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Advances in fluorescent protein technology
Nathan C. Shaner1, George H. Patterson2 and Michael W. Davidson3

The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development (NICHD), Bethesda, MD 20892, USA
National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, Tallahassee, FL 32310, USA


Journal of Cell Science

Accepted 31 October 2007
Journal of Cell Science 120, 4247-4260 Published by The Company of Biologists 2007

Current fluorescent protein (FP) development strategies
are focused on fine-tuning the photophysical properties of
blue to yellow variants derived from the Aequorea victoria
jellyfish green fluorescent protein (GFP) and on the
development of monomeric FPs from other organisms that
emit in the yellow-orange to far-red regions of the visible
light spectrum. Progress toward these goals has been
substantial, and near-infrared emitting FPs may loom over
the horizon. The latest efforts in jellyfish variants have
resulted in new and improved monomeric BFP, CFP, GFP
and YFP variants, and the relentless search for a bright,
The rapid rise of fluorescent proteins (FPs) as a fundamental
staple of biomedical research has often been termed a
revolution, the first phase of which began just over a decade
ago when the original Aequorea victoria jellyfish wild-type
green fluorescent protein (GFP) was elegantly used to highlight
sensory neurons in the nematode (Chalfie et al., 1994). Since
then, the race has been on to produce new and improved
versions that are brighter, cover a broad spectral range, and also
exhibit enhanced photostability, reduced oligomerization, pH
insensitivity, and faster maturation rates. The wild type
(wtGFP) was quickly modified to produce variants emitting in
the blue (BFP), cyan (CFP) and yellow (YFP) regions (Fig. 1)
(Heim et al., 1994; Ormö et al., 1996; Tsien, 1998), but the
orange and red spectral regions have remained unachievable
with Aequorea GFP and were surprisingly elusive until the
unexpected discovery of the first red FP from a nonbioluminescent reef coral (Matz et al., 1999). This
breakthrough launched a second phase of the revolution, the
relentless quest for the holy grail of fluorescent proteins, a
bright and photostable monomeric red-emitting derivative,
whose performance matches that of the best GFP variants. The
search continues on today. So far, progress has generally been
very encouraging, although there still remains a significant
performance gap in many FP spectral classes.
Despite the recent advances in FP technology, most groups
still use the enhanced version of wild-type GFP (EGFP), as
well as the original cyan and yellow derivatives (ECFP and
EYFP), for the majority of their imaging applications (Olenych
et al., 2006; Patterson, 2007). The reluctance of many
researchers to transition to newer FP variants is fueled by the
checkered availability of various FPs, coupled with (often
justified) uncertainties about reported claims of improved
brightness, photostability, monomeric character and utility in

monomeric and fast-maturing red FP has yielded a host of
excellent candidates, although none is yet optimal for all
applications. Meanwhile, photoactivatable FPs are
emerging as a powerful class of probes for intracellular
dynamics and, unexpectedly, as useful tools for the
development of superresolution microscopy applications.
Key words: Fluorescent proteins, Mutagenesis, Optical highlighters,
Photoactivation, Photoconversion, Photoswitching, Live-cell

fusions. In many cases, simply finding a source for a new FP
can be a time-consuming and discouraging challenge. 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
Here, we attempt to address frequent misconceptions that
occur during the transition to new FP variants, including the
issue of oligomerization. In addition, we discuss recent
advances in protein engineering strategies as well as
improvements to the color palette and the development of the
current armament of photoactivatable FPs. Finally, we provide
suggestions for the best FP choices in single- and multi-color
imaging, and potential avenues for obtaining these proteins.
New additions to the FP color palette
The broad range of FP genetic variants developed over the past
several years feature fluorescence emission profiles spanning
almost the entire visible light spectrum (Chudakov et al., 2005;
Shaner et al., 2005; Verkhusha and Lukyanov, 2004). Several
guiding motifs have emerged regarding the fundamental
origins and manipulation of the emission color (Remington,
2006). Local environmental variables around the chromophore,
including the position of charged amino acid residues,
hydrogen bonding networks and hydrophobic interactions
within the protein matrix, can produce blue or red spectral
shifts in the absorption and emission maxima of as much as 40
nm. Larger spectral shifts, which distinguish the general FP
spectral class (CFP, GFP, YFP, etc.), are generally attributed to
differences in the covalent structure and the extent of ␲-orbital

␤-sheets are numbered and depicted as thin. 2002). 2007. its low brightness and poor photostability have long made it an unattractive option for most researchers.D. All of these new BFP variants could potentially be made truly monomeric by addition of the A206K mutation. (4) YFP....W. 2007) rather than the trans isomer that is common to CFP and related variants. The monomerizing mutation A206K is useful for all known GFP derivatives. Mena et al. 3A). is an excellent fluorescence resonance energy transfer (FRET) donor (M. Three groups (Ai et al. 2005). shared and monomerizing (gray). YFPs (yellow).. Y39N.. Kremers et al. 2006) have recently reported improved blue Aequorea FP variants that feature significantly higher brightness and photostability compared with EBFP. Sapphire (violet).. EBFP2. the task of genetically engineering more finely tuned color variants and broadening the spectral range of useful proteins should become easier. 8 and 10. whereas ␣-helices are depicted as gray cylinders. these novel variants offer the first real hope for successful long-term imaging of live cells in the blue spectral region. Furthermore. In general. and chromophore structures of common Aequorea FP derivatives. Named Azurite. unpublished) for EGFP in live cells. Note that almost 75% of the mutations are located in the central helix and in ␤-sheet strands 7. 2007. (B) Aequorea GFP mutation map showing common mutations superimposed on a topological layout of the peptide structure. Kremers et al. wavelength-specific mutations occur near the central helix containing the chromophore. As further studies into the complex characteristics of FP chromophores yield clues about the structure-function relationship with the polypeptide backbone. 2007) (M.. Several of the sfGFP folding mutations (S30R. Even though all three probably exhibit weakly dimeric character in highly concentrated microenvironments (Zacharias et al. the brightest and most photostable blue FP (see Table 1). Although EBFP was one of the first spectral variants derived from Aequorea GFP. (2) CFP. (3) EGFP. Mutations are color-coded to represent the variants to which they apply: BFPs (blue).D.. GFPs (green). folding. whereas folding mutations occur throughout the sequence. (1) BFP. unpublished) and can be readily imaged with standard BFP and 4⬘. but is replaced by A206V in sfGFP and EBFP2. which is unlikely to have any substantial negative impact on their properties. they function effectively in fusions with subcellular localization targeting proteins (Ai et al. green cylinders with an arrow pointing towards the C-terminus. recent BFP and CFP variants have dramatically strengthened the potential for imaging in these regions as well. Many of the cyan and yellow FP mutations introduced near the termini of the proteins resulted during the CyPet and YPet mutagenesis efforts (Nguyen and Daugherty. .. CFPs (cyan). F99S and N105T) also occur away from the chromophore. The tryptophan residue (Trp66) in (2) is illustrated in the cis conformation as occurs for Cerulean derivatives (Malo et al.6-diamidino-2phenylindole (DAPI) filter sets. SBFP2 (strongly enhanced blue fluorescent protein) and EBFP2 (Fig. 1. conjugation of the chromophore (Figs 1 and 2). Portions of the chromophores that are conjugated and give rise to fluorescence are shaded with colors corresponding to the emission spectral profile.W.4248 Journal of Cell Science 120 (24) Journal of Cell Science Fig. (A) FP ␤-barrel architecture and approximate dimensions. 2007. Blue and cyan FPs Although most attention now focuses on the orange-to-far-red spectral regions.

