Probes Sted Fpalm Storm

7/30/2019 Probes Sted Fpalm Storm

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Fscc micscpy has bcm a sstia tt sty bigica mcs, pathways a tsi iig cs, tisss a aimas. Cmpa withth imagig tchiqs, sch as electron microcopy,th mai aatag f fscc micscpy is itscmpatibiity with iig cs, which aws yamica miimay iasi imagig xpimts. Th maiwakss f fscc micscpy, hw, has bits spatia sti, which was imit t ~200 m fmay yas.

At f th sti spctm, poitron-emiion tomography, magnetic reonance imaging aoptical coherence tomography pi a-tim a-t fm aima hma sbjcts, bt thy catisc tais sma tha ~1 mm, ~100 m a~10 m, spctiy(FIG. 1). At th ppsit , c-t micscpy pis a mca- spatiasti, bt cs mst b fix, which is iasi

a pts yamic imagig. Btw ths twsti xtms, fscc micscpy pisa ag f spatia a tmpa stis. Th mstwiy s fscc imagig mths, confocalmicrocopy a wide-field microcopy, ca s c-tai ca gas (f xamp, th cs,th pasmic ticm a th Ggi appaats)a ca tack ptis a th bimcs i ics. Th spatia sti imit, hw, ptsth sti f sig syaptic sics pais fitactig ptis. May fis f bigy wbfit fm imp cmbiatis f spatia atmpa sti. F xamp, a syaptic

sics a ~40 m i siz a sigaig ccs th miiscs timsca. Bactia a y 15 mi siz, a sbca fats a iffict t sby ctia f scc micscpy.

W fcs th ct mgc f w fa-fifscc imagig tchiqs, which thticayha imit t thi spatia sti. W scibth ct imitatis i tms f spatia a tmpasti, a iscss hw sm f ths might bcm thgh impmts i fsct pbtchgy. I ga, tw casss f pbs a sf sp-sti imagig: fsct ptis (FPs)a -gticay c pbs, sch as gaicsma-mc fphs a qatm ts. Wscib th si chaactistics, stgths awaksss f ach pb cass. W as sggst ftimpmts t pb sig a tagtig, whichmight hp t big s cs t mca-sti

imagig i i cs i a tim.

Overcoming the diffraction limit

I 1873, Abb bs that fcs ight aways sts ia b iffact spt, a th siz f th sptpacs a famta imit th miima istac atwhich w ca s tw m fats1(TIMELINE).This spt is cmmy pst by th point preadfunction (PSF). Th mathmatica xpssi f Abbsfiig is that th sti f a fscc mic-scp is imit t /2nsi i th fca pa (xy)a 2/nsi2 ag th ptica axis (z), wh is thwagth f th ight s a nsi is th mica

*Department of Chemistry,

Massachusetts Instituteof Technology,

77 Massachusetts Avenue,

Cambridge, Massachusetts

02139, USA.Center for Engineering in

Medicine, Massachusetts

General Hospital,

114 16th Street, Charlestown,

Massachusetts 02129, USA.

e-mails:

[email protected];

[email protected]

doi:10.1038/nrm2531

Publihed online

12 November 2008

Electron microscopyA focued electron beam i

ued to illuminate the ample.

Electron microcope ue

electrotatic and

electromagnetic lene to formthe image by focuing the

electron beam in a manner

that i imilar to how a light

microcope ue gla lene

to focu light.

Fluorescent probes for super-resolution imaging in living cellsMarta Fernndez-Surez* and Alice Y. Ting*

Abstract | In 1873, Ernst Abbe discovered that features closer than ~200 nm cannot be

resolved by lens-based light microscopy. In recent years, however, several new far-field

super-resolution imaging techniques have broken this diffraction limit, producing, for

example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial

resolution. Current research is focused on further improving spatial resolution in an effortto reach the goal of video-rate imaging of live cells with molecular (15 nm) resolution.

Here, we describe the contributions of fluorescent probes to far-field super-resolution

imaging, focusing on fluorescent proteins and organic small-molecule fluorophores.

We describe the features of existing super-resolution fluorophores and highlight areas of

importance for future research and development.

nATure revIeWS |molecular cell biology voluMe 9 | deCeMBer 2008 |929

REVIEWS
mailto:[email protected]:[email protected]:[email protected]:[email protected]


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|

PET

MRI or US

OCT

WF or TIRF

Confocal

4Pi or I5M

GSD

SSIM

STED

PALM or STORM

NSOM

EM

Seconds

Seconds

Seconds

Milliseconds

Milliseconds

Milliseconds

ND

ND

Seconds

Seconds

NA

NA

Fluorescencemicroscopy

Supe

rresolution

1 nm 1 m 10 m 100 m 1 mm 1 cm 10 cm10 nm 100 nm

Protein Nucleus Mammalian cell

VirusGolgi Bacterial cellSynaptic

vesicle Yeast cell Mouse brain MouseER

Mitochondria

Temporalresolution

Positron-emissiontomographyAn in vivo imaging technique

that detect the location of

poitron-emitting iotope by

the pair of-ray that are

emitted when the poitronencounter electron. The mot

common can i produced by

imaging the metabolic activity

of fluorodeoxyglucoe, a

radioactive analogue of

glucoe.

Magnetic resonanceimagingA medical imaging technique in

which the magnetic nuclei

(epecially proton) of a

ubject are aligned in a trong,

uniform magnetic field, aborb

energy from tuned radio

frequency pule and emit

radio frequency ignal a their

excitation decay.

Optical coherencetomographyAn in vivo imaging technique

that end out femtoecond

infrared pule and ue

optical interference to ene

reflection from tiue

inhomogeneitie.

Confocal microscopyA mode of optical microcopy

in which a focued laer beam

i canned laterally along thex

and y axe of a pecimen in arater pattern. Point-like

illumination and point-like

detection reult in a focal pot

that i narrower than that

obtained in wide-field

microcopy.

Wide-field microscopyThe mot popular mode of

light microcopy, in which the

entire pecimen i bathed in

light from a mercury or xenon

ource, and the image can be

viewed directly by eye or

projected onto a camera.

apt f th s. As mst ss ha a micaapt f


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Point spread function(PsF). A meaure of the

performance of an optical

ytem. The PsF define the

apparent hape of a point

target a it appear in the

output image. For a

fluorophore, PsF i a Gauian

function, whoe full-width at

half maximum (FWHM) define

the patial reolution of the

imaging ytem.

Multiphoton microscopyA form of laer-canning

microcopy that ue the

imultaneou aborption of

two or more photon of a long

wavelength to excite

fluorophore that are normally

excited by a ingle photon of

horter wavelength. Thi i anonlinear imaging technique

that enable deep penetration

into thick tiue and reduce

light damage.

