Single-Molecule Localization Super-Resolution Microscopy: Deeper and Faster

Single-Molecule Localization Super-ResolutionMicroscopy: Deeper and Faster

Sébastien Herbert,1,2 Helena Soares,3 Christophe Zimmer,1,* and Ricardo Henriques1,*

1Institut Pasteur, Groupe Imagerie et Modélisation, CNRS URA 2582, 25 rue du Docteur Roux, 75015 Paris, France2Frontiers in Life Sciences PhD Program, University Paris Diderot, 5 rue Thomas-Mann, 75013 Paris, France3Institut Pasteur, Lymphocyte Cell Biology Unit, CNRS URA 1961, 28 rue du Docteur Roux, 75015 Paris, France

Abstract: For over a decade fluorescence microscopy has demonstrated the capacity to achieve single-moleculelocalization accuracies of a few nanometers, well below the ;200 nm lateral and ;500 nm axial resolution limitof conventional microscopy. Yet, only the recent development of new fluorescence labeling modalities, theincrease in sensitivity of imaging hardware, and the creation of novel image analysis tools allow for theemergence of single-molecule-based super-resolution imaging techniques. Novel methods such as photoacti-vated localization microscopy and stochastic optical reconstruction microscopy can typically reach a tenfoldincrease in resolution compared to standard microscopy methods. Their implementation is relatively easy onlyrequiring minimal changes to a conventional wide-field or total internal reflection fluorescence microscope. Therecent translation of these two methods into commercial imaging systems has made them further accessible toresearchers in biology. However, these methods are still evolving rapidly toward imaging live samples with hightemporal resolution and depth. In this review, we recall the roots of single-molecule localization microscopy,summarize major recent developments, and offer perspective on potential applications.

Key words: single molecule, super-resolution, fluorescence, microscopy, PALM, STORM

INTRODUCTION

Fluorescence microscopy is one of the main methods forthe study of cell biology. Through fluorescent tagging,molecules such as DNA, RNA, and protein can be readilydifferentiated within their cellular environment and ob-served in a noninvasive manner. Notwithstanding, opticaldiffraction in standard microscopes restricts resolution to;200 nm laterally and ;500 nm axially ~Abbe, 1882!. Inthe last decades, major efforts have been made to improveboth microscopy instrumentation and image analysis inorder to push the boundaries of imaging resolution, depth,and speed ~Rino et al., 2009!. In recent years, these effortsgave rise to the field of super-resolution microscopy: a novelstream of approaches focused on achieving resolutions offew nanometers ~typically 1–100 nm axially in two or threedimensions!, while keeping most properties of classicalfluorescence microscopy ~Yildiz et al., 2003; Huang et al.,2009!. Single-molecule localization microscopy ~SMLM!, afamily of super-resolution methods, relies on the capacity todiscern and pinpoint individual molecules even withindensely labeled environments ~see Figs. 1, 2!, achievingmolecular localization accuracies at the nanometer level inexperimental settings ~Yildiz et al., 2003!. This family ofmethods edges toward the ,1 nm resolving power ofelectron microscopy ~EM! and atomic force microscopy

~AFM! ~Giessibl, 1995; Erni et al., 2009!, while keeping themolecular-specific labeling of fluorescence imaging.

Only recently did SMLM transition from a developmen-tal phase to true biological applications. Several novel stud-ies have demonstrated the capacity of SMLM to provide ananoscale view into ultrastructure not easily differentiatedwith classical optical microscopes, such as lysosomes ~Betziget al., 2006!, the Golgi apparatus ~Betzig et al., 2006!,microtubules ~Huang et al., 2008b!, clathrin-coated pits~Huang et al., 2008b!, viral proteins and structures ~Manleyet al., 2008; Lelek et al., 2012!, bacterial structures ~Biteenet al., 2008; Greenfield et al., 2009!, bacterial vacuole rup-ture ~Ehsani et al., 2012!, nuclear pores ~Löschberger et al.,2012!, and synaptic vesicles ~see Fig. 3!.

Here, we review the evolution of SMLM, from itsinception to its current state, as it progresses toward fast,live-cell, and deep imaging.

THE RESOLUTION LIMIT

In microscopy, photons follow the optical path from thelight source into the sample and are finally collected by adetector. Within this route they will interact with differentphysical media such as lenses, filters, the sample itself, andits embedding medium. Each of these factors will contrib-ute to light scattering and visual blurring of the observedsample. Additionally, the limited aperture of microscopeobjectives will induce for each visible fluorophore a spatialprofile featuring a central spot with a series of concentric

Received June 2, 2012; accepted August 9, 2012*Corresponding authors: E-mail: [email protected], [email protected]

Microsc. Microanal. 18, 1419–1429, 2012doi:10.1017/S1431927612013347 MicroscopyAND

Microanalysis© MICROSCOPY SOCIETY OF AMERICA 2012

REVIEW ARTICLE

rings, a silhouette known as the Airy diffraction pattern,generally referred to as the point spread function ~PSF, seeFig. 1!. These effects can be easily interpreted when observ-ing spatial distinct fluorophores. In the 19th century, E.Abbe described this effect analytically and defined the reso-lution limit as the minimal distance allowing for subdiffrac-tion points to be differentiated. This distance can becalculated through Abbe’s formulas: l/~2NA! perpendicularto the optical axis and ~2lh!/~2NA!2 along the optical axis,where l is the wavelength of the light, h is the refractionindex of the medium, and NA the numerical aperture of theobjective lens in the imaging system ~Abbe, 1882!. In biolog-ical systems, molecules of a few nanometers in size aretypically organized within molecular aggregates below Abbe’slimit ~roughly 200 nm!. Standard microscopes are thusincapable of accurately resolving most structures and inter-actions at the molecular scale. Electron microscopy andAFM are able to achieve resolutions under 1 nm ~Andoet al., 2008; de Jonge et al., 2009!, but they lack the mainadvantages of fluorescence optical microscopy such as live-cell imaging, molecular specific multicolor labeling, andrelatively simple experimental protocols. The ideal micros-copy technique would combine these features with thesubnanometer resolution of EM and AFM.

Figure 1. Point spread functions and the resolution limit: ~A!representative PSF profile for a wide-field or TIRF microscopeviewed in the xy-plane ~scale bar � 200 nm! and xz-plane ~scalebar � 600 nm!; ~B! two particles at resolvable and unresolvabledistances as seen in a microscope, red arrows demonstrate whenthe centers of the particles are resolvable, green line demarks theintensity profile shown in the plots, dotted red and blue linescorrespond to the profile of the upper and lower particles, blackline to the summed profile of both particles.

Figure 2. Acquisition and analysis of a biological structure through single-molecule localization methods. In this familyof methods, the microscope acquires a diffraction-limited sequence of images featuring subsets of temporarily activeand spatially distinct fluorophores, switched-on from a larger population of nonactive fluorophores. Generally, the firstframes show an unwanted base level of naturally active photoswitchable fluorophores, impeding the immediatediscrimination of single molecules ~Acquisition – t0!. The subsequent switch-off ~mainly by bleaching! of thesefluorophores leads to a reduced number of active fluorophores ~Acquisition – t1!. The constant illumination by anexcitation beam in combination with either a pulsed or continuous activation light ~not always required! allows a smallnumber of active fluorophores in each frame to be maintained ~Acquisition – t1, t2, . . .!. Analysis of these frames permitsaccurate fluorophore detection and localization ~Analysis – t1, t2, . . .!. The superposition of the calculated particlepositions will generate a super-resolution reconstruction ~Analysis – Reconstruction!. This example makes use of asynthetically generated structure for illustration purposes.