monomerizing mutations (green) and shared mutations (gray). advanced CFP-YFP FRET biosensors exhibiting high dynamic range for the detection of metabolites. that has even higher brightness levels. 2003). when combined with either yellow or orange FPs. Thr.. Note that unlike the cluster of mutations surrounding the chromophore for Aequorea GFP variants (Fig. Mutations are color-coded to represent the variants to which they apply: mRFP1 (red). . 2005) and mCerulean (Fig. (2) eqFP611.and C-termini due to the addition of amino acids derived from GFP to improve fusion performance are shaded in light green. folding efficiency. mTFP1 has spectral characteristics that are slightly red-shifted with respect to most CFPs (thus the name teal rather than cyan). The residue at position 66 can be Met. 2.. The high quantum yield of mTFP1 (see Table 1) provides an excellent alternative to the cyan derivatives mECFP (Shaner et al. 2006). Ca2+ fluctuations. which generally feature the heteroaromatic amino acid tryptophan at position 66 in the chromophore. ␤-sheets are numbered and depicted as thin.. as well as a host of other intracellular processes. but less than twofold brighter in mammalian cells. one of the mFruit proteins (Shaner et al.Fluorescent protein advances A Red fluorescent proteins 4249 trans-chromophore Tyr67 Try64 Gly68 (1) Gly65 (2) eqFP611 cis-chromophore R66 = Met. mCherry (cyan).. Derived from a tetrameric Clavularia soft coral protein. features a novel threering chromophore that is created when the lysine residue at position 66 cyclizes with its own ␣carbon to form a tetrahydropyridine ring conjugated to the chromophore (Remington et al. Allele Biotech). 2005). (B) DsRed mutation map showing mutations of useful variants superimposed on a topological layout of the peptide structure. these high-performance FPs should be useful for fusion tags and for creating new. 1). Cys or Glu Met63 Tyr67 Tyr67 Gly68 Gly68 (3) (4) mOrange ZsYellow Lys66 B C-terminus N-terminus GFP residues R2A Journal of Cell Science L225A H222S N6D H41T V7I DsRed derivative mutation map F224G L223T K5E K83L R153E Y192A V194K-N T147S T217A β-2 β-3 V195T-V β-11 V44A K45R V16E C117E V156A L150M N42Q β-1 Thr66 Backbone cleaved D196N β-10 R17H Q66M F65I S197I V71A M182K β-8 β-7 I161M H162K K163M-Q T21S β-9 β-4 K166R mRFP1 mCherry mPlum dTomato Shared Monomer β-6 I180T L124V S179T F124L-M I125R F177V V127T A164R V22M β-5 V105L V175A L174D The cyan spectral region (~470 nm to 500 nm) was dominated by progeny of the original Aequorea ECFP (Table 1) until the introduction of a monomeric teal-colored variant. Recently. a red variant derived from Entacmaea quadricolor. 2004) as a FRET donor Fig. 2006). orange.. 1) for enhanced brightness. Thr. 3B) (Rizzo et al. red FP mutations are distributed throughout the sequence. (A) Chromophore structural variation in yellow. 2004). termed SCFP and SYFP. also features a three-ring chromophore where Thr66 cyclizes with the preceding carbonyl carbon to yield a partially conjugated oxazole ring (Shu et al. Extensions of the N. protein and enzyme phosphorylation. 2006). dTomato (yellow). Nevertheless. mPlum (violet).. Substituting tyrosine for tryptophan reduces the broad fluorescence emission spectral width from ~60 nm to a narrower and more manageable 30 nm. acid insensitivity and greater photostability (Ai et al. mTFP1 contains a tyrosine residue at this location. which reduces bleedthrough in multicolor and FRET experiments.. mTFP1 requires a specialized filter set for optimal imaging.. and red FPs. but it can still produce suitable signal levels with a standard ECFP filter set. The resulting super cyan and yellow fluorescent derivatives. whereas ␣-helices are depicted by gray cylinders. (1) FPs derived from DsRed and other reef coral organisms thought to have a cis-chromophore. are significantly brighter than the parent proteins when expressed in bacteria. On the downside. Unlike other CFPs. solubility and FRET performance (Kremers et al. pH changes. a comprehensive site-directed mutagenesis approach has been applied to optimize previously identified mutations in monomeric variants of ECFP and EYFP (Fig. mTFP1 (Fig. Gln. Gln. derived from the button polyp Zoanthus. (3) ZsYellow (also zFP538). is the only known FP featuring a trans-chromophore (Petersen et al. red cylinders with an arrow pointing towards the C-terminus. Cys or Glu. 3C. (4) mOrange.

**Measured and normalized per standard photobleaching protocol (see Shaner et al. 1999 Heim et al. 1995 Ai et al. 2007 Shaner et al. 2006 Cubitt et al... 2005). For specialized applications.6 5. 2004 Karasawa et al.. *Common literature FP abbreviation. 1999 Pédelacq et al. § Recommended common filter sets and custom FP sets available from aftermarket manufacturers..0 4. 2005). 2002 Merzlyak et al. 2002 Miyawaki et al.. 2007 Reference Physical properties for the recommended FPs in each spectral class.3 4. we suggest choosing filter combinations that closely match the spectral profiles (see Shaner et al... ‡Literature values except as noted..0 53 3.5 96 166¶ 4.7 4. 2004 Nguyen and Daugherty.2†† <4.462 Cyan-green Green Green Green mTFP1 mEGFP mEmerald sfGFP 517 548 Yellow Yellow mCitrine YPet Far-red mPlum 590 588 587 649 635 610 607 584 581 559 530 529 528 527 510 157¶ 6. Photobleaching represents the time to bleach from an emission rate of 1000 photons per second to 500 photons per second (t1/2) in a widefield fluorescence microscope.. 1995 Ai et al.5 <4..7 †† 37** 98 122 49 49 15 6... Physical properties of useful fluorescent proteins Journal of Cell Science 4250 Journal of Cell Science 120 (24) . ¶Measured in live cells with mEGFP (t1/2=150 seconds) as a control.0 5. Red Far-red mKate Red mRFP1‡‡ mCherry 555 Orange TagRFP 584 554 Orange Orange mKO tdTomato 516 515 mVenus 514 Yellow Yellow EYFP‡‡ 485 487 488 433/445 Cyan mCerulean 448 475/503 383 433/445 Emission peak (nm) Excitation peak (nm) Cyan Color of spectral class Blue ECFP‡‡ EBFP2 Protein* Table 1.0 4...5 <4. ‡‡Included for reference. 2004 Cubitt et al.5 101¶ 60 6.9 6. 2004 Shcherbo et al..7 6...5 48 95 31 80 59 53 51 54 39 509 54 34 492 507 27/24 475/503 13 18 Brightness† Monomer Monomer Monomer Monomer Monomer T-dimer Monomer Weak dimer Monomer Monomer Weak dimer Weak dimer Monomer Monomer Monomer Monomer Weak dimer Weak dimer Association state‡ MYG MYG MYG QYG MYG MYG CYG GYG GYG GYG GYG TYG TYG TYG AYG TWG TWG SHG Chromophore TxRed TxRed TxRed TxRed TRITC/DsRed TRITC/DsRed TRITC/DsRed FITC/YFP FITC/YFP FITC/YFP FITF/YFP FITC/GFP FITC/GFP FITC/GFP CFP CFP CFP DAPI/BFP Filter set§ Wang et al. 2001 Nagai et al.. ††Averages of literature values..7 5. †Product of the molar extinction coefficient and the quantum yield (mM cm)–1.0 5.7 5. 2004 Campbell et al. 2006 Rizzo et al.3 pKa‡ 174 110 36 64 55 Photostability‡ 12. 2007 Shaner et al.0 15 17 8. 2005 Griesbeck et al.

