Topic Introduction

Fluorescent Protein Tracking and Detection: Fluorescent Protein Structure and Color Variants

Adapted from Live Cell Imaging, 2nd edition (ed. Goldman et al.). CSHL Press, Cold Spring Harbor, NY, USA, 2010 (in press).

INTRODUCTION

The rapidly growing arsenal of genetically encoded fluorescent proteins (FPs) obtained from sea creatures has launched and fueled a revolution in live cell imaging. The diverse array of applications benefiting from FPs ranges from markers targeted at organelles and protein fusions designed to monitor intracellular dynamics to reporters of transcriptional regulation and in vivo probes for whole-body imaging and detection of cancer. FPs have enabled the creation of highly specific biosensors to monitor a wide range of intracellular phenomena, including pH and metal-ion concentration, protein kinase activity, apoptosis, membrane voltage, cyclic nucleotide signaling, and tracing neuronal pathways. The purpose of this article is to provide a description of the wide variety of FPs that are currently available.

RELATED INFORMATION

Fluorescent Protein Tracking and Detection: Applications Using Fluorescent Proteins in Living Cells (Rizzo et al. 2009) provides an introduction to applications of FPs in live cells and tips on how FPs can be used with success in live cell imaging. Several examples of use of FPs for intracellular monitoring are described in Lalonde et al. (2005), Li et al. (2006), and Wang et al. (2008).

OVERVIEW

The discovery of the original green fluorescent protein (GFP) can be traced back to the early 1960s, when researchers studying the bioluminescent properties of the jellyfish Aequorea victoria isolated a blue-emitting, Ca⁺⁺-dependent bioluminescent protein they named aequorin (Shimomura et al. 1962). Alongside this purification, another protein was found that was not luminescent, but that fluoresced green under ultraviolet light and was eventually named green fluorescent protein. Over the next couple of decades, it was determined that aequorin and GFP work together in the light organs of A. victoria to convert Ca⁺⁺-induced luminescent signals to the green luminescence that was characteristic of the species (Shimomura 2006). Although the cDNA for GFP was eventually cloned in 1992 (Prasher et al. 1992), its potential as a molecular probe in virtually any species was not fully appreciated until GFP was first used for tracking gene expression in the sensory neurons of the nematode Caenorhabditis elegans (Chalfie et al. 1994). GFP has since been engineered to produce a vast number of useful blue, cyan, and yellow mutants that are broadly referred to as FPs. More recently, FPs from a variety of other species have been identified, resulting in further expansion of the color palette into the orange and red spectral regions (Matz et al. 1999). With the continued development in FP technology, genetically encoded probes are now becoming fully appreciated for a wide variety of applications.

Features of A. victoria GFP

One of the most important things to appreciate about GFP is that the entire 27-kDa structure is essential to the development and maintenance of its fluorescence (Remington 2006). Remarkably, the principal chromophore is derived from just three amino acids: Ser65, Tyr66, and Gly67 (Fig. 1 ). Although this simple amino acid motif is commonly found throughout nature, it does not generally result in fluorescence. The feature unique to GFP and other autofluorescent FPs is that these critical amino acid residues are located very near the center of a remarkably stable barrel-shaped structure consisting of 11 β-sheets that surround a central -helix (Fig. 1A; Ormö et al. 1996). The β-sheets are linked through less-ordered proline-rich loops, and the amino acid side chains in each sheet alternately project into the protein interior or outward from the surface. Unlike most soluble proteins, many interior residues are charged or polar, and they bind numerous water molecules in place by hydrogen bonds to the amino acids.

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Figure 1. Structure of the GFP β-barrel, EGFP chromophore formation, and general FP chromophore structures. (A) β-Barrel architecture and approximate dimensions; (B) steps in the formation of the EGFP chromophore; (C) EBFP chromophore; (D) ECFP chromophore (the tryptophan residue [Trp66] is illustrated in the cis conformation as occurs for Cerulean derivatives); (E) EGFP chromophore; (F) EYFP chromophore; (G) FPs derived from DsRed and other Anthozoa organisms thought to have a cis-chromophore. The residue at position 66 can be Met, Gln, Thr, Cys, or Glu. (H) eqFP611, a red variant derived from E. quadricolor, is the only known FP featuring a trans-chromophore. (I) ZsYellow (also zFP538), derived from the button polyp Zoanthus, features a novel three-ring chromophore that forms when the lysine residue at position 66 cyclizes with its own -carbon to form a tetrahydropyridine ring conjugated to the chromophore. (J) mOrange, one of the mFruit proteins, and mKO also feature a three-ring chromophore in which Thr66 (or Cys65) cyclizes with the preceding carbonyl carbon to yield a conjugated oxazole (mOrange) or thiazole (mKO) ring.

In the context of this special environment, a reaction occurs between the carboxyl carbon of Ser65 and the amino nitrogen of Gly67 that results in the formation of an imidazolin-5-one ring (Fig. 1B). Further oxidation results in conjugation of the imidazoline ring with Tyr66 and maturation of the fluorescence species. Importantly, the native GFP chromophore exists in two states: a predominant, protonated state with an excitation maximum at ~395 nm and a less prevalent, non-protonated state with an excitation peak at ~475 nm (Tsien 1998). Regardless of the excitation wavelength, fluorescence emission is maximal at ~507 nm.

The mechanism of chromophore formation is thought to be similar in every FP so far discovered, regardless of the source (Remington 2006). Examination of the amino acid sequences for more than 100 naturally occurring variants from many species reveals that only four residues are absolutely conserved. These are Tyr66 and Gly67, which form the chromophore, as well as Arg96 and Glu222. The location of Gly67 is critical to enable formation of the cyclized chromophore through nucleophilic attack. Therefore, unsurprisingly, the addition of a side chain to this amino acid completely destroys the ability of FPs to create their chromophore. Considering the fact that any aromatic residue can replace Tyr66, it is curious that this amino acid is so highly conserved, but it may be necessary to ensure complete and successful maturation of the chromophore. Arg96 and Glu222 are catalytic residues positioned near the chromophore that are essential to the maturation process (Sniegowski et al. 2005).

