Characterization and development of photoactivatablefluorescent proteins for single-molecule–basedsuperresolution imagingSiyuan Wanga, Jeffrey R. Moffitta, Graham T. Dempseya, X. Sunney Xiea, and Xiaowei Zhuanga,b,c,1

Departments of aChemistry and Chemical Biology and bPhysics, Harvard University, cHoward Hughes Medical Institute, Cambridge, MA 02138

Contributed by Xiaowei Zhuang, April 10, 2014 (sent for review February 7, 2014)

Photoactivatable fluorescent proteins (PAFPs) have been widelyused for superresolution imaging based on the switching andlocalization of single molecules. Several properties of PAFPsstrongly influence the quality of the superresolution images.These properties include (i) the number of photons emitted perswitching cycle, which affects the localization precision of individ-ual molecules; (ii) the ratio of the on- and off-switching rate con-stants, which limits the achievable localization density; (iii) thedimerization tendency, which could cause undesired aggregationof target proteins; and (iv) the signaling efficiency, which de-termines the fraction of target–PAFP fusion proteins that is de-tectable in a cell. Here, we evaluated these properties for 12commonly used PAFPs fused to both bacterial target proteins,H-NS, HU, and Tar, and mammalian target proteins, Zyxin andVimentin. Notably, none of the existing PAFPs provided optimalperformance in all four criteria, particularly in the signaling effi-ciency and dimerization tendency. The PAFPs with low dimeriza-tion tendencies exhibited low signaling efficiencies, whereasmMaple showed the highest signaling efficiency but also a highdimerization tendency. To address this limitation, we engineeredtwo new PAFPs based on mMaple, which we termed mMaple2 andmMaple3. These proteins exhibited substantially reduced or unde-tectable dimerization tendencies compared with mMaple but main-tained the high signaling efficiency of mMaple. In the meantime,these proteins provided photon numbers and on–off switching rateratios that are comparable to the best achieved values among PAFPs.

STORM | PALM | fPALM | photoconvertible | photoswitchable

Photoactivated localization microscopy, stochastic optical re-construction microscopy, and related imaging methods take

advantage of photoswitching and imaging of single molecules tocircumvent the diffraction limit of spatial resolution in lightmicroscopy (1–3). In these methods, only a subset of the fluo-rescent labels in the sample is switched on at any given time suchthat the positions of individual fluorophores can be localizedfrom their images with high precision. Iteration of this processallows numerous fluorescent labels to be localized and an imagewith sub–diffraction-limit resolution to be reconstructed fromthe fluorophore localizations. Fluorescent proteins that can beactivated from dark to fluorescent or converted from one colorto another are widely used for such imaging approaches (4, 5).Although photoactivatable fluorescent proteins (PAFPs) aregenerally dimmer than photoswitchable dyes (6, 7) and hencegive lower image resolution, the ease and high specificity of la-beling protein targets in living cells with fluorescent proteinsmakes PAFPs highly appealing probes for imaging the dynamicsof cellular structures (4, 8).For single-molecule–based superresolution imaging methods,

several properties of PAFPs are particularly important for theimage quality. Here, we focus on four such key properties. (i)The first property is the photon budget, defined as the averagenumber of photons emitted in each switching event. Given thatthe error of localizing an individual fluorophore approximatelyscales with the inverse square root of the number of detected