as a spectral class. unpublished) (Merzlyak et al. amphioxus (Deheyn et al. and even the best offer no clear advantage over EGFP (Table 1. when coupled with cyan. Several of the major problems with the original Discosoma DsRed FP (slow maturation. 2007) (Fig. the high extinction coefficient should render mKO an excellent FRET acceptor for cyan and teal FPs. TagRFP. TurboRFP (commercially available from Evrogen). green and yellow spectral classes. The resistance to acidic environments afforded by YPet is superior to Venus and other YFP derivatives. Fig.. 2).. Merzlyak et al. 2002). Merzlyak et al. Although Emerald is somewhat more efficient than EGFP. 2004).. it has a fast photobleaching component that might affect quantitative imaging in some environments [this artifact is likely to be reduced in monomeric A206K variants (N. 3F) are currently the most useful probes in the yellow class (see Table 1). are among the brightest and most versatile genetically encoded probes yet developed. Emerald contains the F64L and S65T mutations featured in EGFP. however. Often termed red fluorescent proteins (RFPs – perhaps wishful thinking on the part of the discoverers). 3E) (Pédelacq et al. Yellow FPs Yellow FPs. Fig. which has similar properties to those of its EGFP parent (Cubitt et al. Regardless of the color designation. unpublished)]. as well as monomers from proteins in other Anthozoa species.C. Orange FPs In contrast to the hundreds of FPs engineered in the cyan. 2007). 2007).. 2004). 2007... proteins in the orange spectral class are readily imaged in multicolor scenarios. but neither is commercially available. Fig.S. brightness and pH resistance.. A total of 33 residue alterations to the DsRed sequence were required for the creation of the firstgeneration monomeric red FP (mRFP1) (Campbell et al. TagRFP (from Evrogen). 2007. only few probes have been developed so far that emit in the orange and red wavelengths (~560 nm to 650 nm). 2004) actually have emission profiles that are clearly more orange than red. However.. Table 1). including different Aequorea species (Labas et al.. Both the tetrameric and monomeric variants of Kusabira Orange are now commercially available (from MBL International). A bright new monomeric orange protein. Vinkenborg et al. 2002). its photostability in our hands is not as high as was originally claimed (Merzlyak et al. but also has four additional point mutations that improve folding.. 2007) and reef corals (Matz et al. speculate that TagRFP will be an excellent FRET acceptor when fused to GFP and YFP donors. Merzlyak et al. Even so. copepods (Shagin et al. Although optimized for FRET. but the construction of true monomeric DsRed variants. YPet is the brightest YFP variant yet developed and demonstrates very good photostability (Fig. although still widely used. named after the birthstone Topaz (Cubitt et al. 2007. an intermediate green state. and the obligate tetrameric character) were overcome through sitedirected and random mutagenesis efforts. EYFP (Table 1). 2006). which has absorption and emission maxima at 584 nm . The resulting FP has an absorption maximum at 548 nm and emits bright yellow-orange fluorescence centered at 559 nm.. However. 1999. the existing proteins in these 4251 spectral classes (see Fig. Shaner et al. green and red FPs. 3D.. Another potentially useful YFP. Note.. there remains a serious doubt as to the origin of YPet’s increased performance. which makes it an excellent choice for long-term imaging experiments (see Table 1). the newly developed SYFP (discussed above) should become a useful member of the yellow palette once its performance in fusions expressed in mammalian cells is confirmed. has very favorable photophysical properties (Table 1) and performs well in a wide variety of fusions expressed in mammalian cells (M. mKO exhibits a brightness value similar to that of EGFP and its photostability in widefield-fluorescence illumination is among the best of any FP. TagRFP. 2005. In addition. the original variant. and tdTomato (Matz et al. Invitrogen). The most significant addition to the GFP palette in the past several years is superfolder GFP (Fig. 3H) was subsequently created by introduction of an additional 20 mutations.. while simultaneously performing random mutagenesis to rescue the folding properties. featuring high photostability. Furthermore. 2002.. However. 1999). which is likely to be due simply to enhanced dimerization with its co-evolved partner CyPet (Ohashi et al. 2007) (see Table 1). is far from optimal owing to its high pKa and sensitivity to halides... that superfolder GFP may generate higher background noise levels when one images fusions in which a significant number of the proteins fail to target correctly but still produce bright fluorescence. 2 and Table 1).W. Rizzo and Piston.D. 3G... YPet (yellow fluorescent protein for energy transfer).Journal of Cell Science Fluorescent protein advances Green FPs Numerous proteins emitting in the green (500 nm to 525 nm) spectral region have been discovered from a wide range of sources. subsequent random mutagenesis yielded a rapidly maturing variant. a similar but less well characterized Aequorea derivative. exhibit oligomerization artifacts. A point of confusion exists in the nomenclature for FPs in the orange region. has recently been introduced as a promising candidate for localization and FRET studies (Merzlyak et al. probes such as DsRed. mutation rate at 37°C and brightness. Originally cloned as a dimer from the sea anemone Entacmaea quadricolor. but that remains to be demonstrated.. 2004). 2005). Perhaps the best current choice for live-cell imaging is the GFP derivative Emerald (Fig. can be purchased from Invitrogen. On the basis of the crystal structure of a closely related protein from the same species (eqFP611. 3J). was derived from synthetic DNA shuffling coupled with fluorescence-activated cell sorting (FACS) to enhance the pairing of cyan and yellow proteins for FRET (Nguyen and Daugherty. The monomeric YFP variants mCitrine and mVenus (Fig.. however. 3M-Q). using a standard tetramethyl-rhodamine isothiocyanate (TRITC) filter set. Kusabira Orange was originally derived as a tetramer from the mushroom coral Fungia concinna through site-specific mutagenesis of a cDNA clone in which ten residues were added to the N-terminus of the protein (Karasawa et al. which can efficiently fold even when fused to insoluble proteins and is slightly brighter and more acid resistant than either EGFP or Emerald (Table 1). 1999). which will enhance the utility of this probe in biosensor combinations targeted at acidic organelles. exhibit the potential to be useful in a variety of imaging scenarios. A monomeric version (mKO. 1999). Verkhusha and Lukyanov. has proven to be a difficult task (Campbell et al. which have all been isolated from coral species. Shcherbo et al. The final variant. Most. replaced several key amino acid residues involved in dimerization.