1420 Sébastien Herbert et al.

PUSHING THE BOUNDARIES OFFLUORESCENCE MICROSCOPY

Recent years have witnessed a boom in attempts to push thecurrent resolution boundaries of fluorescence microscopy,focused on three main areas: imaging hardware, cell label-ing, and image analysis. Many super-resolution methodsdifferent from SMLM have been developed. Below, we give abrief historical overview of their development before focus-ing in SMLM approaches.

In one of the earliest attempts to achieve super-resolution microscopy, Cremer and Cremer ~1978! pro-posed the use of interference to confine the excitation beamPSF into a single spot smaller than the diffraction limit.Once combined with point scanning, this method allowedfor super-resolution imaging.

Later in 1995, the development of the 4Pi microscopeallowed for 140 nm axial and 210 nm lateral resolutions tobe achieved. In this setup, two opposing objectives lensesare used to coherently illuminate and detect the same spot~Hänninen et al., 1995!.

The first applications of total internal reflection fluores-cence ~TIRF! appeared in the early 1980s. This methodgenerates an evanescent light wave that restricts sampleillumination to the first couple of hundred nanometersdirectly above the coverslip ~Axelrod, 1981!. TIRF is idealfor live cell imaging, providing images with a strong signal-to-noise ratio by constraining the fluorescence excitationdepth in the z-axis. It has proved to be a powerful tool inthe study of cell membrane dynamics, cytoskeleton, or

cell-substrate contact regions ~Hu et al., 2007; Mattheyseset al., 2010!. Initially TIRF microscopy required the use ofprisms, to induce an evanescence illumination field directlyover the coverslip, and an objective opposed to the prism, inan upright-type microscope setup. The increase in the max-imum NA ~1.4 to 1.65! of objectives has allowed for theevanescence field to be generated directly by the objectivelens and thus facilitates the imaging of biological cells.However, TIRF is restricted to a limited penetration depthclose to the surface of the samples ~depth , 200 nm!.

Structured illumination microscopy ~SIM! is a morerecent super-resolution approach. In this method, the illu-mination beam is shaped by a diffraction grating to create asinusoidal-type illumination pattern directly in the sample.A series of images is then acquired while varying theposition and rotation of this pattern. Computational recon-struction methods then yield a super-resolution image byexploring the additional frequency information extractedfrom the acquired images. This method virtually doublesthe NA of the system, hence doubling the lateral resolutionto around 100 nm ~Gustafsson, 2000!. SIM relies only onoptics and computational processing; the sample prepara-tion and labeling are exactly the same as in standardfluorescence microscopy ~Kner et al., 2009!.

Another super-resolution method, stimulated emissiondepletion ~STED!, uses two independent lasers to sharpenand confine the effective excitation pattern to a subdiffrac-tion area. To this end, a first focused laser excites thefluorophores of a diffraction limited area in the sample, anda second laser, engineered into a doughnut-shaped intensityprofile, forces the excited fluorophores at the periphery ofthe diffraction limited spot back into the ground state. Onlythe fluorophores at the center of the doughnut will be ableto emit light and be detected ~Hell & Wichmann, 1994; Klaret al., 2000!. STED has been demonstrated to achieve 30–40 nm in lateral and axial resolution within biologicalsamples ~Schmidt et al., 2008!.

The super-resolution optical fluctuation imaging ~SOFI!method is able to analytically improve the optical resolutionof a dataset by analyzing the temporal uncorrelated fluctua-tion in the emission of fluorophores ~Dertinger et al., 2009!.Although SOFI in biological samples is generally limited to100 nm lateral resolution, it has been demonstrated to workwith both conventional epifluorescence or TIRF microscopes.High speed imaging with standard fluorophores has beendemonstrated in live cell imaging ~Dedecker et al., 2012!.

SMLM approaches have been developed in parallel tothese approaches. In many cases, SMLM can be used as analternative or a complement to the mentioned super-resolution methods. For the remaining part of the review,we will focus on SMLM.

BYPASSING THE RESOLUTION LIMIT WITHSINGLE-PARTICLE LOCALIZATION

In fluorescence microscopy, an individual subdiffractionparticle can be localized with accuracy below the diffraction

Figure 3. 3D direct STORM ~dSTORM! image of fixed hippocam-pal neurons stained with antibodies against synaptic markers:~A! dSTORM image of an isolated axonal track ~gray: VGlut1, asynaptic vesicle reporter; green: bassoon, a presynaptic site re-porter!; ~B! super-resolution z-depth reconstruction of an individ-ual synaptic vesicle from blue inset of image A; ~C! particledetection profile from demarcated red line in images B, shows;20 nm resolution. Cell preparation and labeling as described inHenriques et al. ~2010!.

Single-Molecule Super-Resolution 1421

limit if its spatial profile does not overlap with that of otherimaged objects ~Thompson et al., 2002! ~see Fig. 1!. In thiscase, the center of the PSF—corresponding to the fluoro-phore location—can be computed with an accuracy that islimited mostly by the detected fluorophore signal relative tothe noise ~Gelles et al., 1988; Thompson et al., 2002; Oberet al., 2004! ~see Fig. 1!.

Several analytical methods have been developed forlocating the center of a particle with subdiffraction accuracy~Crocker & Grier, 1996; Thompson et al., 2002; Rogers et al.,2007; Chao et al., 2009a!. Although the calculation oftwo-dimensional ~2D! coordinates ~x, y! requires a singleimage with the visible particle ~Ober et al., 2004!, thedetermination of the z coordinate with a low localizationerror demands an optical alteration of the PSF shape toencode the position of the particle in the z-axis ~Kao &Verkman, 1994! or multiple-focal planes to be acquired~Juette et al., 2008; Chao et al., 2009b! ~detailed later in theHigh-Resolution Single-Molecule Localization in 3D sec-tion of this review!. Various different algorithms can beemployed for single-particle localization ~Cheezum et al.,2001!, some of the most accurate 2D and three-dimensional~3D! particle localization methods involve fitting the parti-cle intensity profile to a Gaussian function approximationof the PSF. High-precision fitting near the theoretical reso-lution limit can be achieved through nonlinear least-squaresoptimization ~Cheezum et al., 2001!, or even more accu-rately through a maximum-likelihood estimator ~Abrahamet al., 2009!. Notwithstanding, fitting does entail a consid-erable computational cost due to the multiple iterativecycles needed for the position of the particles to be re-trieved. Constrained center-of-mass ~Henriques et al., 2010!and radial symmetry ~Ma et al., 2012; Parthasarathy, 2012!have also been used for high-speed calculation of theparticle center with reduced computational burden whencompared to fitting methods, although at the cost of aminor increase in the localization error.

High photon output of fluorophores is critical toachieve localization accuracy bellow the diffraction limit~Thompson et al., 2002!. Synthetic fluorescent dyes tend toemit a considerably higher number of photons ~typicallytenfold more! than genetically encoded fluorophores ~Hen-riques & Mhlanga, 2009!, allowing better localization accu-racy. However, the use of large linkers such as antibodies totag the molecules of interest leads to an ;10 nm spacingrelative to the fluorophore ~Jares-Erijman & Jovin, 2003;Ries et al., 2012!, thus incorporating an additional error tolocalization of the molecule of interest. However, a solutionis provided by the use of small linkers to label proteins ofinterest with dyes, such as nanobodies ~Ries et al., 2012!, thefluorescein arsenical helix binder ~FLAsH! ~Lelek et al.,2012!, or small peptide tags ~Klein et al., 2010; Wombacheret al., 2010!.