photons, a higher photon budget leads to higher localizationprecision and hence higher image resolution (7, 9). (ii) Thesecond property is the on–off switching rate ratio (on–off ratio),defined as the ratio between the on-switching (activation) andoff-switching or photobleaching rates under the illumination ofthe imaging light only (7). Even in the absence of activation light,the imaging light itself can also switch on the PAFPs, albeit ata low rate. Thus, the ratio between the on-switching and off-switching rates under this condition determines the lower boundof the fraction of PAFP molecules in the on-state at any giventime. The presence of activation light would increase this frac-tion. When the product of this fraction and the density of fluo-rescent labels reaches approximately one fluorophore perdiffraction-limited volume, it becomes difficult to resolve andprecisely localize the activated fluorophores. Hence the on–offratio limits the density of fluorescent labels that can be localized,which in turn affects the effective image resolution based on theNyquist sampling theorem (10). (iii) The third property is thedimerization tendency. Many PAFPs have a weak tendency toform dimers; this could even be true for the PAFPs that arereported as being monomeric. When these proteins are fused totarget proteins that also tend to polymerize, they may causeundesired aggregation of the target proteins and distort the na-tive distribution of the protein of interest. (iv) The fourthproperty is the signaling efficiency, defined as the ratio betweenthe number of detectable PAFP-fusion molecules per cell andthe expression level of the fusion protein. Fluorescent proteinsdo not necessarily fold with 100% efficiency. Among the foldedmolecules, not all of them will become mature at the time ofimaging. Among the matured PAFP molecules, only a subset canbe photoactivated and imaged. Because of these deficiencies, thenumber of fusion molecules detected could be substantiallylower than the expression level of the fusion protein. PAFPs withhigher signaling efficiencies will lead to higher localizationdensities for a given target protein, which will in turn increase theeffective image resolution.In this work, we measured the above properties of 12 commonly

used PAFPs, including PAGFP (11), Dendra2 (12, 13), mEos2

Significance

Photoactivatable fluorescentproteins (PAFPs) are importantprobesfor superresolution fluorescence microscopy, which allows thespatialorganizationofproteins in livingcells tobeprobedwithsub–diffraction-limit resolution. Here, we compare four properties ofPAFPs that are critical for superresolution imaging and report twonewPAFPs that exhibit excellentperformance inall fourproperties.

Author contributions: S.W., J.R.M., and X.Z. designed research; S.W., J.R.M., and G.T.D.performed research; S.W. and J.R.M. contributed new reagents/analytic tools; S.W., J.R.M.,X.S.X., and X.Z. analyzed and interpreted data; and S.W., J.R.M., G.T.D., X.S.X., and X.Z.wrote the paper.

Conflict of interest statement: A US provisional patent application has been filed for thenew fluorescent proteins developed in this work.1To whom correspondence should be addressed. E-mail: zhuang@chemistry.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1406593111/-/DCSupplemental.

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(14), mEos3.2 (15), tdEos (16), mKikGR (17), PAmCherry (18),PAtagRFP (19), mMaple (20), PSCFP2 (13, 21), Dronpa (22), andmGeosM (23). From this screen, we found that none of thesePAFPs was simultaneously optimal in all four criteria describedabove. For example, PAtagRFP and mEos3.2 exhibited thehighest photon budgets among PAFPs, excellent on–off ratios, andundetectable dimerization tendencies, but showed poor signalingefficiencies. Alternatively, mMaple provided excellent signalingefficiency and on–off ratio with a photon budget nearly equal tothose of PAtagRFP and mEos3.2, but had a substantial di-merization tendency. To address this limitation, we developed twonew PAFPs based on mMaple that exhibited substantially reducedor undetectable dimerization tendencies while maintaining thehigh signaling efficiency, high photon budget, and low on–off ratioof mMaple. These PAFPs will substantially facilitate super-resolution imaging of cellular structures.

ResultsPhoton Budget of Photoactivatable Fluorescent Proteins. To evalu-ate the properties of PAFPs under conditions similar to typicalsuperresolution imaging experiments, we fused each PAFP tovarious target proteins and expressed these fusion proteins ineither mammalian cells or bacteria. To measure the photon bud-get, we fused the PAFPs to the mammalian focal adhesion proteinZyxin, transiently transfected BS-C-1 cells with the fusion con-structs, and imaged the cells using the superresolution imagingmode in which individual activated proteins were imaged. Thedistributions of the photon numbers detected per activation eventwere determined for all 12 PAFPs. Four example distributions,for mEos3.2, mMaple, PSCFP2, and PAGFP, are shown in Fig.1. The mean photon numbers determined from these dis-tributions are listed in Table 1.Fewer photons were detected from the green PAFPs (PAGFP,

PSCFP2, Dronpa, and mGeosM) than from the red ones (Dendra2,mEos2, mEos3.2, tdEos, mKikGR, PAmCherry, PAtagRFP, andmMaple). However, within the same color group, the differencein photon budget was less than twofold. In addition to theabove-listed fluorophores, we also imaged rsFastLime (24) andrsEGFP (25). Both of these proteins gave relatively low photonbudget (<60 photons per switching event), which would lead torelatively poor localization precision. We thus did not furthercharacterize these proteins. It is, however, worth noting thatthese proteins are excellent choices for a different mode ofsuperresolution imaging [reversible saturable optical fluorescencetransitions (RESOLFT)] due to the large number of switchingcycles that they exhibit before photobleaching (24, 25).