Shaner et al. 3I) is extremely bright and. rendering it much less useful than analogous monomeric GFPs and YFPs. 3.. (C) mTFP1-actin-C-7 (human ␤-actin. (L)mPlum-␣-actinin-N-19 (human nonmuscle. These mFruits together with mKO and TagRFP. respectively. microtubules). essentially fill the gap between the most red-shifted jellyfish FPs (such as YPet) and the multitude of oligomeric red coral FPs that have been reported and are now commercially available. which may interfere with fusion-protein packing in some biopolymers. have successfully been applied in the search for orange. (I) tdTomato-zyxin-N-7 (human zyxin. Extensive mutagenesis efforts (discussed below). gap junctions). mitochondria). However. 2) (Patterson. (G) YPet-EB3-N-7 (human microtubule-associated protein. (M-Q) Fusion of mEGFP with human histone H2B (mEGFP-H2B-N-6).and C-termini. Because of its twin chromophores. (E) superfolder GFP-lamin B1-C-10 (human lamin B1. (J) TagRFP-tubulin-C6 (human ␣-tubulin. focal adhesions). The FP fusion terminus and number of linker amino acids is indicated after the name of the targeted organelle or fusion protein. RP/EB family). Red FPs The quest for a well-behaved red-emitting FP has long been the goal for live-cell and whole-animal imaging. (A-L) Subcellular localization of selected monomeric FP fusions (listed in Table 1) with targeting proteins imaged in widefield fluorescence. 2004). generated during the breakup of the tetrameric DsRed protein (Campbell et al. mCherry is far more photostable than mStrawberry and is the best choice to replace mRFP1 for long-term imaging experiments. The major drawback of tdTomato is its larger size. (B)mCerulean-paxillin-N-22 (chicken. which is intended to increase tolerance to fusion proteins and reduce potential localization artifacts. moreover.. 2002). (A) EBFP2mito-N-7 (human cytochrome C oxidase subunit VIII. (O) prometaphase. is also exceptionally photostable under widefield illumination (Table 1). The resulting monomeric FPs exhibit maxima at wavelengths from 560 nm to 610 nm and are named after common fruits that bear colors similar to their emission profiles. red and farred FP variants that further reduce the tendency of these potentially advantageous biological probes to self-associate while simultaneously pushing emission maxima towards longer wavelengths The brightest FP in any spectral class is the tandem version of dimeric (d) Tomato. (F)mVenus-Cx43-N-7 (rat ␣-1 connexin43. respectively. and the fact that longer excitation wavelengths generate less phototoxicity and can probe deeper into biological tissues. (K)mCherry-vimentin-N-7 (human vimentin. Although several mFruits lack the brightness and photostability . However. Images are pseudocolored to match the FP emission profile. (P)metaphase. Golgi complex). (Q) anaphase.4252 Journal of Cell Science 120 (24) Journal of Cell Science Fig. (N)prophase. 2004). this derivative exhibits significantly reduced fluorescence emission compared with the native protein and photobleaches very quickly. filamentous actin). the resulting tdTomato (Fig.. intermediate filaments). intermediate filaments). A tandem-dimer version (effectively a monomer) contains two copies – head-to-tail – of dTomato with a 12-residue linker. which have brightness levels of ~75% and 50% of EGFP. (H) mKO-Golgi-N-7 (Nterminal 81 amino acids of human ␤-1. nuclear envelope). (D)mEmerald-keratin-N-17 (human cytokeratin 18. and 607 nm. 3K. primarily because of the requirement for red probes in multicolor imaging experiments. focal adhesions). an orange derivative that was one of the original ‘Fruit’ proteins (Shaner et al. Perhaps the most dramatic development on this front has been the introduction of new FPs derived from mRFP1 through directed mutagenesis targeting chromophore residues that are known to play a key role in determining the spectral characteristics (Fig. (M) interphase. This variant contains the first and last seven residues from GFP on its N.4glactosyltransferase. dTomato was derived from an intermediate termed dimer2. 2004. emission peaks at 596 nm and 610 nm). cytoskeleton). Among the best red members in the Fruit series are mStrawberry and mCherry (Table 1. Fig.