Other factors influencing resolution include fluoro-phore density, optical system stability, sample drift, and thetype of fluorophore localization. Due to these features, thelocalization accuracies can vary considerably, typically rang-

ing between 1 to 40 nm ~Yildiz et al., 2003; Pertsinidis et al.,2010; Schermelleh et al., 2010!.

The principle of subdiffraction accuracy single-particlelocalization has been the foundation for seminal studiesincluding the work of Gelles et al. ~1988! in the movementof kinesin-coated beads and of Yildiz et al. ~2003! on themovement of Myosin V along actin filaments.

DETECTING INDIVIDUAL MOLECULESWITHIN THE ENSEMBLE

To minimize the visible fluorophores spatial overlap re-quired for high accuracy particle localization, the aforemen-tioned methods are limited to single-molecule imaging of avery low density of emitting fluorophores. In general, how-ever, molecules to be imaged are present at unknown densi-ties, typically too high to be resolved by standard microscopy.Accurate single-molecule localization is impossible whenclosely packed fluorophores emit in the same spectral rangesimultaneously ~Fig. 1!; however, they could be individuallyresolved if made to emit in different colors ~Betzig, 1995! orat different points in time ~Lidke et al., 2005; Betzig et al.,2006; Hess et al., 2006; Rust et al., 2006!. The concept ofhigh-accuracy localization of neighboring fluorophores withdistinct colors was experimentally demonstrated in 1998~Van Oijen et al., 1998; Lacoste et al., 2000!. In 2004, theprinciple of differentiating and localizing sequentially disap-pearing fluorophores ~“bleached”! from crowded regionswas introduced by subtracting consecutive images in atime-lapse sequence ~Gordon et al., 2004; Qu et al., 2004!.Later in 2005, Lidke et al. used the stochastic blinking ofquantum dots as a method to transiently distinguish andlocalize individual fluorophores in space and time ~Lidkeet al., 2005!. It was in 2006 that the independent work ofBetzig et al. ~2006! in photoactivated localization micros-copy ~PALM!, Hess et al. ~2006! in fluorescence PALM~FPALM!, and Rust et al. ~2006! in stochastic optical recon-struction microscopy ~STORM! led to a breakthrough insingle-molecule super-resolution methodology. These groupsdemonstrated that the use of photoswitchable fluorophoresallows control of the number of actively emitting fluoro-phores in each acquired image; therefore, it is possible tominimize the probability that in any given image two ormore fluorescent molecules spatially overlap. Photoswitch-able fluorophores are able to reversibly or irreversibly switchbetween two or more spectrally distinct states. The changebetween states is generally induced by light and can bepartially controlled via the microscope sample illumination.PALM, FPALM, and STORM require the majority of photo-switchable fluorophores to stay in a nonvisible state. This isgenerally achieved by a high-intensity excitation of thevisualized fluorescent state, forcing fluorophores to be revers-ibly or irreversibly bleached. A small fraction of these fluo-rophores can then be transiently switched to a visible stateby either naturally recovering from reversible photobleach-ing or by photoactivation with a low-intensity light ofcompatible wavelength. By acquiring a sequence of images


while the photoactivation of subsets of fluorophores occur,each frame will capture a small number of emitting individ-ual fluorophores compatible with single-molecule localiza-tion ~see Figs. 1, 2!. A super-resolution representation of thedataset can then be recreated by plotting the positions ofthe molecules localized in each frame of the sequence ofimages ~see Fig. 2!. As each frame needs to have a suffi-ciently small number of emitting fluorophores to keep theoverlapping probability low, hundreds to thousands of im-ages commonly need to be acquired to accumulate enoughparticle detections to accurately represent molecular andcellular structures. The final resolution of the reconstruc-tion depends on the localization accuracy of each individualfluorophore, the density of localized molecules, and thecapacity to correct for imaging artifacts such as unwantedmotion ~drift! of the sample during the acquisition. Typi-cally, SMLM approaches based on PALM and STORM willlead to a 10–20-fold increase in resolution when comparedto the classical resolution limit ~;200 nm!, as seen in theexample of Figure 3.

PALM and STORM rely on photoswitchable propertiesinherent or conferred to fluorophores. Photoswitchablefluorophores can stochastically switch to an excitable statewhen illuminated by activating light @e.g., near-ultraviolet~UV! light applied to photoactivatable green fluorescentprotein ~PA-GFP!# and to disable it through high-poweredexcitation light ~e.g., a strong irradiation of 488 nm laserwill switch-off the PA-GFP fluorescence, typically by irrevers-ibly bleaching the molecule!. Different SMLM methodsrely on different types of photoswitchable fluorophores.PALM-based approaches tend to use genetically encodedphotoswitchable fluorescent proteins ~e.g., PA-GFP, mEos2!,while STORM-based methods tend to rely on the induciblephotoswitching properties of synthetic dyes when coupledin certain dye pairs ~e.g., Cy3-Cy5!, where the excitation ofone dye ~Cy3! will induce the photoactivation of thecomplementary fluorophore ~Cy5!. The direct-STORM~dSTORM! approach further demonstrated the possibilityof inducible photoswitching properties in simple classicalfluorophores ~e.g., Cy5! without the requirement of a pairof fluorophores ~Heilemann et al., 2009!. Several studieshave shown that special embedding media prevent theirreversible photobleaching of fluorophores, granting photo-switchable properties to fluorophores ~Folling et al., 2008;Steinhauer et al., 2008; Vogelsang et al., 2008; Heilemannet al., 2009!.

HIGH-RESOLUTION SINGLE-MOLECULELOCALIZATION IN 3DEven though SMLM approaches allow localization of indi-vidual molecules with subdiffraction lateral accuracy in 2Dimages, the axial position of these particles needs to beobtained through additional methods. Several approacheshave attempted to encode the axial position of fluorophoresinto their observable 2D spatial profile ~see Fig. 4 andTable 1!. These include astigmatism ~Huang et al., 2008a!,

BiPlane detection ~Juette et al., 2008!, double-helix PSFformation ~Pavani et al., 2009!, and dual-objective interfer-ometry ~Shtengel et al., 2009; Aquino et al., 2011!.

Astigmatism relies on the introduction of a cylindricallens in the emission optical path, as a result of which afluorescent particle will appear as a fluorescent spot elon-gated along either the X or the Y direction depending on itsaxial coordinate Z. The Z coordinate is thus encoded in theellipticity of each spot and can be computationally retrieved~Huang et al., 2008a!. Huang et al. ~2008a! applied thismethod to STORM microscopy, resolving the morphologyof mitochondria and mitochondria-microtubule contacts.Combining astigmatism with multiple acquisitions at differ-ent z-depths, they imaged entire cells in 3D ~typically 50 mmwide and 3 mm thick! with a resolution up to 25 nm in xyand 67 nm in z. A recent study ~Xu et al., 2012! combinedastigmatism with a dual-objective scheme. This system hasachieved resolutions up to ;10 nm in lateral direction and

Figure 4. Principles of 3D single-molecule super-resolution meth-ods: ~A! retrieving the z-position of molecules from their observ-able spatial-profile—astigmatism changes the fluorescent spotsellipticity, DH-PSF converts the normal spatial profile into tworotating lobes and BiPlane simultaneously acquires images fromdifferent focal planes—z-coordinates for each spot can be esti-mated through the analysis of their shape; ~B! relation between 3Dmethods applied to single-molecule super-resolution, the axialthickness of the optical slices, and the possible complementarytechniques for super-resolution z localization.