On–Off Ratio of Photoactivatable Fluorescent Proteins. To de-termine the on–off ratio, we measured the rates for switching on

and switching off (or photobleaching) the PAFPs in the presenceof imaging light only. By definition, the on-switching rate is theincrement in probability of the on-switching events per unit time.To measure this quantity, we imaged the Zyxin-PAFP–express-ing cells in the superresolution mode for a short period withoutany activation light (with imaging light only). The samples werethen imaged to completion with an additional activation light at405 nm. The ratio of the total number of activation events ac-cumulated by a certain time during the period without activationlight over the total number of activation events by the end of theimaging process was determined. The slope of this cumulativeactivation probability against time then gave the on-switchingrate (Fig. 2 A and B, and Table S1). The off-switching rate wasdetermined from the inverse of the mean lifetime of the on-stateof each PAFP in the presence of the imaging light (Fig. 2 C andD, and Table S1). The ratios between the on- and off-switchingrates were determined for all 12 PAFPs (Table 1). Because bothon- and off-switching rates scale linearly with the illumination in-tensity, the on–off ratio should be independent of the imaging lightintensity. The on–off ratios were generally very small (10 −5 to10−6) except for PAGFP, Dronpa, and mGeosM, which were∼10−3.From the superresolution images of Zyxin, it is evident that the

PAFP with a smaller on–off ratio gave images with a higher lo-calization density and hence higher image quality (Fig. 2 E and F).This correlation can be understood based on the Nyquist samplingtheorem, which suggests that the final resolution of an image is atleast twice the average distance between the localized probes (10).

Dimerization Tendency of Photoactivatable Fluorescent Proteins.Dimerization or oligomerization of fluorescent proteins maycause undesired aggregation of the target proteins. Here, insteadof measuring the dimerization affinity of PAFPs in vitro, we di-rectly probed whether the PAFPs could cause aggregation ofa target protein using a previously reported method (26). In thisapproach, the fluorescent proteins are fused to the Escherichia coliprotease ClpP, which itself oligomerizes to form a tetradecamericcomplex. It has been suggested that ClpP proteins tend to ag-gregate and form a single visible punctum in E. coli when fused toa fluorescent protein with a substantial dimerization tendency,whereas fusion to a fluorescent protein with a weak or no di-merization tendency tends to display a diffuse localization pattern(26). We thus fused E. coli codon-optimized PAFP sequencesto the chromosomal copy of clpP, and report whether cellsexhibit the single-punctum phenotype (Table 1 and Fig. 3A).Fusion with mKikGR, mGeosM, mMaple, PAmCherry,

PSCFP2, or mEos2 all led ClpP to form a single punctum in atleast a subset of cells, suggesting that these PAFPs exhibit ap-preciable dimerization tendency, whereas Dendra2, mEos3.2,tdEos, PAtagRFP, PAGFP, and Dronpa did not show highenough dimerization tendency to drive detectable ClpP aggre-gation (Table 1 and Fig. 3A). As a cautionary note, becausedifferent target proteins have different expression levels andintrinsic oligomerization or polymerization tendencies, PAFPsthat cause appreciable aggregation of ClpP may not necessarilycause aggregation of other target proteins, and vice versa.To further illustrate the potential effect of PAFP dimerization

on target proteins, we imaged E. coli nucleoid-associated proteinH-NS, E. coli chemotactic receptor Tar, and mammalian in-termediate filament protein Vimentin fused to different PAFPs.In an earlier paper (27), we have reported that H-NS appears asa few large and discrete clusters in cells when fused to mEos2,a previously reported monomeric version of the Eos fluorescentprotein (14, 16). We also observed similar results when H-NSwas fused to PAmCherry (27), another previously reported mo-nomeric PAFP. Fusion with these PAFPs did not appear toperturb the functional activity of H-NS (27). Given the residualdimerization tendency of mEos2 and PAmCherry detected bythe ClpP assay (Table 1 and Fig. 3A), we extended the study ofH-NS using other PAFPs here (Fig. 3B). Notably, H-NS generallyappeared as large and discrete clusters in cells when fused toPAFPs that exhibited appreciable dimerization tendency in the

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Fig. 1. Photon number measurements of PAFPs. The histograms are ex-ample photon number distributions of mEos3.2 (A), mMaple (B), PSCFP2 (C),and PAGFP (D) measured by imaging individual Zyxin-PAFP fusion proteins inlive BS-C-1 cells. The mean photon numbers are indicated.