One feature of GFP that has important implications for its use as a probe is that its fluorescence is highly dependent on the structure surrounding the chromophore. As a result, the photophysics of GFP as a fluorophore are quite complicated, but surprisingly, the molecule can still accommodate quite a bit of modification. The rigid structure and molecular organization of the β-barrel interior is responsible for the unique chemical environment that creates and harbors fluorescence in FPs as evidenced by the fact that synthetic chromophore analogs are devoid of fluorescence (Follenius-Wund et al. 2003). Changes to this environment can produce dramatic variations in spectral characteristics (up to hundreds of nanometers), photostability, acid resistance, and a variety of other physical properties.

Much has been done over the past decade to fine-tune the fluorescence of the native GFP to provide a broad range of molecular probes, but the vast potential of GFP as a starting material for constructing molecular probes cannot be understated. Denaturation of GFP destroys its fluorescence, and mutations to residues surrounding the chromophore can significantly change the fluorescent properties of GFP (Stepanenko et al. 2008). In fact, mutations to residues far removed from the chromophore and seemingly benign to fluorescence can also dramatically alter the chemical and photophysical properties of GFP. The tight packing of residues inside the β-barrel results in a very-high fluorescence quantum efficiency (up to 80%) (Patterson et al. 1997). This rigid protein structure can also confer resistance to changes in pH, temperature, and denaturants such as urea and guanidinium chloride (Ward 2006). Mutations in GFP that affect its fluorescence generally have negative effects on this stability, resulting in a reduction of quantum efficiency and greater environmental sensitivity. Although some of these defects can be compensated for by additional mutations, derivative FPs are generally more sensitive to the environment than native GFPs. These limitations should be considered when designing experiments.

Even though several hundred FP derivatives have now been reported in the literature (Patterson 2007), most research groups use the enhanced green fluorescent protein (EGFP) version of the original A. victoria GFP (Shaner et al. 2007), and many still use the original enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) derivatives (Fig. 1; Cubitt et al. 1995; Patterson 2007). The reluctance of researchers to transition to newer variants derives at least in part from confusion about FP spectral characteristics and availability together with uncertainties about reported claims of improved brightness, photostability, and other properties. The lack of dependable commercial sources often requires researchers to rely on the generosity of the original laboratory, which, in turn, can be bombarded with an avalanche of requests shortly after a new protein or fusion construct has been reported. Barring the implementation of an organized and efficient system for the distribution of FP variants to the scientific community, this situation is unlikely to improve in the near future.

Mutations That Improve Use of FPs for Mammalian Systems

To adapt GFPs for use in mammalian systems, several properties of the original GFP were modified and are now found in all commonly used variants. First, the maturation of the fluorescence was optimized for 37°C. Maturation of the wild-type GFP chromophore is quite efficient at 28°C, but increasing the temperature to 37°C substantially reduces the maturation rate and results in decreased fluorescence (Patterson et al. 1997). A single mutation of Phe64 to Leu (F64L) (Cormack et al. 1996) results in dramatically improved maturation of fluorescence at 37°C, which is at least equivalent to the maturation rate at 28°C. This mutation is present in the most popular varieties of A. victoria-derived FPs, but is not the only mutation that improves folding at 37°C or other critical properties (for a review, see Tsien 1998). Among the other important mutations are F46L, V68L (enhances chromophore oxidation), Q69M (improves chloride and pH resistance and photostability in YFPs), N149K (improves folding rate), F99S, M153T, V163A (reduces hydrophobicity; enhances folding), I167T (reduces thermosensitivity), and S175G (reduces aggregation). Additional folding mutations were uncovered during the construction of a variant known as superfolder GFP (discussed below) (Pédelacq et al. 2006), which was engineered by fusing libraries of shuffled GFP sequences to polypeptides that by themselves exhibit poor folding. In all, six mutations in superfolder GFP were identified that have the potential to enhance folding and maturation in A. victoria GFP derivatives.

In addition to improving maturation rates at 37°C, redesigning the nucleic acid sequence to coincide with codon preferences of the host organism has also improved the utility of GFP derivatives (Yang et al. 1996; Zacharias and Tsien 2006). For example, more than 190 silent mutations were introduced into the coding sequence to optimize expression in human tissues (Yang et al. 1996). Further improvements in performance can be achieved by including the proper protein translation initiation sequence (Kozak 1987). Thus, installing a new codon beginning with a G immediately after the start codon (Met, AUG) is sufficient to produce the Kozak consensus site but introduces an extra amino acid (preferably Val or Ala) into the sequence. In Aequorea variants, the amino-terminal region of the protein is tolerant to such additions, but the same may not hold true for other FPs.

Monomeric FPs

In their naturally occurring states, virtually all FPs are oligomeric (usually dimeric or tetrameric). As an example, wild-type A. victoria GFP is thought to participate in a tetrameric complex with aequorin (Ward 2006). Fortunately, this complex has only been observed in vitro at very high protein concentrations, and the tendency to dimerize in commonly used A. victoria-derived FPs is generally very weak (K_d > 100 µm) (Zacharias et al. 2002). However, when these probes are targeted to specific cellular compartments, such as the plasma membrane, the local concentration can theoretically become high enough to permit dimerization. The A206K mutation on the β-barrel surface has been found to practically eliminate the dimerization of A. victoria GFPs (Zacharias et al. 2002). Eliminating the tetrameric oligomerization state of coral proteins is far more complicated, but efforts in the past several years have led to significant progress in producing monomeric coral FPs (Shaner et al. 2007).

Oligomerization is a significant concern for the proper localization of FP fusions (particularly those involved in the formation of biopolymers, such as microtubules), and it is of paramount importance when performing fluorescence resonance energy transfer (FRET) experiments where even the slightest tendency of an FP to dimerize could confound data interpretation. The fluorescence images presented in Figure 2 illustrate correct subcellular fusion protein localization of FPs from jellyfish and corals that have been rendered monomeric. Collectively, these improvements to the oligomeric character of jellyfish, coral, and anemone FPs have resulted in a very useful armament of probes for live cell imaging of mammalian, plant, insect, and prokaryote cells.