Optical sectioningThe imaging of thin ection of

a ample without the need to

mechanically lice it. Thi i

achieved by eliminating the

excitation and/or detection of

fluorecence that originate in

the out-of-focu plane.

Effectively, the ditance

between the cloet and

furthet object in focu i

greatly reduced to yield a clean

optical ection.

Ground state depletionA mode of REsOLFT

microcopy (ee REsOLFT) that

exploit the aturation of

fluorophore tranition from the

ground tate to the dark triplet

tate. A laer beam with a light

intenity ditribution featuring

one or more zero witche

ome of the fluorophore to

their triplet tate T1

or another

metatable dark tate, while

recording thoe that are till

left or have returned to theground tate s

0.

Saturated structured-illumination microscopyA mode of REsOLFT

microcopy (ee REsOLFT) that

exploit the aturation of

fluorophore tranition from the

ground tate s0

to the excited

inglet tate s1. Thi differ

from sTED in that ultraharp

dark region of molecule are

created with teeply

urrounded region of

molecule in the bright tate.

imagig, th ky t cmig th iffacti imit ist spatiay a/ tmpay mat th tas itibtw tw mca stats f a fph (fxamp, a ak a a bight stat). Sm tchiqsachi sp sti by awig th PSF f asmb imag f may fphs. Ths tch-iqs ic stimat missi pti (STed)16,ground-tate depletion (GSd)17, a aturated tructured-illumination microcopy (SSIM)18,19 a its ct cmbi-ati with I5M (I5S) (REF. 20). oth sp-stiimagig tchiqs tct sig mcs a y th picip that a sig mitt ca b caizwith high accacy if sfficit mbs f phtsa cct21. Ths tchiqs ic phtacti-Ths tchiqs ic phtacti-

at caizati micscpy (PAlM)22, fsccphtactiati caizati micscpy (FPAlM)23a stchastic ptica cstcti micscpy(STorM)24(TIMELINE).

Cell biology imaged at super resolution

W fcs attti STed, PAlM, FPAlM a

STorM tchiqs bcas f ct pts that shwth abiity f ths tchiqs t achi sp s-ti i bigica samps (f a i-pth iw fth high- a sp-sti imagig tchiqss REF. 25). Sa th tchiqs2629 ha ctyb p bt it is t ay t assss thi pttiaf bigy.

Imaging an ensemble of molecules. STed was th fistfa-fi sp-sti imagig tchiq t bappi t c imagig30. T bak th iffacti imit,STed ss spatiay mat a satab tasitisbtw tw mca stats. Spcificay, th samp isimiat by tw as bams: a xcitati as psis immiaty fw by a -shift ps ca thSTed bam (FIG. 2Aa). excit fphs xps tth STed bam a amst istaty tasf backt thi g stats by mas f stimat missi.This ia (ay xptia) -xcitati f thfsct stat by th STed bam is th basis f bak-ig th iffacti imit i STed imagig. Athghbth as pss a iffacti-imit, th STed psis mifi t fat a z-itsity pit at th fcact a stg itsitis at th spt piphy (cat-ig, f xamp, a ght shap). If th tw pssa spimps, y mcs that a cs t thz f th STed bam a aw t fsc, ths

cfiig th missi twas th z. This ffctiyaws th PSF (f xamp, t 65 m i FIG. 2Aa), atimaty icass sti by th iffactiimit. T btai a cmpt sbiffacti imag, thcta z is sca acss th samp. usig thisschm, STed micscpy has achi 20 m s-ti i th fca pa31,32 a, cty, 45 m stii a th imsis33.

Sic its iti i 1994, STed has b appit sa c bigica pbms. STed ssyapttagmi-I i iiia syaptic sics (~40 mi siz), a shw that this pti fms isatcsts p sic fsi34. STed as a th

ig-ik stct f th pti bchpit at syapticacti zs i th Drosophila melanogasterms-ca jcti35, a th siz a sity fsytaxi-Icsts i PC12 cs36. Aitiay, STed has abth isaizati f th ca pti spicig cmp-t-35 (SC35)31, th ictiic actychi cpt37,th tasit cpt pttia cha M5 (TrPM5)38a th ftii-2-ic csts f th amyipcs pti39. rcty, STed was xt ttw-c imagig, abig ccaizati stisf tw mitchia ptis32. This sty qiasca-sti imagig bcas th timitchi is y ~200500 m.

Impssiy, Wstpha a cags pti-at imagig f syaptic sics with 62 m at-a sti (FIG. 2Ab) i i hippcampa s40.usig STed micscpy, th syaptic sics, abwith ATTo647n-cjgat ati-syapttagmi ati-bis, w bs t b highy stict isisyaptic bts. By ctast, sics tsi btsxhibit fast, ia mmt, which might pst

tasit thgh axs (FIG. 2Ab). Tim-aps imagig fi mammaia cs with


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i sig-mc-bas sp-sti mths. I

ach imagig cyc, mst mcs mai ak, bta sma pctag f mcs a stchasticayswitch , imag a th caiz. rpatig thispcss f may cycs aws th cstcti f asp-sti imag.

Sch sp-sti tchiqs ha b s timag ptidnA cmpxs in vitro24 a t imagmca stcts, sch as yssms, mitchiaa ahsi cmpxs22, a mictbs a cath-i cat pits46 i fix cs. Sig-mc-bassp-sti micscpy has as b xt tmtic imagig4648. Tw-c STorM ath gaizati f mictb twks a cathi-cat pits i fix mammaia cs with ~20 msti46(FIG. 2Bb). usig PAlM (FIG. 2Ca), tw-cimagig f acti a ahsi cmpxs i f ix cswas pt48 (s FIG. 2Cb, which shws fibia-ikahsis f paxii ig paa t acti fibs). Atasca sti, itt ap is bs btwacti a paxii, athgh acti bs sy cs-t a sm bt t a paxii ahsis (FIG. 2Cb).This stcta atiship c t b iw sigctia micscpy.

Th-imsia (3d) sp-sti imagighas as b achi with STorM a FPAlM.usig ptica astigmatism, Hag a c-wkspfm STorM imagig with 2030 m s-

ti i th xypa a 50 m sti i th axiaimsi49. usig mtifca pa imagig, Jtta c-wks achi 3d FPAlM imagig with30 m sti i th xypa a 75 m stii th axia imsi50.

rcty, PAlM a FPAlM ha b xt ti-c imagig5153. F xamp, Hss a cagsimag th istibti f th mmba ptihamaggtii fm ifza is i i fibbastswith ~40 m accacy, a tmi a ffcti if-fsi cfficit f 0.1 m p sc51. Mayet al.imag iiia tso45 sica stmatitis is Gpatics a HIv-1 Gag mmba ptis i iig

cs, a btai high-sity mca tacks 52.