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ClpP assay, such as mEos2, PAmCherry, and mMaple. H-NS alsoappeared as large clusters when fused to tdEos, even though tdEosdid not show sufficient dimerization to drive ClpP aggregation.However, fusion of H-NS to Dendra2, mEos3.2, and PAtagRFP,which did not exhibit detectable dimerization tendency by the ClpPassay, appeared much more dispersed in the nucleoids. We alsoobserved the more dispersed phenotype with immunofluores-cence imaging of H-NS, but did not present this comparisonhere because fixation of the bacterial nucleoid is in generaldiscouraged. It is known that H-NS oligomerizes/clusters evenwithout fusion to fluorescent proteins (28, 29), and this intrinsicH-NS clustering effect is important for its in vivo function asa transcriptional silencer (29) and brings gene loci in differentchromosome regions into spatial proximity in wild-type cells(27). However, fusion with mEos2, PAmCherry, mMaple, andtdEos may have exaggerated the H-NS clustering effect. Withoutprior knowledge of the residual dimerization tendency of these“monomeric” PAFPs, we previously interpreted the large clustersof H-NS-mEos2 and H-NS-PAmCherry earlier as a property ofH-NS (27). Results here suggest that this interpretation is likelyinaccurate because the clustering of H-NS is likely enhanced by theunderappreciated dimerization effect of mEos2 and PAmCherry.Although it is formally possible that the more monomeric PAFPscould disrupt intrinsic H-NS clustering by discouraging PAFPself-interactions, we consider this a less likely possibility.As another example, Tar preferentially clustered at cell poles

when fused to mEos2 and mKikGR (Fig. 3C) (30), but spreadout more evenly along the envelope of E. coli when fused to themore monomeric mEos3.2 (Fig. 3C), suggesting that the pref-erential cell pole distribution might have been exaggerated bythe dimerization of mEos2 and mKikGR. Such aggregation ef-fect could also apply to eukaryotic systems. For example, fusion tomEos2 and mKikGR caused Vimentin filaments to cluster intothick bundles in mammalian cells, whereas Vimentin-mEos3.2appeared as thin filaments that were similar to immunofluores-cence images of Vimentin in untransfected cells (Fig. 3D).

Signaling Efficiency of Photoactivatable Fluorescent Proteins. Evenwhen the PAFP is fused to the target protein at its endogenouschromosomal locus, the number of detectable fluorescent proteinsdoes not necessarily reflect the expression level of the fusionprotein. This deficiency not only prevents quantitative analysis ofthe target protein, but may also reduce the resolution of the image

by effectively decreasing the labeling density. Here, we comparedthe relative signaling efficiency of different PAFPs by determiningthe number of single-molecule localizations per cell of the PAFPsfused to a common target protein under endogenous expression.We fused the PAFPs to the E. coli gene hupA, which encodesa subunit of the nucleoid-associated protein HU, at its endoge-nous chromosomal locus and determined the total number ofHU-PAFP localizations per cell (Fig. 4 and Table 1). The four greenPAFPs were too dim to be detected as single molecules in E. coli,whereas the eight red PAFPs showed similar photon budget to thosemeasured in mammalian cells (data not shown). Among the redPAFPs, mMaple provided by far the largest number of HUlocalizations, which was 6- to 32-fold higher than those of otherHU-PAFP fusion proteins (Fig. 4).The difference in the number of HU-PAFP localizations for