Similar to most soluble proteins. Semi-random mutagenesis of five residues surrounding the chromophore led to a red FP that has a bimodal excitation spectrum (peaks at 452 nm and 580 nm) with emission at 606 nm. 2007). Chudakov and associates (Shcherbo et al. 1998). has four additional mutations (Zapata-Hommer and Griesbeck. 2007). named Keima (after the Japanese chess piece). Katushka exhibits the highest brightness levels of any FP in the spectral window encompassing 650-800 nm. emission maximum = 620 nm). 2006). 2004) has yielded two new FPs representing the first true far-red genetically engineered probes (emission wavelength maxima of 625 nm and 649 nm).4) and mCitrine (Förster distance 5. 2007) have applied a combination of site-specific and random mutagenesis to generate libraries encoding TurboRFP variants that contain mutations at key positions surrounding the chromophore. Miyawaki and associates (Kogure et al. An additional four mutations 4253 substantially reduce the 580 nm peak and blue-shifted the other absorption peak to 440 nm. 3 and Table 1). The interior of the protein is so tightly packed that even water molecules are fixed into place by hydrogen bonds to the amino acids and there is little room for diffusion of ions or other intruding small molecules. although there are also patches containing hydrophobic residues. regardless of the spectral class or supposedly monomeric characteristics (Kremers et al. many of the FPs listed in Table 1 can be combined for dual. 2005... emission maximum = 507). photostability and many other physical properties. in most cases there remains no EGFP equivalent. monomeric probes across the entire visible spectrum should eventually be available. Extending the Sapphire strategy to red FPs. Evrogen. in which a large portion of the polypeptide backbone is wound into 11 strands of an extensively hydrogen-bonded ␤-sheet that surround a central ␣-helix containing the chromophore. Although only two-thirds as bright as EGFP. whereas the red and far-red FPs are among the dimmest in all spectral classes. their existence suggests that bright. Notable FPs featuring the MYG chromophore include ZsGreen1 (Clontech. the exterior residues are usually charged or polar. mPlum should be useful in combination with cyan. Introduction of the four principal Katushka mutations into TagRFP generated a monomeric. Several more rounds of mutagenesis produced a dimer (dKeima) that has similar spectral properties. has rather limited brightness (10% of EGFP.Journal of Cell Science Fluorescent protein advances necessary for many imaging experiments (Shaner et al.0). respectively. a region that is important for deeptissue imaging. 1996. This concept is perhaps best exemplified by the fact that the common tripeptide Met-TyrGly (MYG) is able to form chromophores spanning an astonishing 175-nm emission range. in terms of photostability and other crucial areas of performance (with the exception of pH stability). T-Sapphire (T for Turbo). This derivative. green.. bright and photostable additions will become available for all spectral classes. having excitation and emission maxima at 399 nm and 511 nm. After screening for mutants exhibiting far-red fluorescence and conducting additional rounds of random mutagenesis. mKeima exhibits limited brightness (similar to that of mPlum) and requires a specialized filter combination for imaging. . the YFPs still have suboptimal photostability. Given that most of these proteins have only been introduced in the past couple of years. exhibits an emission maximum at 616 nm. Even so. In addition. devoid of the minor excitation peak at 475 nm (Tsien. The rigidly defined structure of the FP interior is responsible for the unique chemical environment that nurtures autocatalytic chromophore formation by three of the amino acids in the central ␣-helix. TagFP635) that has similar spectral characteristics. FP mutagenesis has also targeted the separation distance between absorption and emission maxima (Stokes shift) to generate better probes for FRET. 2003). 2006) used a far more rigorous approach to construct the longest Stokes shift FP variant yet developed (180 nm) using a chromoprotein derived from the Montipora stony coral. Tsien. TagRFP (Merzlyak et al. such as mEmerald (Förster distance 4. yellow and orange FPs for multicolor imaging experiments and as a FRET partner for green and yellow FPs. they obtained a dimeric protein named Katushka (emission maximum of 635 nm. but it is useful in FCCS and multicolor imaging experiments (Kogure et al. several of the mFruit proteins have become commercially available (from Clontech). The photostability of mKate is reported to be exceptional and the protein displays brightness similar to that of mCherry. Sapphire. 1) (Ormö et al. New additions to the blue and cyan region feature substantially improved brightness and photostability. Recently. Although promising candidates are now available in every FP spectral class (see Fig. and any of the orange FPs are excellent choices for long-term multicolor imaging. Introducing the T203I mutation into wtGFP produces a variant. 3L).. although brighter than EGFP. and a monomer (mKeima. 2005). Increasing FP Stokes shift In addition to engineering efforts designed to modulate the emission spectra. available from Evrogen as TurboFP635). Further extension of the mFruit spectral class through a novel technique known as iterative somatic hypermutation (SMH) (Wang et al. mCherry (Shaner et al. we remain optimistic that in the future. 2006. mPlum (Fig. 2001). The ␤sheets are linked together by less-ordered proline-rich loops and the amino acid side chains in each sheet alternately project into the protein interior or towards the surface.. tdTomato (Shaner et al. fluorescence cross-correlation spectroscopy (FCCS) and multicolor imaging. A derivative that displays improved folding and brighter fluorescence. Table 1) but excellent photostability. Advances in FP engineering All of the FPs discovered thus far adopt a similar 3D cylindrical structure. Sapphire exhibits a dramatic Stokes shift of 112 nm.... far-red protein named mKate (see Table 1. The most potentially useful. Yarbrough et al..and triple-color imaging to yield excellent results. with short helical segments protecting the ends of the cylinder (Fig. These variants should be excellent donors in FRET combinations with orange and red proteins because of their ability to be excited in the ultraviolet region. Remington. Changes in this environment give rise to variations in spectral characteristics.. stable. 2004).. but is tetrameric. which makes it an excellent candidate for localization experiments in the far-red portion of the spectrum. Further compounding the problem is the potential for aggregation artifacts due to poorly folding proteins. which is apparently dependent solely upon the nature of the chemical and physical environment provided by the interior of the ␤-barrel.

and mPlum (Wang et al. The generation of GFP derivatives that have improved folding efficiencies when participating in fusion proteins was the basis for the work that produced superfolder GFP (Pédelacq et al. 2007) to a monomeric green variant. The crucial point of the superfolder work is that there is yet more room for engineering improvements even in the highly optimized GFP derivatives. 2004).Journal of Cell Science 4254 Journal of Cell Science 120 (24) 2004). Although the first attempt at FP consensus engineering resulted in a product that is less efficient than the guide protein. These efforts generated a variety of spectral variants that have emission profiles shifted by tens of nanometers to both lower and higher wavelengths. After four rounds of DNA shuffling. differs from mAG by 23 residues and by 76 residues from the most distant relative used to create the consensus (GFP2 from the coral Agaricia fragilis). 2006) by repacking the protein core with bulkier amino acid residues intended to constrain chromophore motions that reduce fluorescence emission through internal conversion mechanisms. pioneered by Roger Tsien and associates. 2007.. probably reflecting the observation that surface residues evolve more quickly than internal residues. after the similarly colored mineral. This methodology was successfully applied to BFP in an attempt to enhance its brightness and photostability (Mena et al. by themselves.. 2006) (see above). the investigators isolated a brightly fluorescent clone that contains six new mutations in addition to the enhanced GFP and folding reporter mutations. which was not part of the design criteria. and the far-red FP. CGP (for consensus green protein).. 2003). which is largely responsible for their superior performance.. CGP exhibits a high level of fluorescence when expressed in bacteria and is monomeric. They wanted to generate red FP derivatives that feature emission wavelengths in the far-red region (>625 nm). mKate (Shcherbo et al. iterative somatic hypermutation (SHM) (Wang et al. this approach will undoubtedly become more useful as the database of known FP sequences grows. Although the chromophore structures have yet to be elucidated for most of the reported FPs harboring the MYG triplet. Vinkenborg et al. 2005). Interestingly. Pédelacq and colleagues started with a derivative consisting of cycle-3 GFP (also known as ‘folding reporter’ GFP) (Crameri et al. there are limits to the logical deductions that can be made from crystal structures and many of the variants generated through random mutagenesis suffer from reduced intensity and folding problems or lack fluorescence altogether.. rendering CGP significantly less bright.. exhibit poor folding. seemingly obvious mutations to alter a specific feature must be followed by one or more rounds of random mutagenesis to rescue other desirable properties. This finding highlights at least one potential limitation of the targeted library approach. Thus. over 85% of the residues that differ from mAG to CGP are predicted to be found on the surface of the protein. Consensus engineering attempts to alter protein properties by modifying the sequence to one that more closely resembles a consensus derived from a large population of similar proteins in a particular family. they used random mutagenesis to identify residues directly influencing FRET efficiency and then subjected these to partial or complete saturation mutagenesis through synthetic shuffling. Azami Green (mAG). Noting that B lymphocytes can introduce point mutations into the .. Nguyen and Daugherty have employed a similar approach. they are likely to exhibit a wide range of diversity. It will be interesting to see whether the approach can be successfully applied to other FP properties. A promising approach that should be useful to fine-tune the properties of virtually any FP involves the coupling of a structure-based library in which specific residues are targeted with quantitative screening for improved or otherwise modified characteristics (Neylon. pH sensitivity and maturation rates. CyPet. The CyPet-YPet FRET caspase-3 biosensor exhibits a 20fold improvement in dynamic range over a similar mCeruleanmVenus pair. At the ends of the MYG spectral range are the cyan FP. As crystal structures of the Aequorea FPs became available. photostability. AQ14 (Shkrob et al. Amino acid substitutions in CyPet and YPet are distributed throughout the proteins. termed evolutionary optimization. Park et al.. but the quantum yield is reduced by almost 40% relative to mAG.. The result. A truly unique approach to genetic engineering of new FPs. oligomerization. 1996). However. which severely limits its use as a stand-alone probe. 2004). as well as those in its immediate vicinity (Fig. All too often. This approach. beneficial mutations occurring both near and far away from the chromophore (Fig. derived from the stony coral genus Galaxeidae (Karasawa et al. emission maximum = 486 nm). protein engineers were able to rationally design new variants that feature an even wider range of mutations affecting spectral properties. This impressive increase in efficiency has recently been challenged (Ohashi et al. 1). CyPet folds very poorly at 37°C. Hampered by the difficulty of using rational design to improve FRET performance. The discovery of red-shifted FPs in corals similarly led to new variants that have an emission spectral range of ~160 nm and feature a high degree of variety in their properties. Ironically. may yield similar results. The final result was YPet (see above) and a cyan variant.. that exhibits superior pH stability and faster maturation than ECFP. AmCyan1 (Clontech. when applied to proteins from coral species in other color classes. However. The construction of new FP variants using guided consensus sequence engineering has been applied (Dai et al. 2007). The best variant exhibited an intrinsic brightness 60% greater than native BFP and was named Azurite... 2004).. 2004. one of the two most beneficial mutations in this study occurred through the unintended incorporation of a new codon that alters position 224. 1997). This variant was engineered by fusing libraries of shuffled GFP sequences to polypeptides that. 1). but this does not outweigh the benefits that arise from reducing the number of experimental iteration cycles and thus the speed with which good candidates can be identified. 2005) (emission maximum = 663 nm). careful consideration to this artifact should be given in the design of experiments using the CyPet-YPet combination. several groups have used innovative techniques to improve design strategies. involves directed evolution using a technique borrowed from the immune system. to optimize CFP and YFP when combined in a FRET biosensor equipped with a caspase3 cleavage site to detect apoptosis (Nguyen and Daugherty. For these reasons. and the F64L and S65T mutations from EGFP (Patterson et al. 2007) by demonstrations that CyPet-YPet biosensors are prone to enhanced dimerization. The first attempts to engineer new FPs logically targeted residues participating in the chromophore. The poorly folding peptides are termed bait proteins and interfere with the correct folding of the GFP moiety when expressed in bacteria.