Shff a cags imag ahsi-cmpx yam-ics i i CHo cs with 60 m sti at a imagigsp f 2560 scs p fam53. Thy bs thmigati f ahsi cmpxs away fm th cg, which ha pisy b s by ctiamicscpy. Hw, sp sti aw thm tbs, f th fist tim, that th a f migatigahsi cmpxs ms fast tha th ft. As,w ahsi cmpxs fm i th c iti aththa at th c g.

Limitations of super-resolution imaging

Sp-sti micscpy has imp bth ataa axia stis, cty achiig ~5070 msti i a th imsis33,49,50. Hw, w afa fm mca sti (15 m), i which ii-

ia mcs i a macmca assmby ca bs. What facts tmi th spatia stiimit f ach f ths mths?

Ensemble super-resolution imaging. I STed, th s-ti ps th xtt f th satati f missipti, bcas this fis th g t which thPSF ca b aw (FIG. 2Aa). This atiship is giby qati 1:

x= (1)

2nsin 1 + I

max 1/2

Isat

Spatial resolution =

I this qati, is th wagth, nsi is thmica apt f th micscp, Imax is th appiitsity f th STed ps a Isat is th STed itsitythat gis 50% pti f th missi31. It thffws that t maximiz th satati f missipti (Imax/Isat) a imp STed sti, s t ith icas th itsity f th STed ps(icas Imax) cas th itsity t s apatica fph t th ak stat (cas Isat). Fxamp, a typica Imax itsity s i ay STed st-is f ~250 MW p cm2 pc a ata sti f

Timeline | Development of super-resolution imaging techniques and applications to cell biology

1873 1928 1957 1980 1990 1992 1994 1995 1998 1999

The first concept to break the

diffraction limit in the near-field

(NSOM) is proposed

Abbe discovers that the spatialresolution of light microscopy

is limited by diffraction to

approximately 200 nm

The concept of confocal

microscopy is described

Multiphoton microscopy images

biological samples, providing optical

sectioning and deep tissue penetration

The concept of breaking the

diffraction limit in the

far-field (STED) is proposed

4Pi allows imaging of fixed cells

with 100 nm axial resolution in a

confocal set-up

Confocal microscopy allows biological

imaging with 200 nm lateral and 450 nm

axial resolution with optical sectioning

4Pi microscopy improves the axial

resolution to 150 nm

NSOM is applied to imaging of fixed mammalian tissue

High axial resolution wide-field

imaging (I5M) is conceived

I5M allows imaging of fixed cells

with 100 nm axial resolution in

a wide-field set-up

FPALM, fluorescence photoactivation localization microscopy; NSOM, near-field scanning optical microscopy; PALM, photoactivated localization microscopy;PALMIRA, PALM with independently running acquisition; STED, stimulated emission depletion; STORM, stochastic optical reconstruction microscopy.

R E V I E W S

932 | deCeMBer 2008 | voluMe 9 www../ws/


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Reversible saturableoptically linear fluorescencetransitions(REsOLFT). A mode of light

microcopy that exploit the

aturation of a reverible ingle

photon tranition from a dark

tate to a bright tate, or vice

vera. A light intenity

ditribution featuring zero

create arbitrarily harp

region of molecule in the

dark or the bright tate; the

bright region allow the

aembly of a ubdiffraction

image. The patial reolution

i no longer limited by the

wavelength of the light in ue,

but rather i determined by the

aturation that can be realized.

PhotoswitcherA molecule that can reveribly

witch between two molecular

tate on irradiation with light

of a pecific wavelength and

intenity. Currently known

fluorecent photowitcher are

photoactivatable moleculethat witch between a dark and

a fluorecent tate upon

illumination.

Total internal reflectionfluorescenceA microcopy technique that i

deigned to probe the urface

of fluorecentlylabelled living

cell with an evanecent wave.

Thi wave i generated by a

light beam that trike between

two media of differing

refractive indice at an angle

beyond the critical angle.

5070 m f th fsct sma mc ATTo532

(REF. 31). Icasig Imax ab this a was t pssibbcas th pb phtbach t qicky. lat, ths f STed pss f g ati (t c g-stat mtipht abspti)54 a f w fqcy(t aw th tipt stat t ax a ai tipt-statxcitati)31 st i a mak cas i f-ph phtbachig. Ths impmts ab thicas f Imax t ~2,200 MW p cm2 a st i1520 m sti f th sam y31.

Bcas th wi aways b a pp imit t Imaxthat is imps by pb phtbachig a sampamag, a atati appach is t cas Isat (aspaat chaactistic f ach fph). Isat fisth itsity at which th at f stimat pti isfast tha th cmptig itstat tasitis, sch asfscc missi xcitati t high (sigt tipt) xcit stats, a Isat is isy pptia tbth th fscc iftim f th fph a itscss-scti f missi pti. Ths, g STedys a chaactiz by high qatm yis, missispcta that match th STed wagth, hacphtstabiity, g fscc iftims (>0.8 s)55 aa w css-scti f mtipht abspti a fabspti by th xcit stats.

Ath appach t cas Isat is t chag that f th bight a ak stats f th pb. Wscib ab a stimat pti that bigs th

mc fm th xcit stat S1 (bight) t th gstat S

0(ak), bt STed ca as wk if, f xamp,

th tw stats a tw ifft mca stats f aphtswitchab pb. This m ga appach tsp-sti imagig is tm reverible aturableoptically linear fluorecence tranition (reSolFT)56, whichappis t a smb tchiqs bas a stimattasiti btw ay tw mca stats (f xam-p, STed, SSIM a GSd). Bcas th sptasitstat tasiti is amst -xistt wh sigphotowitcher , Isat is mch sma a thf th s-ti ca b imp, with w as itsitis.This was fist shw by Hfma et al.56, wh btai

50100 m sti i th fca pa sig th pht-switchab pti FP595 (isat fm Anemoniasulcata; BOX 1) with a STed pw f y 100600 Wp cm2, which is six s f magit w tha thats f ATTo532 (REF. 31), a simia t th itsitiss, f xamp, i FPAlM imagig51.

It is as aatags t maximiz th sp f sp-sti imagig, t aw th sty f yamic p-csss i iig cs. Iitia acqisiti tims f PAlM,FPAlM, STed a STorM sp-sti tchiqsw f th f mits t hs, which stictthm t th imagig f fix samps. rcty, STed wasappi t imagig f syaptic sic mmts i iigcs at i at, 28 fams p sc40. Th imagigsp i STed is stict by th miimm mb fphts that ca b cct p pix a p it tim.Th high imagig sp i STed was btai at th cstf icasig as itsity (t 400 MW p cm2) acig th mb f phts cct p imagigcyc, which cas a cti i spatia sti (t62 m) as w as fi f iw (t 2.5 m 1.8 m). Th

ga is t achi this sam i-at imagig sp whimaximizig spatia sti a sig as itsitisthat a m apppiat f iig cs.