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Figure 2. Fluorescence imaging of FP-fusion constructs targeting subcellular locations. Construct images are designated as FP-fusion partner-amino (N)- or carboxy (C)-terminal (with respect to the FP) number of linker amino acids. (A) mCherry-H2B-N-6; (B) mWasabi-mitochondria-N-7; (C) mCitrine-Cx43-N-7; (D) mCerulean-cytokeratin-N-17; (E) mApple-annexin (A4)-C-12; (F) mEmerald-vinculin-C-23; (G) mEGFP-EB3-N-7; (H) mKO-Golgi-N-7; (I) mCherry-vimentin-N-7; (J) mTagBFP-lysosomes-C-20; (K) mCerulean-lamin B1-C-10; (L) mKO2-farnesyl-C-5; (M) mTFP1-β-actin-C-7; (N) mOrange2-peroxisomes-C-2; (O) mApple-VASP-C-5; (P) mEmerald- -tubulin-C-6; (Q) mCherry-clathrin (light chain)-C-15; (R) mEGFP-VE-cadherin-N-10; (S) TagRFP-T-endosomes-C-14; (T) mVenus-CENPB-N-22; (U) mCerulean-zyxin-N-6; (V) mKO-fibrillarin-C-7; (W) mECFP-endoplasmic reticulum-N-5; (X) mApple--actinin-N-19; (Y) mEmerald-LC-myosin-N-10; (Z) mPlum--tubulin-N-17; (AA) EGFP-β-catenin-N-7; (AB) mApple-profilin-C-10; (AC) mKO-Pit1-N-6; (AD) mEGFP-TPX2-N-10.

THE FP COLOR PALETTE

A broad range of FP variants has now been produced that spans nearly the entire visible spectrum (Table 1). Mutagenesis of A. victoria GFP has resulted in FPs that range in color from blue to yellow, covering an 80-nm spectral range (Fig. 1C-F). New orange and red FPs from reef coral and anemone species have been discovered and adapted for use in mammalian systems. These FPs feature a more diverse family of chromophores with emission spanning a much larger 200-nm spectral region (Fig. 1G-J). Two guiding principles regarding the manipulation of FP emission color have emerged in the past few years (Remington 2006). First, local environmental variables in the chromophore neighborhood can induce blue or red spectral shifts in the absorption and emission spectra by 20-30 nm. These variables include the position of charged amino acid residues, hydrogen-bonding networks, and hydrophobic interactions within the protein matrix. Second, larger spectral shifts that are used to distinguish the general FP color class (i.e., cyan, green, yellow, etc.) occur because of significant differences in the chromophore covalent structure or the degree of -orbital conjugation that extends into the polypeptide backbone.

In addition to expanding the range of available colors, the brightness, acid resistance, oligomeric character, and photostability of FPs have been enhanced to improve their overall usefulness. The following sections provide an overview of the many different FP variants and highlight some of the more important practical considerations for their use.

Blue FPs

Blue variants of GFP result from the substitution of histidine for tyrosine at position 66 in the chromophore (Fig. 1C) and feature blue emission with a maximum at ~450 nm (Heim et al. 1994). Unfortunately, the original blue variant and an enhanced blue fluorescent protein (EBFP) version containing secondary mutations to increase folding efficiency and brightness still lack the necessary photostability for use in live cell imaging (Patterson et al. 2001). Recent mutagenesis efforts in blue variants of A. victoria GFP have resulted in improved FPs named Azurite, SBFP2, and EBFP2 (Mena et al. 2006; Ai et al. 2007; Kremers et al. 2007), which are brighter and more photostable than EBFP but remain less than half as bright as EGFP. The most promising BFP is derived from a sea anemone and is named TagBFP (Subach et al. 2008). Almost equaling the brightness of EGFP, TagBFP matures faster than blue A. victoria variants and demonstrates similar photostability. Although long-term imaging in live cells using the ultraviolet wavelengths necessary to excite BFPs remains a problem because of phototoxicity, these derivatives offer significant potential as FRET donors to GFPs and YFPs in shorter-term investigations. None of the A. victoria BFP derivatives are commercially available, but TagBFP can be purchased from Evrogen. BFPs can be imaged using a standard DAPI (4'6-diamidino-2-phenylindole) filter set, although a set designed specifically for these FPs can improve imaging performance.

Cyan FPs

Replacing Tyr66 in the chromophore of GFP with tryptophan produces a cyan-emitting variant CFP (Fig. 1D) with a bimodal emission spectrum featuring a major peak at ~480 nm with a shoulder at ~500 nm (Heim et al. 1994). Several CFP variants have been constructed to improve maturation and photostability. The most notable is Cerulean (Rizzo et al. 2004), which is twice as bright as CFP and currently is one of the best choices for imaging in this spectral region. Furthermore, Cerulean is also useful as a FRET donor for yellow and orange FPs in biosensors and has seen extensive application in this area (Piston and Kremers 2007).

A recently described monomeric teal-colored FP (mTFP1) derived from coral exhibits even higher brightness, acid insensitivity, and photostability than any of the cyan A. victoria variants (Ai et al. 2006). mTFP1 has spectral characteristics that are slightly red-shifted with respect to most CFPs and features Tyr rather than Trp at position 66, which reduces the broad fluorescence emission spectral width to ~30 nm from the ~60 nm observed with cyan GFP variants.

Studies to optimize the folding of monomeric A. victoria variants have resulted in supercyan derivatives (Kremers et al. 2006) that are significantly brighter than the parent proteins. These FPs along with mTFP1 are potentially useful for fusion tags and in creating new, advanced CFP-YFP FRET biosensors exhibiting high dynamic range. ECFP can be purchased from Invitrogen, and several coral CFP derivatives are available from commercial sources (Allele Biotechnology, Evrogen, Clontech, and MBL International Corporation). CFPs require a special filter set for observation and imaging, but these are readily available from the microscope and filter manufacturers.

GFPs

Following the discovery of GFP in A. victoria, several analogous FPs emitting in the green spectral region have been isolated from a variety of species, including reef corals and sea anemones (Matz et al. 1999), copepods (Masuda et al. 2006), and amphioxus (Deheyn et al. 2007). Although native GFP is brightly fluorescent and reasonably photostable, the higher peak in the bimodal excitation spectrum (395 nm) lies at the border of the ultraviolet range. Unfortunately, ultraviolet light requires special optical considerations in microscopy and can be damaging to cells during imaging. Early on in the mutagenesis efforts of GFP, Tsien and associates discovered that a single-point mutation (S65T) (Heim et al. 1995) could be introduced to eliminate the 395-nm peak and shift the excitation maximum to 488 nm. This mutation is featured in the most popular variant of GFP, the EGFP that is used by most investigators (Fig. 1E). As an added bonus, EGFP can be imaged using commonly available fluorescein isothiocyanate (FITC) filter sets and is among the brightest and most photostable of the currently available GFPs. These features have made EGFP both the most popular FP and the best choice for most single-label FP experiments. The only drawbacks to the use of EGFP are slight pH sensitivity and a weak tendency to dimerize.