;20 nm in axial direction and allowed resolution of indi-vidual actin filaments. This boost in resolution was sup-ported by the near doubling of photon collection throughthe two opposed objectives, improved objective stabilizationand reduced noise sensitivity thanks to the redundancy ofthe images obtained through the two objectives.

In contrast, in BiPlane FPALM ~BP FPALM! two detec-tion planes are imaged at slightly different axial positionssimultaneously ~;700 nm!. Particle z coordinates are re-trieved by analyzing the changes in their PSF profile withinthe two focal planes. Authors reached 30 nm lateral and75 nm axial resolutions while imaging 4 mm diameter beads~Juette et al., 2008!. BiPlane has the benefit of keeping arelatively stable axial resolution in Z, while in astigmatismthe resolution improvement diminishes with distance tothe focal plane. Both astigmatism and BiPlane are remark-ably efficient at retrieving the axial subdiffraction posi-tions of particles within a z-range of ;1 mm, without theneed of changing the focal position of the objective. Con-sequently, no axial scanning is necessary to cover thisvolume within a sample, thus allowing for fast 3D acquisi-tion. Both methods also can be implemented with minormodifications to either a TIRF or wide-field microscopyapparatus.

Double-helix point spread function ~DH-PSF! standsas a third and alternative method providing axial subdiffrac-tion resolution. Using a spatial light modulator, this tech-nique reshapes the PSF of the emitted light into a doublehelix pattern, leading to two visible lobes in a 2D image.These two lobes are rotated around their midpoint by anangle that depends on the particle Z coordinate. Particlescan then be accurately localized axially by measuring thisangle, while the midpoint between the centroids of eachlobe provides the lateral position ~Pavani et al., 2009!. Thismethod has experimentally achieved 30 nm lateral and50 nm axial resolution. While these resolutions are verysimilar to the other methods, the z-range of DH-PSF isslightly higher reaching ;2 mm.

Axial interference has been used to improve localiza-tion in the z-axis by an approach featuring two opposingobjectives referred to as “interferometric PALM” ~iPALM!~Shtengel et al., 2009!. This method achieves 3D super-resolution localization of fluorophores in parallel to opticalsectioning. In iPALM, the fluorophore emission signal ismade to interference with itself, thereby encoding thez-position of the emitter through an envelope of oscillationsof the resulting signal. In this way, authors correlate theaxial depth of the emitter and the relative intensity detectedby each camera to arrive at particle localization with aresolution up to 10 nm axially and 23 nm laterally within a250 nm thickness layer. However, this system suffers froma limited working range problem and periodic artifacts ~Xuet al., 2012!. Recently, the axial working range problem wasimproved by combining 4pi with PALM microscopy ~Aquinoet al., 2011!. Here, a resolution of ;8–22 nm laterally and;6 nm axially was achieved in an enlarged optical layer of;650 nm around the focal plane.Ta

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CONSTRAINING ILLUMINATION IN3D SPACE

The combination of SMLM with TIRF has been widely usedto constrain the z localization of fluorescing moleculesto an axial region of space below the diffraction limit~,200 nm! ~Betzig et al., 2006; Rust et al., 2006!. Yet, TIRF isrestricted to imaging the sample surface directly over thecoverslip, preventing any deeper objects from being imaged.In contrast, wide-field-based illumination of the sample per-mits deeper imaging but at the cost of a considerable excita-tion of fluorophores below and above the focal plane, thusincreasing unwanted fluorescence background and limitingresolution. Alternative sample illumination methods compat-ible with 3D SMLM have been recently developed. Theseaim to excite only a small in-focus volume ~see Fig. 4 andTable 1!, improving the signal-to-noise ratio, while both de-creasing sample photodamage and phototoxicity in the caseof live cells ~see the Live-Cell Imaging section below!.

Highly inclined and laminated optical sheet ~HILO! is az-sectioning approach that can be performed with minimaladaptations to a microscope capable of TIRF. In this method,the illumination beam is moved to the lateral periphery ofthe back focal plane of the objective lens just before induc-ing a TIRF evanescent field. The resulting beam out of theobjective is at a subcritical angle with the coverslip. Thelarge refraction angle occurring at the surface of the cover-slip shapes the excitation light into a highly inclined andlaminated thin optical sheet incident within the sample.The thickness of the sheet can be changed with the diameterof the illumination beam and has been shown to varybetween 5 and 10 mm ~Tokunaga et al., 2008!. Baddeleyet al. ~2011! applied with success HILO illumination forsuper-resolution imaging.

Vaziri et al. ~2008! used two-photon temporal focusingto confine activated fluorophores to a single optical sliceroughly 2 mm deep. This activation step is followed bystandard PALM imaging using wide-field illumination ofthe entire sample. Only the photoactivated fluorophores inthe two-photon activation plane will emit fluorescence,safeguarding nonactivated fluorophores in the out-of-focusregion from bleaching. This method has been successfullyapplied and improved by York et al. ~2011! to image themitochondrial network within cells at a 50 nm lateral and100 nm axial resolution, up to 8 mm deep in the sample

Similarly, a remarkable combination of selective-planeillumination microscopy ~SPIM! and FPALM, named indi-vidual molecule localization–selective plane illuminationMicroscopy ~IML-SPIM!, has been demonstrated by Zanac-chi et al. ~2011!. Using a first laser, a single thin layer ofphotoactivatable fluorophores is activated ~ranging between2–4 mm!; this region of space is then excited by a secondlaser, allowing the emitted fluorescence to be acquired witha second objective aligned perpendicularly to the excitedplane. This study demonstrates a lateral and axial resolutionof 63 and 140 nm, respectively, 150 mm deep into thesample, without significant influence of scattering and opti-

cal aberrations. As stated by the authors, this method couldbe further improved by using two-photon excitation forselective plane illumination ~Truong et al., 2011!.

SPIM, two-photon activation, and HILO all providez-sectioning capabilities to SMLM approaches. Neverthe-less, these methods alone cannot constrain the sample illu-mination or photoactivation to subdiffraction regions, asthe induced optical slices are thicker than 1 mm ~Tokunagaet al., 2008; York et al., 2011; Zanacchi et al., 2011!. Thus,other methods such as those mentioned in the previoussection must be combined with these approaches to obtainsuper-resolution along the axial direction deep in the sample.

LIVE-CELL IMAGING

Live-cell imaging becomes difficult when dealing with meth-ods such as PALM and STORM, where a high number offluorophores need to be collected in a sufficiently fast man-ner to correctly represent the structures of interest within atime interval ~Henriques et al., 2011!.

Over the last years, dynamic live-cell imaging has beenone of the main points of focus for developments in singlemolecule super-resolution microscopy. One of the key diffi-culties is motion blur, which can arise if the molecules moveover distances larger than the spatial resolution during thetime taken to acquire sufficient frames for a meaningfulsuper-resolution reconstruction ~Shroff et al., 2008!.