Single-molecule-based super-resolution imaging. IPAlM, FPAlM a STorM, spatia sti is t-mi by th mb f phts that ca b cct fmach fph a by th backg fscc,as gi by qati 2:

x= + (2)k1

N1/2

k2b

N

I this qati, xis th caizati pcisi; k1

a k2

a tmi by th xcitati wagth, thmica apt f th bjcti a pix siz; Nisth mb f cct phts (a facti f th ttamitt phts); a b is th backg is ppix21. Ths, t maximiz sti, th aim is t mii-miz backg is a maximiz pht tpt fth fph. F xamp, i th absc f back-g, if 10,000 phts ca b cct fm a sigfph mc bf it bachs is t ff,its caizati ca b tmi t ~2 m pcisi, a400 phts ca pi 1020 m caizati accacy57.Backg ca ais fm samp atfscc, asw as fm sia fscc f sig pbmcs i th ak stat. F sig-mc-bas

sp-sti imagig, it is th siab f f-phs t ha a high ctast ati, which is fi asth missi itsity ati btw th bight ss akstats. This is bcas at w ctast atis, th cctifscc fm ak mcs ca bsc th sigafm th sma mb f bight mcs ig achimagig cyc. o way t c backg is by siga total internal reflection fluorecence (TIrF) micscp,bt this sticts imagig t th c mmba. T maxi-miz spatia sti, it is as imptat t maximizth siga fm fphs i th bight stat. Ths,bight fphs with high xticti cfficitsa high qatm yis a siab.

2000 2003 2006 2007 2008

STED images

membrane

structures with

approximately

100 nm all-directional

resolution in live

yeast cells

STED-4Pi enables imaging of fixed

intracellular structures with 50 nm

axial resolution

Fixed-cell imaging with 20 nm resolution is extended tomultiple colours using PALM, STORM and PALMIRA

Live-cell imaging of membrane proteins is achieved at 40 nm

lateral resolution using FPALM

Three-dimensional imaging of fixed cells with 20 nm

lateral and 50 nm axial resolution using STORM

Imaging of intracellular adhesion-complex dynamics

in live cells with 60 nm lateral resolution using PALM

Video-rate imaging of synaptic vesicles in live

neurons with 60 nm resolution by STED imaging

Biomolecular complexes

and intracellular structuresare imaged with 20 nm

lateral resolution (using

STED, PALM and STORM)

R E V I E W S

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635 nm 780 nm 65 nm

277 nm

Aa Ab

Ba Bb

Ca Cb561 nm405 nm

561 nm405 nm

561 nm405 nm

488 nm405 nm

488 nm405 nm

488 nm405 nm

488 nm

Inactive Eos

Inactive Dronpa

Activated Eos

Activated Dronpa

Bleached Eos

Bleached Dronpa

1 2 3 4

5 6 7 8

100 nm

500 nm

200 nm2 m

Actin

Paxillin

Microtubules

Clathrin-coated pits

PSF of excitationbeam

Effective PSF ofSTED microscope

PSF of STEDpulse

Hot spots Tracks

74nm

11,000 11,000 11,000250 nm

Switch on fewmolecules(red dots)

Switch off allmolecules(gray dots)

Image, localizeand switch off(white crosses)

This f w backg a high pht t-pt highights sm f th mai iffcs btwsig-mc a smb a-t schms. A maiaatag f th sig-mc-bas sp-sti

statgy is that th mak mcs a t fc tg sa phtswitchig cycs this is thcas f smb-bas sp-sti imagig, iwhich phtbachig is a maj pbm. Hw, th

Figure 2 | c fs sp-s qs. a | Point spread function (PSF) of stimulated

emission depletion (STED) microscopy. The focal spot of excitation light (bright red) is overlapped with a doughnut-shaped

red-shifted light (dark red), which quenches excited molecules in the excitation spot periphery. This confines emission to a

central spot. Scanning this central spot (called the zero) across the sample results in a subdiffraction image. a | Synapticvesicle movement imaged with STED. Synaptic vesicles were immunolabelled in live neurons, and the movement of

each vesicle was individually recorded; the sum of 1,000 individual movie frames (11,000) depicts the movement of

various synaptic vesicles. b | Stochastic optical reconstruction microscopy (STORM). The fluorescence image is

constructed from highly precise localization of single molecules. In each imaging cycle, all fluorescent molecules in the

field of view are switched off by, for example, a strong red laser. Only a small percentage of them are then switched on

(green light) such that their images do not overlap, and their emission is recorded (red light) and used to localize their

positions (white crosses) with nanometre accuracy. b | Multicolour and three-dimensional (3D) STORM imaging.

Conventional (left panel) and STORM (right panel) images of immunostained microtubules (green) and clathrin-coated pits

(red) in the same region of a BSC-1 cell. A 3D STORM image of a clathrin-coated pit is inset. Anxycross-section and an

xz cross-section of the pit are shown in perspective.c | Photoactivated localization microscopy (PALM). PALM follows the

same principle as STORM. To perform two-colour PALM imaging, the orange emitters (Eos fluorescent protein) are

sequentially activated (405 nm light), imaged (561 nm light), localized and bleached until a subdiffraction image can be

constructed (steps 13). After bleaching the remaining Eos molecules (step 4), the many active green emitters (Dronpa

fluorescent protein) are first deactivated with a strong 488-nm light (step 5). Then, the green emitters are activated, imaged

and bleached (steps 68). c | Two-colour PALM images show the nanostructural organization of cytoskeletal actin (green)and the adhesion protein paxillin (red) in an HFF-1 cell. Actin bundles densely cluster around some (arrowheads) but not all

(full arrows) paxillin adhesions. Images in part a modified, with permission, from REF. 32 (2007) The Biophysical Society.

Images in part amodified, with permission, from REF. 40 (2008) American Association for the Advancement of Science.

Part bmodified, with permission, from Nature MethodsREF. 24 (2006) Macmillan Publishers Ltd. All rights reserved.Images in part bmodified, with permission, from REF. 46 (2007) American Association for the Advancement of Science.Inset image in part b modified, with permission, from REF. 49 (2008) American Association for the Advancement ofScience. Parts c,c modified, with permission, from REF. 48 (2007) National Academy of Sciences.