Other than EGFP, there are several other GFPs currently in use. The best of these in terms of photostability and brightness is a variant known as Emerald that contains S65T in addition to five mutations (F64L, S72A, N149K, M153T, and I167T) that improve maturation speed and overall performance (Cubitt et al. 1999). The popular EGFP-cloning vectors are no longer available from Clontech, but Emerald is commercially available from Invitrogen under the trade name EmGFP. Other companies sell humanized variants of GFP that offer distinct advantages for FRET experiments. For example, PerkinElmer offers GFP² (F64L), which retains the 400-nm excitation peak and can be used as a FRET partner for EYFP (Zimmermann et al. 2002).

Several GFPs derived from reef corals have demonstrated satisfactory performance and include Azami Green (Karasawa et al. 2003), mWasabi (Ai et al. 2008b), ZsGreen (Matz et al. 1999), and TagGFP (Xia et al. 2002). All of these FPs are commercially available, but none match the performance of EGFP or Emerald. The most significant addition to the green palette in the past several years is superfolder GFP (Pédelacq et al. 2006), which can fold very efficiently even when fused to insoluble fusion proteins and is slightly brighter and more acid-resistant than either EGFP or Emerald. This high-performance version of GFP is not yet commercially available and may suffer from higher background-noise levels due to improperly folded fusion partners that fail to target correctly but still produce bright fluorescence.

YFPs

The first YFPs were created after the crystal structure of GFP revealed that Thr203 was positioned very close to the chromophore (Ormö et al. 1996). Mutation of this residue to Tyr was introduced to stabilize the excited-state dipole moment of the chromophore and resulted in an ~20-nm redshift of both the excitation and the emission spectra (Ormö et al. 1996). Further development led to the generation of EYFP, which is one of the brightest and most popular FPs (Fig. 1F). The brightness and fluorescence spectrum make EYFP well suited for fluorescence microscopy and multicolor FP experiments. It is also useful for energy-transfer experiments when paired with ECFP or GFP2. However, EYFP is far from perfect. The FP is very sensitive to acidic pH and becomes only ~50% fluorescent at pH 6.5 (Patterson et al. 2001). In addition, EYFP is also sensitive to chloride ions and photobleaches much more quickly than the GFPs.

Many of the problems with EYFP have been solved through mutagenesis. The Citrine variant (Griesbeck et al. 2001) is brighter and more resistant to photobleaching, acidic pH, and other environmental effects, whereas Venus (Nagai et al. 2002) is the fastest maturing variant discovered to date. A commercially available derivative (Topaz; Invitrogen) behaves similarly to Citrine, and the brightest YFP (YPet) (Nguyen and Daugherty 2005) was obtained during efforts to enhance YFP performance as an acceptor in FRET pairs with CFPs. YFPs derived from corals are commercially available, but none exhibit performance comparable to the jellyfish derivatives.

YFPs hold a unique position in the color palette in that they can serve as both a FRET donor to red and far-red FPs and as a FRET acceptor to BFPs and CFPs. However, YFPs should be used with caution in photobleaching experiments (i.e., fluorescence recovery after photobleaching [FRAP] and FRET-acceptor photobleaching) because they can spontaneously regain significant levels of fluorescence after apparently being photobleached (Sinnecker et al. 2005; Shaner et al. 2008) or even switch to a cyan-colored species (Valentin et al. 2005; Kirber et al. 2007). YFPs can be observed using a standard FITC or GFP filter set, but for optimal live cell imaging, a set specially tuned for the emission profiles of these variants is recommended.

Orange FPs

FPs exhibiting emission in the orange wavelengths (~560-585 nm) have all been derived from the Anthozoa species and now feature some promising candidates for tracking and dynamics as well as FRET acceptors. Although many of these probes are referred to as red FPs, popular variants such as DsRed (Matz et al. 1999) and TagRFP (Merzlyak et al. 2007) actually have emission profiles that are clearly more orange than red. Regardless of nomenclature discrepancies, FPs in the orange spectral class are readily imaged in multicolor experiments using a standard TRITC (tetramethyl rhodamine isothiocyanate) filter set and are efficiently excited with the common 543-nm and 561-nm lasers found on modern confocal instruments.

Currently, the most useful orange FPs are mKusabira Orange (mKO) (Fig. 1J; Karasawa et al. 2004) and its faster folding derivative mKO2 (Sakaue-Sawano et al. 2008), TagRFP (Merzlyak et al. 2007), mOrange2 (Shaner et al. 2008), and tdTomato (a tandem dimer) (Shaner et al. 2004). mKO is slightly less bright and photostable than EGFP, but mKO2 is brighter and matures much faster. TagRFP, although relatively bright, is marginal with respect to photostability but has a closely related derivative, TagRFP-T (Shaner et al. 2008), that is among the most photostable FPs yet developed. Either version works well in fusion tags. mOrange2 and tdTomato are members of the mFruit family pioneered by Tsien and coworkers (Shaner et al. 2004) (discussed below) and perform quite well as fusions. Counting its two intrinsic fluorophores, tdTomato is the brightest FP of any color, but it contains two copies of the dimeric Tomato FP joined with a 12-residue linker. The larger size of tdTomato may interfere with fusion protein packing in some biopolymers, but this FP works well in fusions to targeting signals that do not require strict biomolecular organization (such as peptide sequences that localize the FP to the membrane, mitochondria, and Golgi).

Orange FPs all feature reasonably high-extinction coefficients and should perform well as FRET acceptors when paired with cyan-, green-, and yellow-donor FPs with high quantum yield. Because all of these proteins were originally derived from the Anthozoa species, their oligomeric character has been significantly altered through (usually extensive) mutagenesis, which may affect a wide variety of other properties. In multicolor experiments, orange FPs can be paired with BFPs, CFPs, GFPs, and far-red FPs with reasonable separation of emission spectral profiles using commercially available filter sets.