Shroff et al. ~2008! argued that according to the Nyquistcriterion the final reconstruction resolution will be limitedto 2/j 1/D, where j is the spatial density of localized mol-ecules and D the number of spatial imaging dimension~typically D � 2 or 3!. To achieve a significant resolution,the density needs to be high, which requires the acquisitionof a large number of frames. In contrast, the amount offrames needs to be sufficiently low to represent a single timepoint with minimal cell motion artifacts. This problem canbe partially solved by streaming images at high frequencies,but this possibly leads to smaller signal-to-noise ratios ofthe detected particles, unless higher laser illumination poweris used, which can however affect cell viability.

To attain high molecular localization densities withinshort time periods, three key factors come into play: ~1! sen-sitivity of the imaging hardware’s and its capacity to reduceunwanted background, such as through 3D sectioning ~seethe previous section!; ~2! the algorithmic capacity to local-ize and distinguish a large number of particles per frame~see the next section!; ~3! fluorophore photon output andcontrast ratios ~note that some photoswitchable fluoro-phores maintain residual fluorescence when in an off-state! ~Henriques & Mhlanga, 2009!. The choice ofhigh-performance fluorophores ~point 3! becomes criticalwhen planning live-cell experiments. Although most fluo-rescent photoswitchable proteins have high contrast ratios,they supply relatively small photon outputs when comparedto fluorescent dyes. For example, a single Cy5 dye emits;6,000 fluorescence photons compared to ;500 photonsfor mEos2 ~Lukyanov et al., 2005; Bates et al., 2007;


Henriques & Mhlanga, 2009!. For this reason, synthetic dyesare attractive candidates as super-resolution labels. How-ever, antibody labeling of live cells is restricted to themembrane surface due to cell impermeability. Early live-cellsingle-molecule super-resolution studies mostly relied ongenetically encoded photoswitchable fluorophores, allowingresolutions of 40 to 70 nm in 2D within 30–60 s timeframes ~Biteen et al., 2008; Shroff et al., 2008!. Currently, amajor effort is being focused on exploring live-cell friendlydye-labeling approaches such as by directly linking dyes toproteins ~Jones et al., 2011! and small peptides sequences~Klein et al., 2010; Wombacher et al., 2010!. For example,the small FLAsH dye has been used to image the integraseenzyme of HIV with 30 nm resolution, resolving morpho-logical traits of the virus and applied to live-cell super-resolution imaging ~Lelek et al., 2012!. In another instance,anti-GFP nanobodies ~small, high-affinity antibodies! wereused to tag GFP proteins expressed within live hippocampalneurons, achieving high density particle tracking whileminimizing the localization error ~;10 nm! stemming fromthe larger standard antibodies ~Ries et al., 2012!. Remark-ably, Jones et al. demonstrated live-cell imaging of clathrincoated pits and transferrin with 1–2 s time resolution in 3Dspace featuring 30 nm lateral and 50 nm axial resolution, byeither direct coupling of fluorophores to proteins or viaSNAP-tags ~Gautier et al., 2008! coupled to photoswitchablecyanide dyes ~Jones et al., 2011!. Manley et al. ~2008!showed the possibility of combining particle tracking withstochastic photoswitching. Their technique named single-particle tracking PALM ~sptPALM! is based on the lowexcitation of photoactivated fluorophores, allowing them tofluoresce for a few consecutive frames until bleached whilekeeping a low amount of active fluorophores in each frame.It becomes possible then to analyze the short trajectories ofeach particle during its activated state. Based on this ap-proach Manley et al. ~2008! acquired more than a thousandtrajectories in a single cell at 20 frames per second allowingto combine spatial and dynamic information of proteinclustering in the cell membrane.

Obviously, an important aspect in live-cell imaging iscell survival. In live-cell SMLM experiments, two main fac-tors can promote cell death or abnormal behavior: ~1! chem-ical toxicity of buffers that enhance photoswitching and ~2!photodamage caused by sample illumination. Genetically en-coded photoswitchable proteins do not generally require suchbuffers and can thus be directly used in physiological sampleembedding settings. One exception is the work of Matsudaet al. ~2010!, where standard GFP was shown to photocon-vert from green to red when stimulated by blue light in thepresence of reduced riboflavin. Although this buffer allowedsuccessful use of GFP for PALM on fixed cells, live-cell imag-ing was not possible due to the impermeability of cell mem-branes to riboflavin and the need for anaerobic conditions.SMLM using synthetic dyes will generally demand specialimaging buffers to induce photoswitching of synthetic fluo-rescent dyes. These can have different degrees of chemicaltoxicity, as recently compared in a review from Henriques

et al. ~2011!. SMLM methods can also cause considerablephototoxicity through extensive use of high intensity illumi-nation, normally required to maintain fast fluorophore photo-switching cycles. This becomes even more critical whennear-UV light is needed to induce fluorophore photoactiva-tion, as for the majority of photoswitchable genetically en-coded protein ~Henriques & Mhlanga, 2009!. Therefore,techniques that constrain light illumination in 3D space suchas TIRF ~see the previous section! or diminish the require-ment for strong illumination intensities—for example, the3B method ~Cox et al., 2011! described in the next section—are extremely advantageous.

SPEEDING UP RECONSTRUCTION ANDIMAGING

One important aspect of single-molecule localization micros-copy is the computational burden of analyzing thousands tomillions of detectable fluorophores in a timely manner. Oneinitial focus in the field was to develop highly efficientalgorithms achieving real-time analysis of PALM orSTORM data concurrent to the acquisition itself ~Heddeet al., 2009; Henriques et al., 2010; Smith et al., 2010; Wolteret al., 2010!. Thanks to such software, researchers are able togenerate super-resolution reconstructions of the samplebeing imaged while images are streamed from the camera.This feature allows optimal acquisition settings based on thereconstruction—for example, adjusting the illuminationintensity to maintain a constant number of molecule detec-tions over time.

When imaging dynamic structures in live cells, the bal-ance between the number of frames needed to generate asuper-resolution time point and the correspondent acquisi-tion time is crucial to resolve cellular ultrastructure mini-mally corrupted by sample movement. The initial requisitethat individual fluorescent spots do not overlap, which se-verely restricts the number of visible particles in each frame,imposes a crucial limiting factor for the imaging speed. Aseries of new algorithms have challenged this requisite andhave enabled a steep reduction of the number of imagesneeded to generate a super-resolution reconstruction. Thesenew algorithms allow simultaneous detection of multipleoverlapping spots from nearby fluorophores, while distin-guishing them from high and complex backgrounds. Oneclass of recently proposed methods includes fitting paramet-ric models consisting of multiple, rather than a single, PSFfunctions ~Holden et al., 2011; Huang et al., 2011! andsparcity-based reconstruction ~CS-STORM! ~Zhu et al.,2012!. Both methods enable up to a tenfold increase inmolecular densities compared to standard PALM and STORMparticle detection methods ~Henriques et al., 2010!. CS-STORM is able to achieve slightly higher particle density foreach frame when compared to multiple PSF fitting but at theexpense of a minor loss in resolution ~;20%!. Yet, these ap-proaches are still highly sensitive to the signal-to-noise ratio.

Another class of reconstruction methods includesbleaching/blinking assisted localization microscopy ~BaLM!