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|

405 nm405 nm orthermal 488 nm

488 nm

503 nm 518 nm

Dark Bright BleachedBleachedGreen Orange

Reversible photoactivation of DronpaIrreversible photoshifting of Eosa b

569 nm

506 nm 390 nm516 nm 569 nm 581 nm

fsct stat mst pc gh phts taw its pcis caizati (which is ptmii STed imagig). I aiti, th sig-mcappach qis stict ct th maximm -sity f phtactiat mcs, a ps thiiab caizati agaist a iffs backg.

Spatia sti sh b imp witht saci-ficig tmpa sti. li-c PAlM was ctys t sty ahsi-cmpx yamics53, bt thiswas ma pssib by th sw itisic mti f ah-si cmpxs. At a imagig at f 2560 scsp fam, may th bigica mmts w

appa t b b. T imp th tmpa s-ti f PAlM, FPAlM a STorM, it is cssay tmaximiz th mb f phts that ca b cctp it aa p it tim. Mayet al.52 pt thathigh-sity sig-patic tackig with PAlM wasbst achi sig esFP, which has th agst c-tast ati a highst pht tpt f a th kwphtshiftab FPs22,48.

Ath csiati is that wh sig is-ib fphs, tmpa sti is as imit byth phtbachig at. I ths cass, ss phtstabpbs a si, athgh a baac mst b mt

Box 1 | Fluorescent proteins used for super-resolution imaging

Fluorescent proteins (FPs) that are used for super-resolution imaging can

be divided into three classes: irreversible photoactivatable FPs (PA-FPs),

whose fluorescence can be turned on with light of a specific wavelength;

photoshiftable FPs (PS-FPs), whose fluorescence excitation and emission

spectra shift following illumination; and reversible PA-FPs (also known as

photoswitching FPs), whose emission can be reversibly switched on and

off with light. For example, exposure to ultraviolet (UV) or blue lightcauses an irreversible spectral shift in the PS-FP Eos110from a green state

to an orange state (see figure, part ). As another example, the reversiblePA-FP Dronpa111fluoresces green in its bright state (see figure, part ).Prolonged or intense irradiation with green light leads to a

non-fluorescent form with absorption maximum at 390 nm, which can

then be reversibly photoactivated back to the green-emitting form

by irradiation with 405 nm light. Dronpa can undergo 100 cycles of

activationquenching with only a 25% loss of its original fluorescence59.

The photophysical properties of PA-FPs and PS-FPs used for

super-resolution imaging vary widely (see table). Activating light refers to

the irradiation used for the photoactivation or photoshifting event;

quenching light reverts the FP to its dark state (this is only applicable to

reversible proteins); pre/post colours refer to the colours before and after

photoshifting (or photoactivation). The following photophysicalproperties all correspond to the fluorescent form of the FPs after

activation or shifting: ex

, excitation wavelength; em

, emission

wavelength;abs

, extinction coefficient;fl, fluorescence quantum yield;

contrast, fold increase in fluorescence at em

after photactivation or

photoshifting;N, number of detected photons per single molecule of FP

in each activation or shifting imaging cycle; NA, not applicable; ND, not

determined.

Fsp

a

Q

P/pss

x

()

() s

(m11)

f

(%) cs N os

rfs

Irreversible photoactivatable fluorescent proteins

PA-GFP UV-violet NA Dark/green 504 517 17,400 79 200 ND Monomer 51,61,62

PA-RFP1-1 UV-violet NA Dark/red 578 605 10,000 8 ND ND Monomer 60,61,64

Photoshiftable fluorescent proteins

PS-CFP2 UV-violet NA Cyan/green 490 511 47,000 23 >2,000 260 Monomer 48,61,69,112

Kaede UV-violet NA Green/orange 572 582 60,400 33 2,000 ~400 Tetramer 22,61,65

KiKGR UV-violet NA Green/red 583 593 32,600 65 >2,000 ND Tetramer 61,66

MonomericEos

UV-violet NA Green/orange 569 581 37,000 62 ND ~490 Monomer 22,48,52,53,61,68

Dendra-2 Blue NA Green/orange 553 573 35,000 55 4,500 ND Monomer 68,112

Reversible photoactivatable fluorescent proteins

FP595 Green 450 nm Dark/red 590 600 59,000 7 30 ND Tetramer 56,70

Dronpa UV-violet 488 nm Dark/green 503 518 95,000 85 ND 120 Monomer 48,59,61

Padron Blue 405 nm Dark/green 503 522 43,000 64 ND ND Monomer 71

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Extinction coefficientThe (molar) extinction

coefficient (ab

)of a pecie

i defined by the equation

A = bc, whereA i the

aborbance of the olution,

b i the path length and c i theconcentration of the pecie.

The fluorecence brightne of

a pecie i proportional to the

product of it molar extinction

coefficient and fluorecence

quantum yield.

Fluorescence quantum yieldThe ratio of photon emitted to

photon aborbed. The

fluorecence brightne of a

pecie i proportional to the

product of it molar extinction

coefficient and fluorecence

quantum yield.

btw fast phtbachig a aqat phtst achi high caizati accacy. I th ws,isib pbs sh b y bight bt t cs-saiy phtstab. Th atati is t s sibfphs sch as cyai (Cy) ys58 th PA-FPdpa59, which ca b t ff a ths t hat b phtbach.

FPs for super-resolution imaging

Athgh simp FPs, sch as g FP (GFP) a ywFP (YFP), ha b s i STed imagig 41,55, mstsp-sti imagig tchiqs xpit thitisic abiity f ctai FPs t chag thi spc-ta pptis iaiati with ight f a spcificwagth. Th a tw mai casss f FPs s isp-sti imagig: ths that ct fm aak stat t a bight fsct stat (PA-FPs), aths that chag fscc wagth ia-iati (phtshiftab FPs (PS-FPs)) (iw iREFs 60,61) (BOX 1). A kw PS-FPs isiby shiftthi wagth bt PA-FPs ca phtactiat ith

siby isiby.

Desired characteristics. FPs f sp-sti imagigsh b as bight as pssib (that is, ha ag extinc-tion coefficient (

abs) a ag fluorecence quantum yield

(f)), t maximiz th mb f tctab phts

p mc (n) a thy sh ha high ctastatis. Aitiay, th sptas itcsiats it a t f th actiat (fsct) statmst b w cmpa with th ight-ct actia-ti at. Th ats f phtbachig (f isibPS-FPs) actiati (f sib switchs) shb baac with th actiati at t s that, ya sma mb f mcs a i th fsct statat ay gi tim, s that aag thy a spaatby m tha th iffacti imit, a as t sthat ach actiat mc mais i th fsctstat f g gh t gi sfficit phts faccat caizati. Fiay, f ca stis, th FPsh b mmic t miimiz ptbati f thtagt pti. As s bw, y cty aaiabmatab FP has at ast awback.