8 4.. MYG. 2006 Dendra2 (N) 507 TRITC FITC/GFP HYG HYG Tandem dimer Tandem dimer 5.. 2006 Dronpa (P) 518 TRITC/DsRed MYG Tetramer NA 4.. Lukyanov et al. §Recommended common filter sets and custom FP sets available from aftermarket manufacturers. 2002 Chudakov et al.. **Photoactivated or photoconverted conformation.5 5.. After 23 rounds. ideal for investigation of protein dynamics in live-cell imaging (reviewed in Lippincott-Schwartz et al.8 578 Red PA-mRFP1 (P) 605 CFP FITC/GFP SYG SYG Monomer Monomer 6. 2006). which typically possess a common set of problems.1 Red KFP1 (P) 600 Gurskaya et al. Given that many of the amino acid triplets so far uncovered in chromophores can give rise to huge variations in emission color (e. 2006 553 Red Dendra2 (P) 580 TRITC HYG Monomer 6.4 516 581 Red tdEos (P) 506 Green tdEos (N) 569 Chudakov et al. 177 nm.1 4.3 Nienhaus et al. 2006 490 Green 573 FITC/GFP HYG Monomer 6. and the FP also exhibits an unusually large Stokes shift of 59 nm. TYG. we suggest choosing filter combinations that closely match the spectral profiles (see Shaner et al.5 19. two areas that are crucial to high performance in acidic organelles and for long-term imaging experiments. 2005. Table 2..8 55.5 Nienhaus et al. if not all. enhance folding and optimize FRET efficiency. 91 nm. Physical properties of useful optical highlighter fluorescent proteins Journal of Cell Science Fluorescent protein advances . oligomerization and photostability through the approaches described above will no doubt ultimately yield better probes than simply scouring the oceans for new candidates. Fine-tuning existing proteins by improving their folding. Ideal optical highlighters (see Table 2) should be readily photoactivatable/photoconvertable to generate a high level of contrast. ‡Literature values except as noted. Remington. photoactivatable or photoconvertible FPs can be used in the direct and controlled highlighting of distinct molecular pools within the cell.. Perhaps more appropriately termed molecular or optical highlighters. The mPlum emission spectrum maximum (649 nm) is shifted by 37 nm relative to the parent. The recent burst of activity in the development of FP engineering technology has led to several new variants that probably would not have been discovered by traditional methodology. their lifetime and behavior can be followed independently. 80 nm). it appears that there is plenty of room in the FP sequence space for additional mutations that will optimize color and many. and the process was repeated iteratively. 2002 Patterson and Lippincott-Schwartz.5 2. Although mRaspberry is not photostable enough for applications in routine live-cell imaging. 2004 FITC/GFP CYG Monomer 5. brightness. 2005). of the other FP properties. establish greater structural stability. a monomeric far-red-emitting protein named mPlum (see Table 1) was isolated and characterized. Labas et al..g. 2002 Ando et al. These advanced techniques have been used to generate new colors. QYG.6 22. For specialized applications. mRaspberry. mPlum stability is comparable to that of many proteins in the yellow spectral class. which preferentially targets highly transcribed genes. and should be monomeric for optimal performance as Protein* variable domains of antibodies through SHM.9 19. A second clone from an earlier SHM round.8 Gurskaya et al. A derivative of mRFP1 under the control of a tetracyclineinducible promoter was expressed from a retroviral vector in the Ramos cell line and transcription was modulated by doxycycline to control the level of SHM. Because only a limited population of photoactivated molecules exhibits noticeable fluorescence.7 515 Green PA-GFP (N) ¶ 400 Reference Filter set§ Chromophore Association state‡ pKa‡ Brightness† Emission peak (nm) Excitation peak(nm) Color of spectral class Optical highlighter FPs Investigations into the complex photophysical properties of FP variants have led to the generation of chromophores that can be activated either to initiate fluorescence emission from a quiescent state (photoactivation) or to be optically converted from one fluorescence emission bandwidth to another (photoconversion). Perhaps new selection methods will enable the improvement of pH sensitivity and photostability. 2003..0 80. Wang and coworkers demonstrated that this technique could also be used to generate new phenotypes in FPs when expressed in a human B-cell line (Ramos) that hypermutates immunoglobulins. 2004 Verkhusha and Sorkin..3 8. has a shorter wavelength emission maximum (625 nm). †Product of the molar extinction coefficient and the quantum yield (mM cm)–1. These proteins are emerging as a powerful new class of probes. The resulting mutants were enriched by FACS to select clones exhibiting the longest emission wavelengths.. *Common literature FP abbreviation. 2004 DAPI/FITC SYG Weak dimer 4. CYG. 2005 TxRed QYG Monomer 4. 137 nm.4 0.503 Green 4255 Table of physical properties for the monomeric and tandem dimer optical highlighters. ¶Native conformation.5 Weak dimer SYG FITC/GFP Patterson and Lippincott-Schwartz.6 10.8 511 468 400 PS-CFP2 (P) 490 Cyan Green PS-CFP2 (N) 504 Green PA-GFP (P) ** 517 13.