different PAFPs may be attributed to multiple reasons. One ofthe possible reasons could be the difference in the number ofblinking (switching) events per fluorophore. We measured theaverage number of blinking events in fixed HU-PAFP–express-ing cells (Table S2). The average number of blinking eventsspanned only a small range from 1.7 to 3.3, which are similar to theresults derived from purified PAFP in vitro (Fig. S1). The locali-zation numbers divided by the average numbers of blinking eventsyielded the numbers of imaged molecules per cell (Table S2), whichis still much bigger in the case of HU-mMaple–expressing cells (4-to 36-fold higher than in other HU-PAFP–expressing cells).A second possible reason for the difference in localization

numbers could be the difference in HU-PAFP expression levels.However, quantitative Western blot experiments showed thatmost HU-PAFP constructs are expressed at similar levels (TableS2), except for tdEos. We divided the number of imaged mole-cules per cell by the number of expressed fusion proteins per cellto calculate the percentage of PAFP imaged (Table S2). Thepercentage for mMaple imaged was still 5- to 22-fold higher thanthe other PAFPs. Therefore, neither the number of blinkingevents nor the expression level is a major contributing factor tothe difference in the observed localization numbers.A third potential reason is the difference in the fluorescent

protein maturation time. Because E. coli has a relatively shortdoubling time under our growth conditions, a substantial fractionof the PAFPmolecules may not have sufficient time to mature andbecome fluorescent. To test this idea, we measured the in vivomaturation time of several PAFPs by using kanamycin to block

Table 1. Properties of PAFPs

PAFPPreactivation/postactivationemission wavelength, nm* Photon no.

On–off switchingrate ratio ClpP clustering†

No. of localizationsper cell‡

Maturationtime, min§

Dendra2 507/573 686 4.2 × 10−6 − 1,810 38mEos2 519/584 745 2.9 × 10−6 + 1,290 340mEos3.2 516/580 809 2.6 × 10−6 − 1,950 330tdEos 516/581 774 3.2 × 10−6 − 1,800 330mKikGR 515/591 599 4.1 × 10−6 + 3,800 31PAmCherry —/595 706 7.8 × 10−6 + 4,200 61PAtagRFP —/595 906 5.7 × 10−6 − 760 200mMaple 505/583 798 1.9 × 10−6 + 24,000 48mMaple2 506/582 783 1.0 × 10−6 + 21,000 62mMaple3 506/583 675 6.2 × 10−7 − 12,300 49PAGFP —/517 313 1.3 × 10−3 − <10PSCFP2 468/511 223 8.1 × 10−6 +Dronpa —/517 262 5.8 × 10−4 − 25mGeosM —/514 248 4.9 × 10−4 + <10

A 405-nm laser was used for photoactivation. The photon number and on–off switching rate ratio were measured in live BS-C-1 cells. The ClpP clustering,number of localizations per cell, and maturation time were measured in live E. coli cells.*The mMaple2 and mMaple3 emission wavelengths were measured in this work with purified proteins. The other wavelengths are cited from refs. 4 and 16.†The “+” indicates that ClpP-PAFP exhibits clustered distributions in at least a subset of cells, whereas “−” indicates that ClpP-PAFP does not exhibit clustereddistributions in any cells. The results on Dendra2, Dronpa, and mEos2 are consistent with a previous report (26).‡The number of HU-PAFP localizations per E. coli cell.§Maturation time is defined as the half-life of the immature state.

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protein synthesis in E. coli cells expressing HU-PAFP, and thenrecording the increase in cellular fluorescence due to PAFPmaturation (Fig. S2 and Table 1). Indeed, compared with some ofthe PAFPs with low signaling efficiencies, mMaple has a sub-stantially faster maturation time. However, the difference inmaturation time was substantially smaller than and hence notsufficient to account for the observed difference in the localizationnumbers (SI Note and Fig. S3).Other potential contributing factors include the fraction of

properly folded PAFPs at equilibrium, the fraction of foldedPAFPs that can ultimately mature, the fraction of folded andmature PAFPs that can be photoactivated, and the fraction ofactivated PAFPs that are sufficiently bright to image. Given thatdifferent red PAFPs tend to have similar photoactivation effi-ciencies (31) and photon budgets (Table 1), the last two factorsare less likely explanations for the observed range of signaling ef-ficiencies. More experiments are required to test these possibilities.Finally, we measured the signaling efficiency of the PAFPs in

mammalian cells by transient transfection of A549 cells with thezyxin::PAFP constructs. For fast data collection, conventionalfluorescent microscopy was used and cells were imaged until thePAFPs were fully bleached. The number of imaged molecules ineach cell was estimated by dividing the total photon count fromthe cell by the product of the average photon number perswitching event (Table 1) and the average number of blinkingevents for each PAFP (Table S3). The results indicate thatmMaple again offers much higher signaling efficiency (7- to 70-fold higher) than the other red PAFPs, even in mammalian cells.Among the green PAFPs, PSCFP2 offers a comparable signalingefficiency to that of mMaple (Table S3).