Irreversible PA-FPs. Tw isib PA-FPs hab gi: PA-GFP a mmic PA-rFP1-1.PA-GFP was th fist t b gi, p bymtagsis f th igia GFP62. Athgh PA-GFP

has b s i FPAlM imagig t tmi thiffsi cfficit f hamaggtii i i fib-basts51, its g f scc a w ctast atists i high backg, which imits spatia s-ti t ~40 m a cssitats a miimm acqisititim f ~150 ms p fam (abt sf swtha th acqisiti tim qi f sFastlim(askw as dpa v157G), a aiat f th sibPA-FP dpa63). Th w ctast ati a xtmyw qatm yi f th y isib PA-FP,mmic PA-rFP1-1 (REF. 64) (BOX 1), which isi fm dsr, ha ths fa pt its si sp-sti imagig.

Irreversible PS-FPs. May f th atay cciga gi PS-FPs xhibit a shift fm g t missi (BOX 1). Amg ths, th ata PS-FP ka65a th gi KikGr66 a bigat ttams, aa ths t sitab f imagig f ca ptis.esFP is th mst cmmy s PS-FP f sp-sti imagig, as it has th highst ctast abightss a has b gi it a mmicfm that is sitab f pti fsi (es; BOX 1)67.esFP was s i f th fist mstatis fPAlM imagig22, a it was at s i cmbia-ti with PS-CFP2 a dpa i tw-c PAlMimagig xpimts48. It was bcas f th ptimaphtphysica pptis f esFP that May a c-wks c pfm sig-patic tackig f mm-ba ptis i i CoS7 cs at a imagig spf 20 fams p sc52. esFP i its igia imicfm was as s i th atst mstati f PAlMi i cs53. Th mai isaatag f mmic es,hw, is that chmph fmati ccs y attmpats bw 30C, which imits its s i mam-

maia cs67. I this ga, da-2, with simiactast a bightss, c pttiay tpfmmmic es, bcas it mats ppy at 37Ca ca b actiat by b ight (which cass ssamag t tiss tha th tait (uv) ight thatis qi f mmic es)68. Hw, thbightst PS-FP is sti mch imm tha sm sma-mc gaic fphs. F xamp, es p-

is ~490 cct phts p mc48, whasth switchab f ph pai Cy3Cy5 pis~6,000 cct phts p mc p switchigcyc a asts ~200 switchig cycs46,58.

Th y g-mittig PS-FP, PS-CFP2 (REF. 69),is pf i mtic stis bcas it has thhighst ctast ati a yis th agst mb fphts f a f th g matab FPs48(BOX 1).

Reversible PA-FPs. rsib PA-FPs (as kw asphtswitchs) a aatags i sp-stiimagig bcas th sam fph ca b imagmtip tims. rsib phtswitchig is maa-ty i reSolFT imagig, i which ach mc isswitch a ff may tims i t cstcta sbiffacti imag.

Th fist sib PA-FP pt was FP595(REF. 70). Athgh FP595 has w ctast a isttamic, it was sccssfy s by Hfma et al.

t achi 50100 m fca pa sti sigreSolFT imagig56. Th bst-kw sib PA-FPis th atay ccig dpa59 a its may gi- aiats. uftaty, athgh dpa xhib-its a ag xticti cfficit a qatm yi, itsfscc is xcit with 488 m ight, which asiactiats th pti, thf stig i a wmb f cct phts p imagig cyc. Highspatia sti ca b btai with PS-CFP2 thawith dpa48.

T cm this imitati, As a c-wk-s cty gi a dpa aiat with psiti-switchig chaactistics71. I ctast t dpa, this

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w aiat, ca Pa, is actiat a imagby b ight, whas uv ight switchs th ptiff. It mais t b s hw th pti bhas fsp-sti imagig. As a aitia xamp fhw pb pmt cmbi with istmta-ti aacs ca a t impmts i imagig,eg a c-wks ha cty shw that th sf a fast phtswitchig aiat f th FP dpa,sFastlim72, i cmbiati with asychs c-ig, accats imagig acqisiti a imiats th f TIrF i PAlM. Th its ight s i thissttig is mst f th fphs it a ak stat.Iiia fphs th stchasticay a spta-sy t t th bight stat, bify mit a bst fphts, th t t th ak stat. I this schm,th acqisiti tim matchs th ma ati f amissi bst, pcig api acqisiti a wbackg fscc. This pc has btm PAlMIrA (PAlM with ipty igacqisiti)47,63,73.

Fiay, th ct giig f th fist m-

mic sib PA-FP, sChy, has p wpssibiitis f mtic tim-aps imagig. li-c PAlMIrA imagig f th pasmic ticmab with sChy pi imags with ~75 mata sti74. As scib f Pa, th athsha as gi a psiti-switchig si fmmic chy, tm sChyr, which mightfth hac its s, athgh its appicabiity t cimagig has t b shw74.

I smmay, FPs ca b tagt with abst sp-cificity bt thy a gay bigg, imm a ssphtstab tha sma-mc fphs. Thisw bightss say maks it cssay t s a TIrFmicscp t miimiz backg fscc. ewith th bightst PS-FP, esFP, th maximm fam atthat ca b achi (~25 scs p fam) is sti tsw t imag mst bigica pcsss if a sti f~60 m is si. Bight FPs a t icastmpa sti witht sig spatia sti.uftaty, athgh th mchaisms f pht-switchig f sm FPs ha b cty scib61, thstict qimt f chmph fmati isith -ba f FPs maks th giig f bightFPs a iffict task a highights th f th is-cy f w FPs fm th spcis. Aitiay, wmmic ptis f ifft cs a taw ti mtic imagig at sp sti.

Non-genetically encoded probes

Th mai casss f -gticay c pbsha b s i sp-sti imagig: igaicqatm ts (BOX 2), sib phtactiatab f-phs (as kw as phtswitchs) a isibphtactiatab fphs (as kw as pht-cag f phs) (BOX 3). STed imagig iitiays ga sma-mc ys, i which th pbwas imag i its xcit stat a th sigmcs w qch by th STed ps that stthm t g stat. I this typ f imagig schm, thitsity f th STed ps s t b xtmy high

t cmpt with th sptas fscc cay fth pb mcs with bth high qatm yia g fscc iftim (sw fscc cay),sch as th ATTo dY ys, a ia 3032,3438,40,42(BOX 3). lat, STed imagig it th mga reSolFT imagig, which ss phtswitchs,sch as FP595 (REF. 56), a fy fgis75.

