LX imaging system as described in the Example 1 , above. An influence of individual vitamins on EGFP photostability was tested by measurements of EGFP photobleaching curves in the presence of a particular vitamin added to the DMEM-V medium (at concentration characteristic for this vitamin in DMEM). These tests showed the following:

• The addition of folic acid (4 mg/L) or choline chloride (4 mg/L) did not change the photostability of EGFP as compared to that observed in DMEM-V (Fig. 9 and 10). • In contrast, the presence of riboflavin (0.4 mg/L) resulted in a dramatic

(about 3.3-fold) decrease of EGFP photostability (Fig. 11).

• The addition of pyridoxal (4 mg/L) to DMEM-V medium led to considerably (about 1.5-fold) decreased photostability of EGFP (Fig. 12).

• The addition of nicotinamide (4 mg/L) resulted in even higher EGFP photostability compared to DMEM-V (Fig. 13). • From these data, it was concluded that optimal visualization medium VM 1 that increase photostability of green fluorescent proteins must be depleted in riboflavin and pyridoxal, but should contain other vitamins.

Although the foregoing invention has been described in some detai! by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.