New PAFPs with High Signaling Efficiency and Low DimerizationTendency. The above results show that mMaple has a muchhigher signaling efficiency than all of the other tested red PAFPs.However, the dimerization tendency of mMaple could lead toaggregation effects on the target proteins. Dendra2, mEos3.2,and PAtagRFP exhibit undetectable dimerization tendency,but have low signaling efficiency. It is thus desirable to developa new PAFP that has both high signaling efficiency and low

dimerization tendency. To this end, we engineered two newPAFPs by introducing point mutations into mMaple designed todestabilize the dimerization of this protein.We first took inspiration from the two mutations that make

mEos3.2 more monomeric than mEos2: I102N and Y189A (15).Based on a sequence alignment between mEos2 and mMaple, wemade the comparable mutations, I111N and Y198A, in mMaple.We then tested the properties of this protein, which we termedmMaple2, by fusing it to Zyxin, ClpP, and HupA and performingmeasurements on the photon budget, on–off ratio, dimerizationtendency, and signaling efficiency as described above. mMaple2exhibited a similar photon budget, on–off ratio, and signalingefficiency as those of mMaple (Fig. 5 A–C, Table 1, and TablesS2 and S3). ClpP-mMaple2 proteins still formed puncta in somecells. However, the percentage of cells showing single punctumwas significantly reduced in comparison with ClpP-mMaple,suggesting a lower dimerization tendency of mMaple2 (Fig. 5D).To further reduce the residual dimerization tendency of mMaple2,

we screened a series of mutations to residues that are predictedto be solvent-exposed near residues 111 and 198. We focused oncharged residues and switched them to the opposite charge. One ofthe derivatives, with mutations E82R, D83K, and D197K, in ad-dition to I111N and Y198A, exhibited undetectable dimerizationtendency when fused to ClpP (Fig. 5D and Table 1). This derivative,which we termed mMaple3, again had a similar photon budget tothat of mMaple and even a lower on–off ratio compared withmMaple (Fig. 5 A and B, and Table 1). Its signaling efficiency wasonly moderately reduced from those of mMaple or mMaple2 (Fig.5C, Table 1, and Tables S2 and S3).

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Fig. 2. On–off switching rate ratio measurements of PAFPs. Sample data arepresented for PSCPF2 (A, C, and E) and PAGFP (B, D, and F). (A and B) Cumu-lative on-switching probability as a function of time without activation light.The slope of the line gives the on-switching rate. (C and D) Distribution of theon-state lifetime. The blue bars represent measured data, which were fittedwith a binned exponential function (magenta dots) Pn =

R nn−1ð1=mÞe− x

mdx, totake into account that lifetimes are rounded to the next larger integer.m is themean lifetime, the inverse of which gives the off-switching rate. The on–offswitching rate ratio is defined as the on-rate divided by the off-rate. Each framecorresponds to 16ms. (E and F) Representative superresolution images of Zyxin-PSCFP2 and Zyxin-PAGFP in live BS-C-1 cells.

Dendra2B mEos3.2 PAtagRFP tdEos mEos2 PAmCherry

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Fig. 3. Dimerization tendency of PAFPs and its effect on the distributions oftarget proteins. (A) Phase contrast (Left) and conventional fluorescent images(Right) of live E. coli cells expressing ClpP-PAFP fusions. PAFPs with substantialdimerization tendencies (mEos2, mMaple) result in the formation of ClpPpuncta. PAFPs with little to no dimerization tendencies (mEos3.2, PAtagRFP)produce a diffusive ClpP distribution. (B) Overlaid superresolution (magenta)and phase-contrast (gray) images of live E. coli cells expressing H-NS-PAFPfusions. (C) Overlaid superresolution and phase-contrast images of fixed E. colicells expressing Tar-PAFP fusions. (D) Superresolution images of fixed Cos-7cells expressing Vimentin-PAFP fusions or fixed untransfected Cos-7 cells withimmunofluorescent labeling of Vimentin (IM). (Scale bars: 1,000 nm.)