Reversible PA probes. Th sma-mc aagst sib PA-FPs (sch as dpa a sChy74)a phtchmic pbs, icig hamis aiayths, as w as phtswitchab cyais.Th switchig mchaism f a phtchmic ham-i B (PC-rHB) is pict i BOX 3. Iaiati f thcs ism with uv ight with ight (f pstw-pht abspti) sts i tasit fmatif a c a bighty fsct p ism 73.Th acti is thmay t by hat withimiiscs t mits, pig th st.Iaiati f th fsct ism with g ightxcits fscc missi bt s t gat

th -fsct stat. This ppty maks thphtchmic hami spi t dpa (simiat Pa a sChyr), bcas th fsccsiga ca b a witht th si asig siffct, which sts i high pht tpt p switch-ig t. Phtchmic hamis as pi axamp f hw pb pmt ca a t imp-mts i th imagig pcss. Fig a c-wks76pt th sig f a w pb bas ham-i 590 (PC-rH590), whs xta igiity impscss-scti cmpa t th igia PC-rHB73 ftw-pht abspti. efficit tw-pht actiatic as b achi with this pb, a fsctimags with 15 m sti i th fca pa wbtai76.

oth phtchmic mcs, sch as pht-chmic iayths77, c pttiay b s isp-sti imagig, bt thy ha imit watsbiity, which sticts thi bigica tiity.

Phtswitchab cyai ys ha b s ibth PAlMIrA47 a STorM24,46,49 imagig. Athghit has as b s a47, Cy5 is bst s i cm-biati with a scay chmph that faciitatsth switchig46,58. F xamp, wh Cy5 is pai withCy3, th sam as that xcits Cy5 is as st switch th y t a stab ak stat. Sbsqtxps t g as ight cts Cy5 back t

th fsct stat, a this cy at ps th cs pximity f th scay y Cy3 (cath actiat)58. Cy5 switchig ca as b faciitatby th actiat fphs, sch as Axa F405 a Cy2 (REF. 46). Fthm, Cy3 was f tfaciitat switchig f th cyais, sch as Cy5.5 aCy7 (REF. 46). This fiig gaty icass th pattf cs that a aaiab f STorM imagig ahas aw simtas isaizati f mictbsa cathi-cat pits i fix mammaia cs with2030 m ata sti46(FIG. 2Bb). This aaiabi-ity f sa cs ctasts with th ack thff matab FPs. uftaty, th pmt

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Irreversible PA probes. Cag cmps, sch ascag Q-hami79,80, ca as b xpit i sp-

sti imagig (BOX 3). dig cagig, iaiatiwith uv ight cass th as f a ptcti gp asts i a ag icas i th fscc itsityf th y. Phtcag pbs ca b s i PAlM,FPAlM STorM i th sam way as isibPA-FPs: thy ca b cag, caiz with high pci-si a th bach. Cagig ca b a way f gat-ig w PA pbs that a bas fphs withthwis g phtphysica pptis bt withtitisic phtswitchig abiity. Th pttia f cagcmps was fist shw by Btzig a c-wks,wh s PAlM t imag cag hamixta thatha b i gass c sips22. Cag fsci

|

Streptavidin for targeting

RESOLFT image Confocal image

Point spread function

y(+m)0.0 0.2 0.4 0.6

45 nmIfluo

ConfocalRESOLFT

0.6 m 0.6 m

QD core and shell

Passivating layer

a

c

b

f imp switchig pais is cty hi byth fact that th switchig mchaism f ths ys is

kw. Fiay, th cty p Cy3Cy5b cjgat faciitats abig78.

Wh cmpa with thi FP ctpats (FP595,dpa a Pa; BOX 1), phtswitchab ys haag ctast atis, a thy ha high xticticfficits, which sts i bight pbs with highmbs f cct phts p mc. of th pht-switchab pbs, hamis sta t bcas f thipttia f itaca abig i i cs, as thy ammba pmab (sphat cyai ys a t).Impmts a qi, hw, t c th kwaffiity f hamis f itaca gas that iscas by thi hyphbicity a psiti chag.

Box 2 | Quantum dots as probes for super-resolution imaging

Quantum dots (QDs) are a class of non-genetically

encoded probes that are commonly used in

single-molecule imaging owing to their enhanced

photostability and extreme brightness. QDs are inorganic

semiconductor nanocrystals, typically composed of a

cadmium selenide (CdSe) core and a zinc sulphide

(ZnS) shell and whose excitons (excited electron-holepairs) are confined in all three dimensions, which gives rise

to characteristic fluorescent properties. For biological

applications, QDs are coated with a passivating layer to

improve solubility, and are conjugated to targeting

biomolecules, such as antibodies or streptavidin (see

figure, part ). As fluorescent probes, QDs arecharacterized by broad absorption profiles, high extinction

coefficients and narrow and spectrally tunable emission

profiles. Small CdSe QD cores (2.3 nm diameter) emit

blue light, whereas larger crystals (5.5 nm diameter) emit

red light, producing size-dependent optical properties114

(see figure, part ).Irvine and co-workers recently reported the ability to

switch on and off a certain kind of QD, thus rendering this

type of QD suitable for super-resolution imaging115

. Theyshowed that the fluorescence of manganese (Mn)-doped

ZnSe QDs can be reversibly depleted with ~90% efficiency

using a continuous-wave modulation laser of ~2 MW

per cm2. The main novelty of this report is that modulation

is achieved directly by light and relies only on internal

electronic transitions, without the need for an external

photochromic activator or quencher. Thus, this type of QD

can be used in super-resolution imaging in the same way as

small-molecule organic photoswitchers (see the main

text). The figure in part shows a comparison between theimages obtained using RESOLFT (reversible saturable

optically linear fluorescence transitions) (top left panel) or

conventional confocal microscopy (top right panel). The

fluorescence intensity profile through a representative

RESOLFT point spread function (yellow dashed line) showsthat in vitro imaging of the QDs using RESOLFT resulted

in ~45 nm lateral resolution, a vast improvement over the

corresponding confocal image (lower panel). Ifluo

,

fluorescence intensity;y, direction or axis. Part modified,with permission, from Nature MethodsREF. 113 (2008)

Macmillan Publishers Ltd. All rights reserved. Image

(by F. Frankel) in part reproduced, with permission, fromREF. 114 (2007) Chemical Society. Part c, reproduced,with permission, from REF. 115 (2008) Wiley-VCH.