Red FPs

The holy grail of FP development has become an effective red FP that is just as useful as EGFP. The advantages of red FPs range from excellent compatibility with existing confocal-microscope lasers and filter sets to the enhanced ability for high-contrast imaging in cells with low-autofluorescence background, and even deep imaging in whole animals, which are more transparent to red light. Because red-shifting the fluorescent properties of A. victoria FPs beyond yellow has been unsuccessful, several groups have extensively searched for a high-performance red FP among the tropical corals and sea anemones (Matz et al. 1999; Wiedenmann et al. 2002; Merzlyak et al. 2007). To complicate the situation further, virtually all of the red FPs so far discovered exist naturally in an obligate dimeric or tetrameric intermolecular association state, which hampers their use for most applications in live cell imaging. Before a truly high-performance red FP could be developed, the oligomerization problem had to be solved. The Tsien laboratory successfully disaggregated a tetrameric FP derived from Discosoma striata, commonly referred to as DsRed, through the introduction of 33 mutations throughout the β-barrel structure. The result was a rather dim (40% of EGFP brightness) monomeric red FP that was named mRFP1 (Campbell et al. 2002). Although mRFP1 photobleaches rapidly in wide-field illumination (~10% of EGFP photostability), it proved useful in fusions and was the only available monomeric probe in this class for several years.

Continued mutagenesis efforts on mRFP1, including adding several amino- and carboxy-terminal amino acids from EGFP as well as directed targeting of residues surrounding the chromophore, resulted in a series of monomeric FPs exhibiting fluorescence emission maxima at wavelengths from 560 nm to 610 nm (Shaner et al. 2004). Further extension of this work through a novel protein-engineering technique known as iterative somatic hypermutation (Wang et al. 2004) yielded the first true far-red genetically engineered FP, mPlum. Collectively, these FPs were named after common fruits that bear colors similar to their emission profiles and are thus referred to as the mFruits.

Notable members of the mFruit series are mApple, mCherry, and mPlum (Table 1), which exhibit emission maxima at 592 nm, 610 nm, and 649 nm respectively. mApple is one of the brightest-red FPs (Shaner et al. 2008) and features spectral characteristics similar to the popular small organic fluorophore Alexa Fluor 568. mCherry is only half as bright as mApple but is spectrally similar to Texas Red. Both FPs are relatively bright and photostable, perform well in fusions, and thus are excellent candidates for multicolor labeling in combination with BFPs, CFPs, GFPs, YFPs, or orange FPs. mPlum is currently the only choice for a monomeric far-red FP in the >650-nm spectral region. Unfortunately, mPlum is only 10% as bright as EGFP and has proven to be problematic in a number of fusion proteins. Several of the mFruit proteins are commercially available from Clontech.

A wide variety of other red FPs has been isolated from the Anthozoa species, and some may prove useful in live cell and transgenic animal investigations (Table 1). Among these are AsRed2 (Matz et al. 1999), HcRed1 (and its tandem-dimer variant) (Gurskaya et al. 2001; Fradkov et al. 2002), and JRed (Shagin et al. 2004), which are commercially available from Clontech and Evrogen. These FPs are all dimeric in character and will thus probably be limited for live cell imaging applications. In contrast, FPs derived by mutagenesis of the sea anemone Entacmaea quadricolor tetrameric proteins, eqFP578 and eqFP611 (Fig. 1H), are beginning to show promise (Shcherbo et al. 2007, 2009; Kredel et al. 2009). One of these, Katushka (Shcherbo et al. 2007), is dimeric but exhibits the highest brightness level (emission maximum at 635 nm) of any FP in the spectral window above 650 nm, a region that is important for deep-tissue imaging. Recently, a tandem dimer of Katushka was introduced for the construction of fusion tags (Shcherbo et al. 2009).

Introduction of the four principal Katushka mutations into TagRFP generated a monomeric, far-red protein named mKate that features similar spectral properties (Shcherbo et al. 2007), and continued efforts have resulted in mKate2, a brighter and more photostable version (Shcherbo et al. 2009). In a separate effort, extensive mutagenesis of eqFP611 has resulted in a relatively bright FP named mRuby, which exhibits reasonable performance in fusions but is less photostable than other red FPs (Kredel et al. 2009). Although none of the currently available red FPs excels in all properties, the continued incremental improvements suggest that bright, photostable, and monomeric probes in this spectral class should eventually be available.

Long Stokes Shift FPs

Aside from engineering FPs to modulate emission wavelength, acid stability, and resistance to photobleaching, mutagenesis has also targeted the separation distance between excitation and emission maxima (Stokes shift) to generate more suitable probes for FRET and multicolor imaging. In the first example, the substitution of Thr for Ile at position 203 in native GFP (Tsien 1998) eliminated the 475-nm absorption peak and produced a variant named Sapphire that exhibits a dramatic Stokes shift of 112 nm (excitation and emission maxima at 399 nm and 511 nm, respectively). Four additional mutations improved the maturation and brightness to generate T-Sapphire (T for "Turbo") (Zapata-Hommer and Griesbeck 2003), a robust protein that is an excellent donor in FRET combinations with orange and red FPs. A more extensive mutagenesis effort on Aequorea BFP derivatives resulted in a new YFP, named mAmetrine (Ai et al. 2008a), which exhibits a Stokes shift of 120 nm with excitation and emission maxima red-shifted by ~10 nm with respect to Sapphire. mAmetrine was used to demonstrate the simultaneous application of two independent FRET biosensors in live cells.

The longest Stokes shift yet reported in any FP (180 nm) arose from mutagenesis of a chromoprotein derived from the stony reef coral (Kogure et al. 2006). This effort initially yielded several tetrameric FPs, named Keima after the Japanese chess piece, displaying an absorption peak at ~440 nm and emission peaks at ~580-620 nm. Continued rounds of mutagenesis produced a dimer (dKeima) and monomer (mKeima) that feature emission maxima at 580 nm and 620 nm, respectively. The monomeric version of Keima has limited brightness and requires a specialized filter combination for imaging but has been successfully used for fluorescence cross-correlation spectroscopy and simultaneous multicolor imaging with five other FPs. A tandem-dimer version (tdKeima) (Kogure et al. 2008) is brighter than the monomer but twice the size.