(C) Photoswitching of Dronpa involves cis-trans photoisomerization induced by alternating radiation between 405 nm and 488 nm. 2005). . A new protein derived from the jellyfish Aequorea coerulescens. Eos. photoconverts from cyan to green fluorescence upon illumination at 405 nm (Table 2). 2004). exhibits Fig. unpublished). Table 2) contain a chromophore derived from the tripeptide His-Tyr-Gly (HYG) that initially emits green fluorescence. all of which contain the HYG chromophore. However. All of the green-to-red optical highlighters so far reported (including Dendra2. Furthermore. Unfortunately. was derived from wtGFP by the substitution of histidine for threonine at position 203 (T203H) to produce a variant devoid of green fluorescence until activated (Patterson and Lippincott-Schwartz. 4. which either remain invisible or continue to emit the original wavelengths. (A) Photoactivation of PA-GFP (illustrated) and PS-CFP2 is believed to occur due to decarboxylation of Glu222 followed by conversion of the chromophore from a neutral to anionic state. PA-GFP is still the best choice in the green region of the palette and is far superior in terms of dynamic range to the only red variant yet reported. such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). PS-CFP2 has an advantage over PAGFP in that a significant level of cyan fluorescence is present before photoconversion. Of the few photoactivatable probes available. probably because the non-activated state is significantly brighter than that of PA-GFP. this feature is crucial for establishing a high dynamic range for photoactivation. Kaede.. 4A and Table 2). Photoactivation. 5A-C). Nienhaus et al.D. This process requires catalysis by the intact protein and results in a dramatic shift of fluorescence emission to longer (orangered) wavelengths (Mizuno et al. This optical highlighter features the same chromophore as PA-GFP (SYG) and may be photoactivated through a similar mechanism (Fig. PA-GFP is difficult to detect in its non-activated form. the full potential for optical highlighters is yet to be realized. the dynamic range of PS-CFP2 is lower than that of PA-GFP and the probe is inferior to green-to-red optical highlighters in terms of photoconversion efficiency. PS-CFP2 (Chudakov et al. enabling tracking of the dynamics in molecular subpopulations (Fig.W.7 and KFP1.Journal of Cell Science 4256 Journal of Cell Science 120 (24) fusion tags. 2002). which enables easier tracking and determination of regions for selective illumination. KikGR. measurements are not negatively influenced by freshly synthesized or non-converted FPs. (B) Green-to-red photoconversion for Kaede. 2005). They offer a gentler alternative to the relatively harsh photobleaching techniques. 4A). occurs when the FP is illuminated with ultraviolet or violet radiation to induce cleavage between the amide nitrogen and ␣-carbon atoms in the His62 residue leading to subsequent formation of a conjugated dual imidazole ring system. Dendra2 and Eos. A similar isomerization mechanism is suggested to operate in mTFP0. 4B). 2003. and KikGR. The only choice for cyan-to-green photoconversion. Irradiation with intense violet light (390-415 nm) produces a 100-fold increase in green fluorescence (emission peak at 504 nm). Similar FPs based on Emerald and superfolder GFP display a reduced dynamic range (M. PA-mRFP1 (Verkhusha and Sorkin. The unconventional chemistry involved in this chromophore transition provides an excellent foundation upon which to develop more advanced highlighters.. which is perhaps the greatest pitfall in determining regions for examination. photoswitchable (PS)-CFP2. which generally require high laser powers and repeated illumination to completely eradicate active fluorophores from the region of interest. Irradiation with short wavelength visible or long wavelength ultraviolet light induces cleavage between the amide nitrogen and ␣-carbon atoms in the histidine residue with subsequent extension of chromophore conjugation to the histidine side chain (Fig. photoconversion and photoswitching mechanisms for optical highlighter FPs. The first photoactivatable (PA) optical highlighter PA-GFP (Fig... However.