~Burnette et al., 2011!, generalized single molecule high-resolution imaging with photobleaching” ~gSHRImP! ~Simon-son et al., 2011! and Bayesian localization microscopy ~3B!~Cox et al., 2011!. Unlike the algorithms above, these meth-ods use the temporal fluctuations of fluorescence emission~caused by photobleaching or photoswitching! to pinpointeach fluorophore ~in the case of BaLM and gSHRImP! or togenerate a probability density map of fluorophore positions~in 3B!. These approaches have been shown to work withclassical fluorophores, thus removing the need for special-ized photoswitchable fluorophores, and even in some casesdispensing of laser activation. Notably, 3B has been recentlydemonstrated to achieve fast ~4s per image! live-cell imag-ing of mCherry labeled cells, with 50 nm resolution using astandard microscope with a xenon arc lamp.

THE FUTURE OF SINGLE-MOLECULESUPER-RESOLUTION

SMLM faces today a large number of developments inphotoswitchable markers, imaging speed, analysis time, 3Dmolecular localization, and imaging depth. These improve-ments should soon be further expanded through integra-tion with each other. For example, the 3B method could befurther enhanced through optical sectioning, allowing forthe analysis of thicker samples.

Due to the fast pace of improvements in SMLM, it ischallenging for nonexperts to keep up with the state-of-the-art of the field. However, the recent release of commercialsystem dedicated to SMLM by major microscopy compa-nies such as Leica, Carl Zeiss, and Nikon already providebiologists access to base tools for super-resolution micros-copy. We also foresee these companies updating their sys-tems to support major recent SMLM developments, althoughwith some delay.

Super-resolution microscopy is now ready to provideresearchers with a novel view of nanoscale structures notvisible through standard optical microscopy. This thrivingfield unlocks the possibility to study cellular structuresbelow the diffraction limit; for example, viral particles,membrane receptors, protein aggregate formation, protein-protein interactions, and many others. EM has been theirdefault imaging method in the past and still provides aresolution level not yet achievable through SMLM. How-ever, SMLM caries many nice features not possible throughEM such as simple sample preparation, molecular specificlabeling, and live-cell and deep tissue imaging.

The further combination of SMLM methods and stan-dard microscopy modalities ~such as wide-field or confocal!can provide users with a method to observe cells in a fastmanner and then “zoom-in” to cellular structures of interestthrough super-resolution.

ACKNOWLEDGMENTS

We thank E. Fabre and M. Lelek at Institut Pasteur - Parisfor advice in the preparation of the manuscript. We would

also like to thank E.F. Fornasiero ~San Raffaele ScientificInstitute and Vita-Salute University, Milan, Italy! for thelabeling and preparation of cells shown in Figure 3. Thiswork was funded by Institut Pasteur and the Fondationpour la Recherche Médicale. H.S. additionally funded by anEMBO long-term fellowship and by a “financement jeunechercheur” from Sidaction.

REFERENCESAbbe, E. ~1882!. The relations of aperture and power in the

microscope. J Roy Micro Soc 2, 300–309.Abraham, A.V., Ram, S., Chao, J., Ward, E. & Ober, R.J. ~2009!.

Quantitative study of single molecule location estimation tech-niques. Opt Express 17, 23352.

Ando, T., Uchihashi, T., Kodera, N., Yamamoto, D., Miyagi, A.,Taniguchi, M. & Yamashita, H. ~2008!. High-speed AFM andnano-visualization of biomolecular processes. Eur J Physiol456, 211–225.

Aquino, D., Schönle, A., Geisler, C., Middendorff, C.V., Wurm,C.A., Okamura, Y., Lang, T., Hell, S.W. & Egner, A. ~2011!.Two-color nanoscopy of three-dimensional volumes by 4Pidetection of stochastically switched fluorophores. Nat Methods8, 353–359.

Axelrod, D. ~1981!. Cell-substrate contacts illuminated by totalinternal reflection fluorescence. J Cell Biol 89, 141–145.

Baddeley, D., Crossman, D., Rossberger, S., Cheyne, J.E., Mont-gomery, J.M., Jayasinghe, I.D., Cremer, C., Cannell, M.B. &Soeller, C. ~2011!. 4D super-resolution microscopy with con-ventional fluorophores and single wavelength excitation inoptically thick cells and tissues. PLoS One 6, e20645.

Bates, M., Huang, B., Dempsey, G.T. & Zhuang, X. ~2007!.Multicolor super-resolution imaging with photo-switchable flu-orescent probes. Science 317, 1749–1753.

Betzig, E. ~1995!. Proposed method for molecular optical imag-ing. Opt Lett 20, 237–239.

Betzig, E., Patterson, G.H., Sougrat, R., Lindwasser, O.W.,Olenych, S., Bonifacino, J.S., Davidson, M.W., Lippincott-Schwartz, J. & Hess, H.F. ~2006!. Imaging intracellular fluo-rescent proteins at nanometer resolution. Science 313, 1642–1645.

Biteen, J.S., Thompson, M.A., Tselentis, N.K., Bowman, G.R.,Shapiro, L. & Moerner, W.E. ~2008!. Super-resolution imag-ing in live Caulobacter crescentus cells using photoswitchableEYFP. Nat Methods 5, 947–949.

Burnette, D.T., Sengupta, P., Dai, Y., Lippincott-Schwartz, J.& Kachar, B. ~2011!. Bleaching/blinking assisted localizationmicroscopy for superresolution imaging using standard fluores-cent molecules. Proc Natl Acad Sci USA 108, 21081–21086.

Chao, J., Ram, S., Abraham, A.V., Sally Ward, E. & Ober, R.J.~2009a!. A resolution measure for three-dimensional micros-copy. Optics Comm 282, 1751–1761.

Chao, J., Ram, S., Ward, E.S. & Ober, R.J. ~2009b!. A comparativestudy of high resolution microscopy imaging modalities usinga three-dimensional resolution measure. Opt Express 17,24377–24402.

Cheezum, M.K., Walker, W.F. & Guilford, W.H. ~2001!. Quanti-tative comparison of algorithms for tracking single fluorescentparticles. Biophys J 81, 2378–2388.

Cox, S., Rosten, E., Monypenny, J., Jovanovic-Talisman, T.,Burnette, D.T., Lippincott-Schwartz, J., Jones, G.E. &Heintzmann, R. ~2011!. Bayesian localization microscopy re-veals nanoscale podosome dynamics. Nat Methods 9, 195–200.


Cremer, C. & Cremer, T. ~1978!. Considerations on a laser-scanning-microscope with high-resolution and depth of field.Microsc Acta 81, 31–44.

Crocker, J.C. & Grier, D.G. ~1996!. Methods of digital videomicroscopy for colloidal studies. J Colloid Interf Sci 179, 298–310.

Dedecker, P., Mo, G.C.H., Dertinger, T. & Zhang, J. ~2012!.Widely accessible method for superresolution fluorescence imag-ing of living systems. Proc Natl Acad Sci USA 109, 10909–10914.

de Jonge, N., Peckys, D.B., Kremers, G.J. & Piston, D.W. ~2009!.Electron microscopy of whole cells in liquid with nanometerresolution. Proc Natl Acad Sci USA 106, 2159–2164.

Dertinger, T., Colyer, R., Iyer, G., Weiss, S. & Enderlein,J. ~2009!. Fast, background-free, 3D super-resolution opticalfluctuation imaging ~SOFI!. Proc Natl Acad Sci USA 106,22287–22292.

Ehsani, S., Santos, J.C., Rodrigues, C.D., Henriques, R., Au-dry, L., Zimmer, C., Sansonetti, P., Tran Van Nhieu, G. &Enninga, J. ~2012!. Hierarchies of host factor dynamics at theentry site of Shigella flexneri during host cell invasion. InfectImmun 80, 2548–2557.