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We further tested the aggregation effects of mMaple2 andmMaple3 on H-NS, Tar, and Vimentin. H-NS-mMaple3 showedsubstantially more spread out H-NS distributions than H-NS-mMaple, although H-NS-mMaple2 still appeared as a few discreteclusters in each cell (Fig. 5E). Tar-mMaple2 and Tar-mMaple3were substantially less concentrated at the cell poles thanTar-mMaple (Fig. 5F). Vimentin-mMaple2 and Vimentin-mMaple3filaments were much less bundled than Vimentin-mMaple fila-ments (Fig. 5G). All of these observations are consistent with thereduced dimerization tendency of mMaple2 and the undetect-able dimerization tendency of mMaple3.The excitation and emission spectra of mMaple2 and mMaple3

were similar to those of mMaple (Table 1 and Fig. S4).

DiscussionIn this work, we characterized four properties of PAFPs that areimportant for superresolution imaging based on single-moleculeswitching and localization. First, the photon budgets are notsubstantially different for different PAFPs within the same colorgroup, but the red PAFPs provide substantially more photons(600–900 photons per activation event) than the green PAFPs(200–300 photons). Even the red PAFPs’ photon budgets aresubstantially lower than those of the bright photoswitchable/photoactivatable dyes (several thousand to one million photons)(7, 32). Given that the localization precision scales approxi-mately linearly with the inverse square root of the photonnumbers, the PAFPs thus give substantially lower localizationprecision than photoswitchable dyes. Second, the on–off ratiosare also not substantially different for different PAFPs within thesame color group, but the red PAFPs tend to show much loweron–off ratio (10−5 to 10−6) than the green PAFPs (∼10−3).PSCFP2 is a noticeable exception with an on–off ratio similar tothe red PAFPs. The on–off ratios of the red PAFPs are sub-stantially lower than those of the popularly used photoswitchabledyes (10−3 to 10−4) (7), and hence PAFPs can provide sub-stantially higher localization density. Third, natural fluores-cent proteins tend to dimerize or tetramerize. Although mutantfluorescent proteins have been made to reduce dimerization,many of the so-called monomeric fluorescent proteins still havesome residual dimerization tendency and thus can cause undesiredaggregation and mislocalization of the target proteins to which

they are fused. Among the 12 PAFPs tested here, the majorityappeared to exhibit a substantial propensity toward aggrega-tion or bundling of target proteins. The exceptions are Dendra2,mEos3.2, PAtagRFP, PAGFP, and Dronpa, which did not showa detectable aggregation effect for the target proteins tested here.It is important to note that these results do not imply that suchaggregation will not happen when these PAFPs are fused to othertarget proteins with strong intrinsic clustering or polymerizationtendency. As another cautionary note, it is also possible that mo-nomeric fluorescent proteins that discourage self-interactions maydisrupt natural oligomerization of the target proteins. Thus, it isa good practice to verify the spatial organization derived fromPAFPs with alternative approaches, such as immunohistochemistryor by using SNAP/CLIP/HALO or short peptide tags to label

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Fig. 5. mMaple2 and mMaple3 exhibit both high signaling efficiency and lowdimerization tendency. (A–D) Photon budget (A), on–off switching rate ratio(B), signaling efficiency (C), and dimerization tendency (D) of mMaple2 andmMaple3 in comparison with mMaple. (Insets in D) Sample fluorescent imagesof ClpP-mMaple2– and ClpP-mMaple3–expressing E. coli. Error bars are SEMsand are too small to be visualized in A. (E) H-NS appears more spread out in E.coli cells when fused to mMaple3 in comparison with the mMaple andmMaple2 fusion proteins. (F) Tar-mMaple2 and Tar-mMaple3 appear moreevenly distributed along the cell envelope and less concentrated at the polarcaps than Tar-mMaple. (G) Vimentin-mMaple2 and Vimentin-mMaple3are less bundled than Vimentin-mMaple. (Scale bars: 1,000 nm.)