. exhibits unusual photochromic behavior characterized by its ability to toggle fluorescence on and off following illumination with two different excitation wavelengths (Fig. A monomeric variant. 5G-I). 2003a). Table 2). Kindling FP (commercially available from Evrogen as KFP1. Dronpa is driven to the protonated species with a commitment decrease in fluorescence to produce a dim (off) state in which the 390-nm absorption peak predominates. t=20 minutes. t=10 minutes. a monomeric FP derived from Pectiniidae (Ando et al. t=45 minutes. whereas 4257 the minor peak arises from the protonated (neutral) form.. The tandem dimer of the green-to-red highlighter named Eos (Nienhaus et al. (D) Photoconversion of a single mitochondrion (red) in a selected region at 405 nm illumination. there is need for better performers in all categories.. KFP1 does not exhibit fluorescence emission until illuminated with green or yellow light in the region between 525 nm and 580 nm. (G) Photoconversion (red) of the selected region (box) with a 405 nm laser. Another photoswitchable highlighter. (A) Circular region of interest selected with an Olympus FV1000 tornado scanner is illuminated at 405 nm for 5 seconds. (F) Cargo exchange between mitochondria (arrow). the region spelling FV10 was activated with a 405 nm laser. 2002. t=0 minutes. (L) FV10 region photobleached while imaging the actin network at 488 nm. (D-F) Tracking of mitochondria labeled with tdEosmito-N-7 in rabbit kidney (RK-13 cell line) epithelial cells. In the green-to-red class. (I) Photoconverted lamellipod retracts amid increased podosome formation and generation of a new leading edge... respectively. The dim state is readily converted back to the original fluorescent (on) deprotonated state with minimal illumination at 405 nm (Fig. the anionic species emits at a maximum of 518 nm with a relatively high quantum yield of 0. 5JL). (J-L) Photoswitching of the actin cytoskeleton with Dronpa-actin-C-7 in rat thoracic aorta (A7r5 cell line) myoblasts.. (H) The photoconverted channel illustrates podosome formation by photoconverted actin and changes in leading edges. Dronpa has an absorption maximum at 503 nm with a minor peak at 390 nm. in which the dark and fluorescent states have been characterized by crystallography. Upon irradiation at 488 nm. t=0 minutes. Clearly. 2007). 5. 2004. t=0 minutes. both are tetrameric and thus not useful for most experiments. termed mTFP0. has been developed from a non-fluorescent chromoprotein isolated from Anemonia sulcata (Chudakov et al. Tsutsui et al. 4C and Fig. However. 2006. By contrast. t=3 minutes. 2004) is better than Dendra2 in terms of brightness and photostability but twice as large (see Fig.. (K) After completely photoswitching the labeled actin ‘off’ at 488 nm. is probably the best choice for sensitive fusions and FRET studies (see Fig. t=0 minutes. t=5 minutes. 2005). Dendra2 (Gurskaya et al.. (C) Ruffles. (B) The photoactivated actin chimera first translocates to the ruffles at the cellular margins as fluorescence intensity decreases in the activated region. 4C).. Wiedenmann et al.Journal of Cell Science Fluorescent protein advances monomeric character but is hampered by low brightness levels and the artifact of continued photoconversion during imaging. (G-I) Examination of lamellipodia with Dendra2-actin-C-7 in OK cells. cytoplasmic actin pools and the filamentous actin network gain more intensity at t=60 minutes. 2005). (A-C) Photoactivation of mPA-GFP-actin-C-7 in opossum kidney (OK cell line) epithelial cells.85 (Table 2). When irradiated at 488 nm.. Low-intensity light results in transient red fluorescence (termed kindling) that has excitation and emission maxima at 580 nm and 600 nm. the best performers in terms of brightness and conversion efficiency are Kaede and KikGR (Ando et al.. Dronpa. 2005). t=20 minutes. 2006). 5DF). which slowly decays upon cessation of illumination as the protein relaxes back to its Fig. the neutral form of the chromophore is almost non-fluorescent. A new generation of specialized reversible optical highlighters with on-off switching capabilities was heralded by the introduction of Dronpa. which was engineered by directed and random mutagenesis.7 (Henderson et al. Photoswitching of Dronpa occurs by interconversion between the deprotonated and protonated forms (Habuchi et al. Optical highlighter FPs in action imaged with laser scanning confocal microscopy. (J) Actin network imaged with a 488-nm laser. The major peak is due to the deprotonated (anionic) species of the chromophore. Similar behavior has been reported for a teal FP precursor. Habuchi et al. (E) Close approach of a non-converted (green) mitochondrion (arrow). but suffers from rapid photobleaching of the red species during laser scanning confocal imaging and is <50% as bright as the tetramers.

(A) Widefield fluorescence image of multiple focal adhesions labeled with tdEos fused to human vinculin. Andresen et al. Hanson. Fig. By contrast. 2003b. These collective features may constitute a fundamental mechanism that is common to all photoactivatable and reversibly photoswitchable FP derivatives. allowing tight control over fluorescent labeling. 6. Chudakov et al. The unique photoactivation probes. red-to-green photoconversion. high-intensity green illumination (~550 nm) or continued irradiation at moderate levels results in irreversible photoconversion to give a fluorescence intensity ~30-fold greater than that of the non-activated protein. Sergey Lukyanov. These investigators include Eric Betzig. Investigations into the mechanism of FP photoswitching (Andresen et al. (C) PALM view of the focal adhesion structure shown in B. Hell. monomeric derivatives that exhibit high contrast and can be easily photoconverted to display a wide spectrum of emission colors. bright. Henderson et al. Richard N. Hess et al. Daugherty. Verkhusha and Jörg Wiedenmann. 6) and stochastic optical reconstruction microscopy (STORM) rely on low levels of illumination to photoactivate selected individual molecules spaced further apart than the diffraction limit so that an array of magnified diffraction spots can be recorded on a highly sensitive camera. Chudakov. These conformational changes are apparently accompanied by varied protonation states of the chromophore that help determine the fluorescent properties (as discussed above). Jennifer LippincottSchwartz. David W. green-to-red optical highlighters. Day. light-induced photoswitching is probably a manifestation of chromophore planarity and structural rearrangements of internal amino acid side chains within the chromophore cavity. James Remington. FPs useful for optical marking should evolve towards brighter. Finally. Roger Y. coupled with advanced new screening technologies.. Egner et al. 2005. optical highlighters are also valuable tools in superresolution microscopy techniques designed to break the traditional Abbe diffraction barrier (Betzig et al. George T. and photoswitchable variants produced thus far from GFP derivatives and reef coral proteins certainly warrant aggressive efforts to solve the problems associated with aggregation and fine-tune their photoactivation requirements and emission profiles. Irradiation with intense blue light (450-490 nm) completely quenches the red fluorescence immediately. 2007.4258 Journal of Cell Science 120 (24) Journal of Cell Science Fig. Photoactivated localization microscopy (PALM) imaging of focal adhesions near the coverslip surface in paraformaldehyde-fixed Gray fox lung fibroblast cells. will greatly expand the potential applications for this class of probes.. Adjacent molecules are not recorded because they still exist in the dark or inactivated state. Campbell. the engineering of advanced optical highlighter proteins that shift photoconversion illumination wavelengths to the blue and green spectral regions (which are significantly less toxic to living cells than the ultraviolet wavelengths currently required). The reconstructed images feature optical resolutions down to 10 nm. 4C). and derivatives emitting in the far-red or near-infrared regions of the spectrum would be especially useful. Stiel et al. and photostable proteins in every spectral class. should further expand the available color palette and ultimately provide rapidly maturing. state (Fig. the image is assembled one molecule at a time by means of iterative switching cycles. Robert E. 2006).. Konstantin A.. a new group can be photoactivated and read out. Patrick S. (B) Total internal reflection fluorescence (TIRF) summed-molecule image of the focal adhesion within the region indicated by the box in A. as well as shifting emission wavelengths to the yellow through the far-red region. This work would not have been possible without the generous contribution of FPs and technical information from the originating laboratories. S. Dmitriy M. 2007) suggest that cis-trans isomerization of the hydroxybenzilidine chromophore moiety is a key event in the switching process. Vladislav V. Atsushi Miyawaki. 2007. Oliver Griesbeck. improved expression at elevated temperatures. In the future.. which includes the apparent assembly of vinculin into a partial network (arrows). or dark. Piston. Calculation of the exact coordinates of the single fluorescent molecules within the photoactivated population enables precise localization. Furthermore. In addition to their use for selectively labeling subpopulations of fusion proteins for dynamic studies... Tsien. Lukyanov. The major drawback of KFP1 is its obligatory tetramerization. . whereas the trans isomer is adopted by the chromophore in the non-fluorescent. Proteins capable of reversible photoactivation. Thus. 2007. As the development of optical highlighters continues. 2006. which seriously affects its potential for use as a fusion tag or FRET partner. The cis conformation represents the fluorescent state. initial non-fluorescent state. Newly introduced methods such as photoactivated localization microscopy (PALM. 2007. after switching off (or photobleaching) the registered molecules. Conclusions Continuing efforts in protein engineering of the existing FPs.. These techniques are becoming increasingly fast and hold significant promise for live cell imaging at unprecedented resolution.

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