Erni, R., Rossell, M.D., Kisielowski, C. & Dahmen, U. ~2009!.Atomic-resolution imaging with a sub-50-pm electron probe.Phys Rev Lett 102, 96101.

Folling, J., Bossi, M., Bock, H., Medda, R., Wurm, C.A., Hein,B., Jakobs, S., Eggeling, C. & Hell, S.W. ~2008!. Fluorescencenanoscopy by ground-state depletion and single-molecule re-turn. Nat Methods 5, 943–945.

Gautier, A., Juillerat, A., Heinis, C., Correa, I.R., Jr., Kinder-mann, M., Beaufils, F. & Johnsson, K. ~2008!. An engineeredprotein tag for multiprotein labeling in living cells. Chem Biol15, 128–136.

Gelles, J., Schnapp, B.J. & Sheetz, M.P. ~1988!. Tracking kinesin-driven movements with nanometre-scale precision. Nature 331,450–453.

Giessibl, F.J. ~1995!. Atomic resolution of the silicon ~111!-~7�7!surface by atomic force microscopy. Science 267, 68–71.

Gordon, M.P., Ha, T. & Selvin, P.R. ~2004!. Single-moleculehigh-resolution imaging with photobleaching. Proc Natl AcadSci USA 101, 6462.

Greenfield, D., McEvoy, A.L., Shroff, H., Crooks, G.E., Win-green, N.S., Betzig, E. & Liphardt, J. ~2009!. Self-organizationof the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. PLoS Biol 7, e1000137.

Gustafsson, M.G.L. ~2000!. Surpassing the lateral resolution limitby a factor of two using structured illumination microscopy. JMicrosc 198, 82–87.

Hänninen, P., Hell, S., Salo, J., Soini, E. & Cremer, C. ~1995!.Two-photon excitation 4Pi confocal microscope: Enhanced ax-ial resolution microscope for biological research. Appl Phys Lett66, 1698.

Hedde, P.N., Fuchs, J., Oswald, F., Wiedenmann, J. & Nien-haus, G.U. ~2009!. Online image analysis software for photo-activation localization microscopy. Nat Methods 6, 689–690.

Heilemann, M., van de Linde, S., Mukherjee, A. & Sauer, M.~2009!. Super-resolution imaging with small organic fluoro-phores. Angew Chem Int Edit 48, 6903–6908.

Hell, S.W. & Wichmann, J. ~1994!. Breaking the diffractionresolution limit by stimulated emission: Stimulated-emission-depletion fluorescence microscopy. Opt Lett 19, 780–782.

Henriques, R., Griffiths, C., Hesper Rego, E. & Mhlanga,M.M. ~2011!. PALM and STORM: Unlocking live-cell super-resolution. Biopolymers 95, 322–331.

Henriques, R., Lelek, M., Fornasiero, E.F., Valtorta, F., Zim-mer, C. & Mhlanga, M.M. ~2010!. QuickPALM: 3D real-timephotoactivation nanoscopy image processing in ImageJ. NatMethods 7, 339–340.

Henriques, R. & Mhlanga, M.M. ~2009!. PALM and STORM:What hides beyond the Rayleigh limit? Biotechnol J 4, 846–857.

Hess, S.T., Girirajan, T.P. & Mason, M.D. ~2006!. Ultra-highresolution imaging by fluorescence photoactivation localizationmicroscopy. Biophys J 91, 4258–4272.

Holden, S.J., Uphoff, S. & Kapanidis, A.N. ~2011!. DAOSTORM:An algorithm for high-density super-resolution microscopy.Nat Methods 8, 279–280.

Hu, K., Ji, L., Applegate, K.T., Danuser, G. & Waterman-Storer, C.M. ~2007!. Differential transmission of actin motionwithin focal adhesions. Science’s STKE 315, 111.

Huang, B., Bates, M. & Zhuang, X. ~2009!. Super-resolutionfluorescence microscopy. Annu Rev Biochem 78, 993–1016.

Huang, B., Jones, S.A., Brandenburg, B. & Zhuang, X. ~2008a!.Whole-cell 3D STORM reveals interactions between cellularstructures with nanometer-scale resolution. Nat Methods 5,1047–1052.

Huang, B., Wang, W., Bates, M. & Zhuang, X. ~2008b!. Three-dimensional super-resolution imaging by stochastic optical re-construction microscopy. Science 319, 810–813.

Huang, F., Schwartz, S.L., Byars, J.M. & Lidke, K.A. ~2011!.Simultaneous multiple-emitter fitting for single molecule super-resolution imaging. Biomed Opt Express 2, 1377–1393.

Jares-Erijman, E.A. & Jovin, T.M. ~2003!. FRET imaging. NatBiotechnol 21, 1387–1395.

Jones, S.A., Shim, S.H., He, J. & Zhuang, X. ~2011!. Fast, three-dimensional super-resolution imaging of live cells. Nat Meth-ods 8, 499–505.

Juette, M.F., Gould, T.J., Lessard, M.D., Mlodzianoski, M.J.,Nagpure, B.S., Bennett, B.T., Hess, S.T. & Bewersdorf, J.~2008!. Three-dimensional sub-100 nm resolution fluorescencemicroscopy of thick samples. Nat Methods 5, 527–529.

Kao, H.P. & Verkman, A. ~1994!. Tracking of single fluorescentparticles in three dimensions: Use of cylindrical optics toencode particle position. Biophys J 67, 1291–1300.

Klar, T.A., Jakobs, S., Dyba, M., Egner, A. & Hell, S.W. ~2000!.Fluorescence microscopy with diffraction resolution barrierbroken by stimulated emission. Proc Natl Acad Sci USA 97,8206.

Klein, T., Löschberger, A., Proppert, S., Wolter, S., van deLinde, S. & Sauer, M. ~2010!. Live-cell dSTORM with SNAP-tag fusion proteins. Nat Methods 8, 7–9.

Kner, P., Chhun, B.B., Griffis, E.R., Winoto, L. & Gustafsson,M.G.L. ~2009!. Super-resolution video microscopy of live cellsby structured illumination. Nat Methods 6, 339–342.

Lacoste, T.D., Michalet, X., Pinaud, F., Chemla, D.S., Alivisa-tos, A.P. & Weiss, S. ~2000!. Ultrahigh-resolution multicolorcolocalization of single fluorescent probes. Proc Natl Acad SciUSA 97, 9461.

Lelek, M., Di Nunzio, F., Henriques, R., Charneau, P., Arhel,N. & Zimmer, C. ~2012!. Superresolution imaging of HIV ininfected cells with FlAsH-PALM. Proc Natl Acad Sci USA 109,8564–8569.

Lidke, K., Rieger, B., Jovin, T. & Heintzmann, R. ~2005!. Super-resolution by localization of quantum dots using blinkingstatistics. Opt Express 13, 7052–7062.

Löschberger, A., van de Linde, S., Dabauvalle, M.C., Rieger,B., Heilemann, M., Krohne, G. & Sauer, M. ~2012!. Super-


resolution imaging visualizes the eightfold symmetry of gp210proteins around the nuclear pore complex and resolves thecentral channel with nanometer resolution. J Cell Sci 125,570–575.