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proteins with dyes (33). The fourth property that we probed is thesignaling efficiency, which determines the number of detectablemolecules of the PAFP fusion protein for a given target protein.When the signaling efficiency is low, the number of localizations isnot sufficient to map out the fine organization of the target protein.Notably, among the eight red PAFPs tested, mMaple has the highestsignaling efficiency, which is about one order of magnitude higherthan the other PAFPs. The downside of mMaple is, however, itsrelatively high dimerization tendency.To overcome this problem, we developed two new PAFPs,

mMaple2 and mMaple3, which largely maintained the superbsignaling efficiency of mMaple but exhibited substantially re-duced dimerization tendency. In particular, mMaple3 exhibitedno detectable dimerization tendency. The photon numbers andon–off ratios of both are similar to those of mMaple, which areamong the best for PAFPs. Based on the above results, we rec-ommend researchers to label target proteins of interest withmMaple3 when performing single-molecule–based superresolutionimaging using fluorescent proteins. mMaple3 provides excellentperformance in all four key properties described here, includinghigh signaling efficiency, low dimerization tendency, high photonbudget, and low on–off ratio. In the cases that the localizationnumber provided by mMaple3 is suboptimal, one should alsoconsider labeling with mMaple2, which will give substantiallyhigher localization numbers, but it is important to check whetherfusion with mMaple2 has led to undesired aggregation effect. Thelarge number of localizations provided by mMaple2 and mMaple3will not only facilitate mapping out the fine spatial distribution ofthe target protein, but will also allow the localizations to be dividedinto more snapshots to facilitate time-lapsed imaging of the dy-namics of cellular structures. When localization number is less ofa concern, mEos3.2 and PAtagRFP are excellent choices for theirhigh photon budget and undetectable dimerization tendency.The four properties measured in this work are key proper-

ties of PAFPs to consider when imaging the spatial organizationof a target protein with single-molecule–based superresolutionimaging. However, they are not the only important properties to

consider when other experimental requirements need to be takeninto account. For example, spectral properties are essential formulticolor imaging. For two-color imaging, green PAFPs areparticularly useful (when paired with a red PAFP) even thoughthey generally give lower photon numbers and higher on–off ra-tios. Dark-to-fluorescent PAFPs are also more favorable thancolor-changing PAFPs as the former take a smaller spectral space.The number of switching cycles is another important parameter toconsider. For quantifying the stoichiometry of the target proteins,it is desirable to have PAFPs that can be switched on only once,although almost all PAFPs tend to blink more than once (31).However, for mapping of protein spatial organization, a largernumber of switching cycles per molecule would allow the targetstructure to be sampled more times, which is advantageous when itcomes to time-lapsed imaging for probing dynamics. For a differ-ent superresolution imaging method, RESOLFT, reversiblyswitching fluorescent proteins with a large number of switchingcycles (such as rsEGFP) is actually required (25).

MethodsPlasmids were constructed with PCR and isothermal assembly (34). E. colichromosomal insertions were created by lambda RED recombination (35). Alist of the plasmids and strains is provided in Table S4. E. coli cells weregrown to exponential phase in M9 minimal media before imaging. Mam-malian cell lines were transfected with purified plasmids using nucleofectionor lipofection, and incubated for 24–26 h before imaging. Phase-contrast,superresolution, or conventional fluorescence images were collected on anOlympus IX-71 inverted microscope with 405-, 488-, and 561-nm laser lines.Images were analyzed with custom-written software. See SI Text for details.

ACKNOWLEDGMENTS. We thank Michael Davidson, Stefan Hell, StefanJakobs, Alice Ting, Antoine van Oijen, Ethan Garner, Sara Jones, and ChongyiChen for providing plasmids and strains, and David Liu for helpful discussions onthe design of PAFPs. S.W. is supported by a Jane Coffin Childs Fellowship. J.R.M.is supported in part by a Helen Hay Whitney Fellowship. This work is supportedby National Institutes of Health Grants GM096450 and GM068518 (to X.Z.) andGM096450 (to X.S.X.). X.Z. is a Howard Hughes Medical Institute Investigator.

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