In summary, promising candidates now exist in all major FP spectral classes, although in most cases, there remains no EGFP equivalent in terms of photostability, brightness, and utility in fusions. New additions to the blue and cyan palettes have significantly increased the usefulness and performance of these probes, and a significantly expanded selection in the orange and red spectral regions now offers more choices for multicolor imaging. Although issues remain for these red and far-red FPs, we are optimistic that high-performance variants will ultimately become available in this spectral class, given the fact that most of the probes listed in Table 1 have been introduced only in the past couple of years.

Optical-Highlighter FPs

One of the most interesting developments in FP research has been the use of these ubiquitous probes as optical or molecular highlighters (Table 2) to observe cellular dynamics and for emerging super-resolution microscopy techniques (Shaner et al. 2007; Fernández-Suárez and Ting 2008). For example, the photoactivatable green fluorescent protein (PA-GFP) version contains a single-point mutation (H203T) to the native GFP that enables irreversible photoconversion of the excitation peak from ultraviolet to blue by illumination with 400-nm light (Patterson and Lippincott-Schwartz 2002). Unconverted PA-GFP has an excitation peak similar to the wild-type GFP (~400 nm). After photoconversion, the 400-nm peak is substantially reduced, and the excitation spectrum at 488 nm increases ~100-fold. This allows very high contrast between the unconverted and converted pools of PA-GFP and is useful for tracking the dynamics of subpopulations of molecules within a cell. Such photoactivatable proteins serve as a foundation for the generalized overall function of optical highlighters, but many of the new and more advanced FP variants have been engineered to be capable of photoconversion (switching from one emission wavelength to another) and/or photoswitching (the ability to selectively turn fluorescence on and off). Figure 3 illustrates several examples of optical highlighters being used for tracking and dynamics in live cells, as well as their application in fixed cells for super-resolution imaging.

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Figure 3. Optical-highlighter FPs in action imaged with laser-scanning confocal microscopy. (A-C) Photoconversion of gap junctions (GJs) labeled with mEos2-Cx43-N-7 in HeLa cells. (A) Photoconversion of a GJ plaque (red) in a selected region (white box) with 405-nm illumination at t = 0. (B) New plaque growth and fusion of a nonconverted plaque, t = 70 min. (C) Plaque after rotation through ~90° revealing photoconverted area surrounded by new growth, t = 120 min. (D-F) Photoactivation of mPA-GFP-β-actin-C-7 expressed in opossum kidney cells. (D) Circular region of interest (white) is illuminated at 405 nm for 5 sec at t = 0. (E) The photoactivated actin chimera initially translocates to membrane ruffles as fluorescence intensity decreases in the activated region, t = 15 min. (F) The filamentous actin network gains more intensity at t = 60 min. (G-I) Photoswitching of the mitochondria with KFP1-mito-N-6 in fox lung cells. (G) Labeled mitochondria imaged with a 543-nm laser in both fluorescence and differential interference contrasts, t = 0. (H) After completely photoswitching the labeled chimera off with 488-nm illumination, the mitochondria now appear devoid of fluorescence, t = 3 min. (I) KFP1 label in mitochondria, reactivated with illumination at 543 nm, does not significantly photobleach after five rounds of photoswitching. (J-L) Super-resolution (PALM) imaging of tdEos-vinculin-C-14 in fox lung cells. (J) Wide-field total internal reflection fluorescence image of a focal adhesion. (K) Summed PALM image of boxed region in J. (L) PALM image of tdEos fusion.

Photoactivatable FPs

Other than PA-GFP (Fig. 3D-F), there has been limited progress in creating useful photoactivatable FPs in other spectral regions. Among the problems of developing and using these probes is the difficulty of detecting photoactivatable FPs in their nonactivated state. In addition, PA-GFP variants based on Emerald and superfolder GFP exhibit reduced dynamic range, likely because their nonactivated state is significantly brighter than that of PA-GFP. Early attempts to engineer photoactivatable red FPs using mRFP1 (Verkhusha and Sorkin 2005) were marginally successful because of the lack of brightness from the activated species, but variants based on mCherry may prove more useful (Subach et al. 2009). PA-mCherry1 features an absorption maximum at 404 nm before and 564 nm after photoactivation. The FP is about one-half as bright as PA-GFP, is equally photostable as the parent (mCherry), and has been demonstrated to perform well in two-color photoactivation imaging with PA-GFP as well as super-resolution photoactivated localization microscopy (PALM) (Fig. 3J-L).

A unique optical highlighter obtained from jellyfish (Aequorea coerulescens), which photoconverts from cyan to green fluorescence upon illumination at 405 nm, also appears to function through a similar mechanism as PA-GFP (decarboxylation of Glu222). Known as PS-CFP2 (Chudakov et al. 2004), this dual-function FP has an advantage over PA-GFP in that a significant level of cyan fluorescence is present before photoconversion, which enables easier determination of regions for selective illumination. Unfortunately, the low dynamic range of PS-CFP2, coupled with limited brightness, renders this probe inferior in terms of photoconversion performance to the green-to-red highlighters that are discussed below.

Photoconvertible FPs

Most of the photoconvertible optical-highlighter FPs feature a common chromophore derived from the cyclized tripeptide His-Tyr-Gly that initially emits green fluorescence (Fig. 4A,B ). Irradiation with ultraviolet light (~365-410 nm) induces cleavage between the amide nitrogen and -carbon atoms in the histidine residue with subsequent extension of chromophore conjugation to include the histidine side chain (Hayashi et al. 2007). Peptide backbone cleavage requires catalysis by the intact protein and results in a shift of emission to orange-red wavelengths. All of the optical highlighters thus far discovered that undergo an irreversible photoconversion between green and orange-red emission are thought to follow this rather unconventional chemistry.