Lukyanov, K.A., Chudakov, D.M., Lukyanov, S. & Verkhusha,V.V. ~2005!. Innovation: Photoactivatable fluorescent proteins.Nat Rev Mol Cell Biol 6, 885–891.

Ma, H., Long, F., Zeng, S. & Huang, Z.L. ~2012!. Fast and precisealgorithm based on maximum radial symmetry for single mol-ecule localization. Opt Lett 37, 2481–2483.

Manley, S., Gillette, J.M., Patterson, G.H., Shroff, H., Hess,H.F., Betzig, E. & Lippincott-Schwartz, J. ~2008!. High-density mapping of single-molecule trajectories with photoacti-vated localization microscopy. Nat Methods 5, 155–157.

Matsuda, A., Shao, L., Boulanger, J., Kervrann, C., Carlton,P.M., Kner, P., Agard, D. & Sedat, J.W. ~2010!. Condensedmitotic chromosome structure at nanometer resolution usingPALM and EGFP-histones. PLoS One 5, e12768.

Mattheyses, A.L., Simon, S.M. & Rappoport, J.Z. ~2010!. Imag-ing with total internal reflection fluorescence microscopy forthe cell biologist. J Cell Sci 123, 3621–3628.

Ober, R.J., Ram, S. & Ward, E.S. ~2004!. Localization accuracy insingle-molecule microscopy. Biophys J 86, 1185–1200.

Parthasarathy, R. ~2012!. Rapid, accurate particle tracking bycalculation of radial symmetry centers. Nat Methods 9, 724–726.

Pavani, S.R., Thompson, M.A., Biteen, J.S., Lord, S.J., Liu, N.,Twieg, R.J., Piestun, R. & Moerner, W.E. ~2009!. Three-dimensional, single-molecule fluorescence imaging beyond thediffraction limit by using a double-helix point spread function.Proc Natl Acad Sci USA 106, 2995–2999.

Pertsinidis, A., Zhang, Y. & Chu, S. ~2010!. Subnanometresingle-molecule localization, registration and distance measure-ments. Nature 466, 647–651.

Qu, X., Wu, D., Mets, L. & Scherer, N.F. ~2004!. Nanometer-localized multiple single-molecule fluorescence microscopy. ProcNatl Acad Sci USA 101, 11298.

Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H.~2012!. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods 9, 582–584.

Rino, J., Braga, J., Henriques, R. & Carmo-Fonseca, M. ~2009!.Frontiers in fluorescence microscopy. Int J Dev Biol 53,1569–1579.

Rogers, S.S., Waigh, T.A., Zhao, X. & Lu, J.R. ~2007!. Preciseparticle tracking against a complicated background: Poly-nomial fitting with Gaussian weight. Phys Biol 4, 220.

Rust, M.J., Bates, M. & Zhuang, X. ~2006!. Sub-diffraction-limitimaging by stochastic optical reconstruction microscopy~STORM!. Nat Methods 3, 793–795.

Schermelleh, L., Heintzmann, R. & Leonhardt, H. ~2010!. Aguide to super-resolution fluorescence microscopy. J Cell Biol190, 165–175.

Schmidt, R., Wurm, C.A., Jakobs, S., Engelhardt, J., Egner, A.& Hell, S.W. ~2008!. Spherical nanosized focal spot unravelsthe interior of cells. Nat Methods 5, 539–544.

Shroff, H., Galbraith, C.G., Galbraith, J.A. & Betzig, E.~2008!. Live-cell photoactivated localization microscopy of nano-scale adhesion dynamics. Nat Methods 5, 417–423.

Shtengel, G., Galbraith, J.A., Galbraith, C.G., Lippincott-Schwartz, J., Gillette, J.M., Manley, S., Sougrat, R., Wa-

terman, C.M., Kanchanawong, P. & Davidson, M.W. ~2009!.Interferometric fluorescent super-resolution microscopy re-solves 3D cellular ultrastructure. Proc Natl Acad Sci USA 106,3125.

Simonson, P.D., Rothenberg, E. & Selvin, P.R. ~2011!. Single-molecule-based super-resolution images in the presence ofmultiple fluorophores. Nano Lett 11~11!, 5090–5096.

Smith, C.S., Joseph, N., Rieger, B. & Lidke, K.A. ~2010!. Fast,single-molecule localization that achieves theoretically mini-mum uncertainty. Nat Methods 7, 373–375.

Steinhauer, C., Forthmann, C., Vogelsang, J. & Tinnefeld, P.~2008!. Superresolution microscopy on the basis of engineereddark states. J Am Chem Soc 130, 16840–16841.

Thompson, R.E., Larson, D.R. & Webb, W.W. ~2002!. Precisenanometer localization analysis for individual fluorescent probes.Biophys J 82, 2775–2783.

Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. ~2008!. Highlyinclined thin illumination enables clear single-molecule imag-ing in cells. Nat Methods 5, 159–161.

Truong, T.V., Supatto, W., Koos, D.S., Choi, J.M. & Fraser, S.E.~2011!. Deep and fast live imaging with two-photon scannedlight-sheet microscopy. Nat Methods 8, 757–760.

Van Oijen, A., Köhler, J., Schmidt, J., Müller, M. & Braken-hoff, G. ~1998!. 3-Dimensional super-resolution by spectrallyselective imaging. Chem Phys Lett 292, 183–187.

Vaziri, A., Tang, J., Shroff, H. & Shank, C.V. ~2008!. Multilayerthree-dimensional super resolution imaging of thick biologicalsamples. Proc Natl Acad Sci USA 105, 20221–20226.

Vogelsang, J., Kasper, R., Steinhauer, C., Person, B., Heile-mann, M., Sauer, M. & Tinnefeld, P. ~2008!. A reducing andoxidizing system minimizes photobleaching and blinking offluorescent dyes. Angew Chem Int Ed Engl 47, 5465–5469.

Wolter, S., Schüttpelz, M., Tscherepanow, M., Van de Linde,S., Heilemann, M. & Sauer, M. ~2010!. Real-time computa-tion of sub-diffraction-resolution fluorescence images. J Mi-crosc 237, 12–22.

Wombacher, R., Heidbreder, M., van de Linde, S., Sheetz,M.P., Heilemann, M., Cornish, V.W. & Sauer, M. ~2010!.Live-cell super-resolution imaging with trimethoprim conju-gates. Nat Methods 7, 717–719.

Xu, K., Babcock, H.P. & Zhuang, X. ~2012!. Dual-objectiveSTORM reveals three-dimensional filament organization in theactin cytoskeleton. Nat Methods 9, 185–188.

Yildiz, A., Forkey, J.N., McKinney, S.A., Ha, T., Goldman, Y.E. &Selvin, P.R. ~2003!. Myosin V walks hand-over-hand: Singlefluorophore imaging with 1.5-nm localization. Science 300,2061–2065.

York, A.G., Ghitani, A., Vaziri, A., Davidson, M.W. & Shroff,H. ~2011!. Confined activation and subdiffractive localizationenables whole-cell PALM with genetically expressed probes.Nat Methods 8, 327–333.

Zanacchi, F.C., Lavagnino, Z., Donnorso, M.P., Del Bue, A.,Furia, L., Faretta, M. & Diaspro, A. ~2011!. Live-cell 3Dsuper-resolution imaging in thick biological samples. Nat Meth-ods 8, 1047–1049.

Zhu, L., Zhang, W., Elnatan, D. & Huang, B. ~2012!. FasterSTORM using compressed sensing. Nat Methods 9~7!, 721–723.


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