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Protein/peptide-directed labelling Enzyme-mediated protein labelling

Recognitionpeptide or protein Fluorescent probe

Enzyme

Targeting methods for non-genetically encoded probes.Athgh -gticay c pbs gayshw icas bightss a phtstabiity cm-pa with FPs, thy as ha isaatags. Th ackf gtic cig mas that ths pbs qia mas t tagt thm t th bimc f itstisi cs. Ths pbs ha b taitiay ta-gt sig atiby cjgati, athgh this has

may isaatags. Atibis a t mmbapmab, a hc a t sf f itacaabig f iig cs. Atiby staiig as saysts i w abig fficicy a th ag siz fatibis as ctaity (~1020 m) t th spatiaatiship btw th ab a its tagt. Why sabig fficicy matt? Th abtagt ati-ship was cty scib by Shff a c-wks by

r

sq

ez s Sz f

sq ()

c

Fps

s

c ps

rfs

Protein or peptide-directed labelling methods

TetraCys NA 610 Yes Fluorescein (FlAsH), resorufin(ReAsH) andCHoAsH

Membrane* andintracellular

82,83,118,119

HexaHis NA 612 No NTA-I and II, OG488, Cy7 andATTO647 or 565

Membrane 8486

PolyAsp NA 816 No Fluorescein, TMR and Cy5 Membrane 8789

Bungarotoxin-bindingpeptide

NA 13 No Rhodamine Membrane 90,91

FKBP12 (F36V) NA 98 No Fluorescein Membrane 92

DHFR DHFR 157 No Fluorescein, bodipy and

TexasRed

Intracellular 93,94

SNAP-tag andCLIP-Tag

AGT 182 Yes Fluorescein, TMR Cy andSNARF1

Membrane andintracellular

9597

Cutinase Cutinase 200 Yes AlexaFluor and QDs Membrane 98

HaloTag Dehalogenase 296 Yes Fluorescein, TMR, AlexaFluorand OG488


99

Enzyme-mediated labelling methods

SorTag Sortase A 6 Yes AlexaFluor and TMR Membrane 100,101

Q-tag Transglutaminase 7 Yes Fluorescein Membrane 102

A1/S6 AcpS or Sfp PPTases 11 Yes AlexaFluor, Cy and TexasRed Membrane 106

AP Biotin ligase 15 Yes Fluorescein, AlexaFluor andQD

Membrane 103105

LAP Lipoic acid ligase 1217 Yes AlexaFluor, Cy3, coumarin,fluorescein


107,108

*Cell-surface protein labelling using the tetraCys tag requires reducing agents. A covalent version of the polyAsp tag has recently been developed88.

Box 4 | Methods for site-specific targeting of small-molecule probes to cellular proteins

Small-molecule fluorophores can be advantageous over their fluorescent

protein counterparts owing to their enhanced brightness and

photostability. However, they are not genetically encodable, which

complicates their targeting. Several methods have been developed for

targeting of organic fluorophores to specific proteins in live cells (see

table). One approach (see figure, left panel) is to fuse the target protein to

a peptide or protein recognition sequence, which then recruits the smallmolecule. In general, protein recognition domains offer greater targeting

specificity but larger bulk than peptide recognition domains117,118. Another

approach (see figure, right panel) is enzyme-mediated protein labelling.

A recognition peptide is fused to the protein of interest and a natural or

engineered enzyme ligates the small-molecule probe to the recognition

peptide. This approach can give highly specific and rapid labelling, with

the benefit of a small directing peptide sequence.

AGT, O6-alkylguanine-DNA alkyltransferase; AP, biotin ligase acceptor

peptide; Cy, cyanine dye; DHFR, dihydrofolate reductase; FKBP12,

12 kDa FK506-binding protein; LAP, lipoic acid ligase acceptor peptide;NA, not applicable; NTA, nitrilotriacetic acid; OG, oregon green;

PPTases, phosphopantetheine transferases; QD, quantum dot; SNARF1,

seminaphthorhodafluor-1; TMR, tetramethylrhodamine.

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aagy with th Nyquit criterion; th istac btwtw ab mcs mst b sma tha haf f thsi spatia sti53. If w wat a spatia s-ti f 5 m, w t ha ab mcs at asty 2 m. Gi th bk a wak affiity f mayatibis, it is iffict t achi sch abig sity.Ath isaatag f may -gticay cpbs is thi iabiity t pmat thgh th cmmba, which sticts thi s t ith fixcs c-sfac ptis.

I t fy aiz th itisic imagig s-ti f sp-sti tchiqs, w t pstatgis t tagt sma mcs witht sigificatyicasig th siz f th tag a with high abig ffi-cicy. BOX 4 scibs sm f th ct appachsf sit-spcific pti abig i i cs. I a sbstf ths mths, th pti f itst is fs t appti pti sqc that cits th sma m-c. examps f mths that s a ppti tag icth ttaCys82,83,118, hxaHis8486, th pyAsp8789 a thbgatxi-biig ppti90,91 mthgis.

Sm th mthgis s ptis t cit thsma-mc pb, sch as th FKBP12 pti92a th zyms ihyfat ctas (dHFr)93,94,o6-akygai-dnA akytasfas (AGT)9597,ctias98 a hagas99. Th s f pti tags,ista f ppti tags, ca imp th spcificity fth biig wig t th ag itacti sfac aathat thy ca stabish with th cit pb, bt thcst is th icas siz f th tag, which ca ptbpti fcti.

T big th qimts f high abig sp-cificity a miima ptbati f th tagt pti,a sc st f mthgis ss a ppti cgi-ti sqc bt th ss a zym t catays thcat attachmt f th pb t th ppti (BOX 4).usig a zym t catays pb igati imps th

spcificity f th abig a as gis fast a ca-t attachmt f th pb. examps f this scappach ic th mths bas th zymsstas100,101, tasgtamias102, biti igas103105,phsphpatthi tasfass (Sfp a AcpS)106 aipic aci igas107,108.

Conclusions and future perspectives

Ths a xcitig tims i c bigy bcas i-cimagig with mca sti (15 m) is wcs t bcmig a aity. Th ct pmtf sp-sti imagig tchiqs has ab th

isaizati f ca fats with pisy imag-i tai. STed imagig has aw a-tim tack-ig f sig syaptic sics i ct hippcampas40 a has a th aatmy a yamicsf sytaxi-I csts i PC12 cs36. STed, STorM,PAlM a FPAlM aw ca stcts t bimag i 3d a i mtip cs32,33,46-50,109. . At scha imp sti, ccaizati f pti paishas b pt that ctaicts pis pts

bas w-sti imagig48, a iffsi pp-tis f mmba ptis ha b mapp t highsti51,52.

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AcknowledgementsThe authors thank X. Zhuang, E. Betzig, R.Y. Tsien, T.Uttamapinant and P. Zou for useful feedback on themanuscript.

DATABASESUniProtKB:http://www.uniprot.org

bruchpilot | FP595 | SC35 |synaptotagmin-I | syntaxin I |

TRPM5

FURTHER INFORMATIONAlice Y. Tings hompage:http://web.mit.edu/chemistry/

www/faculty/ting.html

all linkS are active in the online PdF

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