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Figure 4. (A,B) Photoconversion and (C,D) photoswitching mechanisms for optical-highlighter FPs. Green-to-red photoconversion for Kaede, KikGR, Dendra2, and Eos, all of which contain the HYG chromophore, occurs when the (A) GFP species is illuminated with ultraviolet or violet radiation, which induces cleavage between the amide nitrogen and -carbon atoms in the histidine 62 residue, leading to (B) formation of a conjugated dual imidazole ring system and red fluorescence. Photoswitching of Dronpa and mKFP1 involves cis-trans photoisomerization of the chromophore tyrosyl moiety induced by alternating radiation. (C) The cis isomer produces fluorescence, whereas (D) the trans isomer is dark.

Among the current photoconvertible green-to-red highlighters, the best choices for cellular dynamics and super-resolution investigations are mEos2 (Fig. 3A-C; McKinney et al. 2009), tandem-dimer Eos (tdEos) (Nienhaus et al. 2006), and Dendra2 (Gurskaya et al. 2006). All of these variants were derived from reef corals and have undergone extensive mutagenesis to create monomeric (or tandem-dimer) derivatives from the original tetramers (Table 2). Green fluorescence emission for these FPs occurs with maxima ranging from 506 nm to 519 nm. Upon irreversible photoconversion, the emission maxima shift to orange wavelengths ranging from 573 nm to 581 nm. mEos2 features the highest brightness in the green state, followed by tdEos and Dendra2. The photoconverted orange state of tdEos is brighter than mEos2, which is brighter than Dendra2. Photostability of the orange state is significantly higher in all three of these highlighters but does not significantly vary between them. A recent addition to the monomeric green-to-red highlighter palette is mKikGR (Habuchi et al. 2008), which is dimmer and less photostable than Dendra2 or the Eos variants. Dimeric and tetrameric variants of Eos (Wiedenmann et al. 2004), Kaede (Ando et al. 2002), and KikGR (Tsutsui et al. 2005) are generally brighter than their monomeric analogs, but are less photostable across the board and are useful only for those applications that tolerate oligomeric FPs.

Photoswitchable FPs

Optical highlighters that can be toggled on or off by illumination with two different excitation wavelengths are referred to as photoswitchable FPs. The most notable member of this group is named Dronpa, which is a monomeric variant derived from a stony coral tetramer (Ando et al. 2004). Dronpa features an absorption maximum at 503 nm (anionic, deprotonated chromophore) with a minor peak at 390 nm (neutral, protonated chromophore). When irradiated at 488 nm, the anionic chromophore emits green fluorescence with a maximum at 518 nm and a brightness level almost 2.5 times that of EGFP. Photoswitching of Dronpa occurs in part by interconversion between the deprotonated (on, bright) and protonated (off, dark) forms, and 488-nm illumination drives Dronpa to the dark species. The FP can be subsequently switched back on by brief illumination at 405 nm. This cycle can be repeated several hundred times before photobleaching becomes a significant factor. The mechanism of FP photoswitching is thought to also arise from cis-trans isomerization of the hydroxybenzilidine (tyrosyl side chain) chromophore moiety that accompanies the changes in the protonation state discussed above (Fig. 4C,D). Similar to other highlighters, Dronpa is useful both for dynamics and for super-resolution studies (Fernández-Suárez and Ting 2008). Variants of Dronpa with reversed photoswitching properties and broader spectra, as well as photoswitchable mCherry derivatives, have also been reported (Andresen et al. 2008; Stiel et al. 2008), but application of these rather dim FPs has so far been limited to evaluating their performance in super-resolution imaging. Future versions will no doubt feature higher brightness and should prove useful in dynamics investigations.

Several photoswitching FPs in other regions of the color palette have been developed from anemones and corals. Kindling FP (Chudakov et al. 2003) (commercially available from Evrogen as KFP1) is a tetrameric highlighter that emits red fluorescence at 600 nm upon illumination with green or yellow light (525-580 nm). Upon cessation of illumination, KFP1 relaxes back to its initial nonfluorescent state (Fig. 3G-I). Irradiation with intense blue light (450-490 nm) completely quenches KFP1 fluorescence immediately, enabling control over the photoswitching. A CFP from coral named mTFP0.7 (an intermediate in mTFP1 mutagenesis) has also been demonstrated to photoswitch but has not been characterized in living cells.

Monomeric photoswitchable variants of mCherry have recently been introduced (Stiel et al. 2008) as potential probes for super-resolution microscopy. These derivatives, termed rsCherry and rsCherryRev, display antagonistic switching modes such that irradiation of rsCherry with yellow light induces the bright state, and blue light drives the FP to the dark state, whereas the reverse is observed with rsCherryRev. Both FPs are only ~10% as bright as mCherry when expressed as ensembles in cells but are equally as bright as mCherry on the single-molecule level.

The optical-highlighter FPs produced thus far from GFP derivatives and reef coral proteins show sufficient promise to warrant aggressive efforts to solve problems associated with oligomerization, limited brightness, and low photostability as well as fine-tuning of their spectral profiles. In the future, engineering of more advanced optical highlighters should be able to shift photoactivation wavelengths to the less phototoxic blue and green spectral regions while simultaneously pushing emission wavelengths into the yellow through far-red bands, efforts that will significantly expand the potential applications of these probes.

FUTURE DIRECTIONS

Currently, the focus of FP development is centered on two basic goals. The first aim is to perfect the ever-growing palette of A. victoria-derived FPs. The second aim is to obtain useful red and deep-red FPs that are essentially equivalent in performance to EGFP. Progress toward these goals has been quite impressive. The latest generation of A. victoria variants has solved most of the deficiencies of the first generation of FPs, particularly for the CFPs, GFPs, and YFPs. The search for a monomeric, bright, and fast-maturing red FP has introduced several new and interesting classes, particularly from corals and anemones. As spectral-separation techniques become better developed and more commonly employed, the expectation is that these newer varieties will supplement the existing palette, especially in the yellow, orange, and red regions of the spectrum.

EXAMPLES

Some examples of the use of various fluorescent proteins in imaging can be seen in Movie 1 , Movie 2 , Movie 3 , Movie 4 , Movie 5 , Movie 6 , Movie 7 , Movie 8 , Movie 9 , Movie 10 , Movie 11 , Movie 12 , Movie 13 , Movie 14 , Movie 15 , Movie 16 , Movie 17 , Movie 18 , Movie 19 , Movie 20 , Movie 21 , and Movie 22 .

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Movie 1. African green monkey kidney epithelial cells (CV-1 line) expressing EGFP fused to human -tubulin imaged with a Nikon swept-field microscope using a 1.45-NA total internal reflection fluorescence microscopy (TIRFM) objective.

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Movie 2. Pig kidney epithelial cells (LLC-PK1 line) expressing EGFP fused to human -tubulin and mCherry fused to human histone H2B imaged with a Nikon C1si laser-scanning confocal microscope.

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Movie 3. Fox lung epithelial cells (FoLu line) expressing EGFP fused to human β-actin imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 4. Fox lung epithelial cells (FoLu line) expressing EGFP fused to human β-actin imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 5. Fox lung epithelial cells (FoLu line) expressing EGFP fused to microtubule EB3 imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 6. Fox lung epithelial cells (FoLu line) expressing EGFP fused to human -tubulin imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 7. Fox lung epithelial cells (FoLu line) expressing EYFP fused to human-light-chain clathrin imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 8. Fox lung epithelial cells (FoLu line) expressing mCherry fused to human β-actin and mKusabira Orange fused to human mitochondria imaged with a Nikon C1si laser-scanning confocal microscope.

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Movie 9. Fox lung epithelial cells (FoLu line) expressing mKusabira Orange fused to microtubule EB3 imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 10. Fox lung epithelial cells (FoLu line) expressing mKusabira Orange fused to a mitochondrial targeting sequence imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 11. Fox lung epithelial cells (FoLu line) expressing YPet fluorescent protein fused to microtubule EB3 imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 12. Human cervical carcinoma epithelial cells (HeLa line) expressing EGFP fused to a peroxisomal targeting sequence (SKL) imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 13. Human cervical carcinoma epithelial cells (HeLa line) expressing EYFP fused to a mitochondrial targeting sequence imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 14. Pig kidney epithelial cells (LLC-PK1 line) expressing mEmerald fluorescent protein fused to microtubule EB3 and mCherry fused to human histone H2B imaged with a Nikon C1si laser-scanning confocal microscope.

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Movie 15. Fox lung epithelial cells (FoLu line) expressing GFP fused to a targeting signal for the Golgi apparatus and DsRed fused to a mitochondrial targeting sequence imaged with a Nikon C1si laser-scanning confocal microscope.

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Movie 16. Human osteosarcoma epithelial cells (U2OS line) expressing mEmerald fluorescent protein fused to an endoplasmic reticulum targeting peptide imaged with a Nikon C1si laser-scanning confocal microscope.

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Movie 17. Opossum kidney epithelial cells (OK line) expressing EGFP fused to human β-actin imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 18. Opossum kidney epithelial cells (OK line) expressing EGFP fused to an endosome targeting signal imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 19. Opossum kidney epithelial cells (OK line) expressing EGFP fused to a targeting signal for the Golgi apparatus imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 20. Opossum kidney epithelial cells (OK line) expressing EGFP fused to LAMP1 (targeting the lysosomes) imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 21. Opossum kidney epithelial cells (OK line) expressing EYFP fused to human β-actin imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

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Movie 22. Opossum kidney epithelial cells (OK line) expressing EYFP fused to a targeting signal for the Golgi apparatus imaged with a Nikon swept-field microscope using a 1.45-NA TIRFM objective.

WEB RESOURCES

Fluorescent Proteins and Microscopy

http://learn.hamamatsu.com/ (Hamamatsu Learning Center.)

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP1.htm (Green fluorescent protein--The GFP site.)

http://www.microscopy.fsu.edu/ (Molecular Expressions.)

http://www.microscopyu.com/ (Nikon MicroscopyU.)

http://www.olympusconfocal.com/ (Olympus FluoView Resource Center.)

http://www.olympusmicro.com/ (Olympus Microscopy Resource Center.)

http://zeiss-campus.magnet.fsu.edu/ (Zeiss On-Line Campus.)

Fluorescence Filters

http://www.chroma.com/ (Chroma Technology Corp.)

http://www.omegafilters.com/ (Omega Optical.)

http://www.semrock.com/ (Semrock.)

Fluorescent Protein Vendors

http://www.addgene.org/ (Addgene. A nonprofit plasmid archive for research scientists. Distributes numerous fluorescent protein vectors.)

http://www.allelebiotech.com/ (Allele Biotechnology. FPs: mTFP1, mWasabi.)

http://www.bdbiosciences.com/ (BD Biosciences. Baculovirus transfer vectors with BFP and YFP variants.)

http://www.clontech.com/ (Clontech Laboratories. Living colors FP line: AcGFP1, AmCyan1, AsRed2, DsRed2, DsRed-Express, DsRed-Monomer, HcRed1, ZsGreen1, ZsYellow1, mFruits.)

http://www.evrogen.com/ (Evrogen. Turbo and Tag FP lines: TurboGFP, YFP, RFP, FP602; TagCFP, GFP, YFP, RFP; PhiYFP, JRed, TagRFP, mKate, dKatushka, PS-CFP2, Dendra2, KFP-Red, HyPer, KillerRed.)

http://www.invitrogen.com/ (Invitrogen. Vivid colors FP line: Emerald, Topaz, CFP, BFP Cycle 3 GFP.)

http://www.lonzabio.com/ (Lonza. Amaxa pmaxFP line: pmaxFP-Green, pmaxFP-Yellow, pmaxFP-Yellow-m, pmaxFP-Red.)

http://luxbiotech.com/ (LUX Biotechnology. NanoLight FP line [UK]: Renilla mullerei GFP, Ptilosarcus GFP, Renilla reniformis GFP.)

http://www.mblintl.com/ (MBL International Corporation. CoralHue FP line: mAzami Green, mKusabira Orange, mKO2, Dronpa, Kaede, Kikume Green-Red, Keima Red, Midoriishi-Cyan.)

http://www.nanolight.com/ (NanoLight Technology. NanoLight FP line [USA]: Renilla mullerei GFP, Ptilosarcus GFP, Renilla reniformis GFP.)

http://las.perkinelmer.com/ (PerkinElmer. BRET² assay vectors FP line: GFP² humanized codon cloning vectors.)

http://www.promega.com/ (Promega. Monster green FP line: phMGFP.)

http://www.stratagene.com/ (Stratagene. Vitality FP vectors: hrGFP and hrGFPII Nuc, Mito, Golgi, Peroxy.)

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