due to the availability of efficient fluorescent probes and labeling recipes , fluorescence microscopy allows the direct observation of cellular processes in fixed and living cells as well as in complete organisms with molecular specificity and high temporal resolution in three dimensions ( giepmans et al .
2006 ; haugland 2005 ; lippincott - schwartz and patterson 2009 ; pawley 2006 ) . due to the wave nature of light ,
however , the spatial resolution is limited to about half of the wavelength of the light in the imaging plane ( abbe 1873 ; rayleigh 1903 ) , that is , conventional confocal and wide - field fluorescence microscopy does not provide insight into the structural and dynamic organization of biological samples such as vital protein assemblies and machineries with a size of a few tens of nanometers . within recent years ,
different far - field microscopic approaches have been introduced that have found a way to bypass the so - called diffraction barrier exploiting concepts to distinguish fluorescence emission of fluorophores in an additional dimension , e.g. , absorption , fluorescence emission , and fluorescence lifetime ( betzig 1995 ; heilemann et al .
1998 ) , and thus localize their positions individually . however , the number of distinguishable spectroscopic characteristics of fluorophores is too low to distinguish thousands of fluorophores per diffraction - limited area that compose a typical biological structure . on the other hand ,
fluorescence emission of fluorophores is distinguishable also in time allowing the temporal separated fluorescence detection of fluorophores .
thus , the key to super - resolution imaging is confinement of the number of simultaneously fluorescing fluorophores .
this is achieved in a deterministic way by generating a light pattern that prevents the emission of fluorophores in a defined area , as used by stimulated emission depletion ( sted ) ( dyba and hell 2002 ; hell and wichmann 1994 ; willig et al .
2006 ) , and structured illumination microscopy ( sim ) ( gustafsson 2000 , 2005 ; heintzmann and cremer 1999 ; kner et al . 2009 ) or in a stochastic way by temporally modulating the emission of individual fluorescent molecules by photoactivation , photoconversion , or photoswitching , as used in photoactivated localization microscopy ( palm ) ( betzig et al . 2006 ; manley et al . 2008 ; shroff et al .
2007 ) , fluorescence photoactivation localization microscopy ( fpalm ) ( hess et al . 2006 , 2007 ) , stochastic optical reconstruction microscopy ( storm ) ( bates et al . 2007
; huang et al . 2008b ; rust et al . 2006 ) , direct storm ( dstorm ) , ( heilemann et al . 2008 , 2009 ; van de linde et al .
2009 ; bock et al . 2007 ; flling et al . 2008 ; geisler et al . 2007
; gunkel et al . 2009 ; lemmer et al . 2008 ; steinhauer et al .
2009 ) ( table  1 ) . historically , localization microscopy started with the efforts of lidke et al .
( 2005 ) to use the fluorescence intermittencies of semiconductor quantum dots for single - molecule localization .
the authors envisioned that a high - resolution image consisting of individually localized points may be reconstructed and termed it  pointillism 
however , because blinking characteristics of quantum dots can be best described by a power law ( kuno et al .
2000 ) , densely labeled structures are difficult to resolve . alternatively , transient binding of fluorescent probes to the target structure ( giannone et al .
2010 ; sharonov and hochstrasser 2006 ) or the conversion of fluorogenic probes by catalysts ( roeffaers et al . 2009 ) can be exploited to bypass the diffraction limit by the reconstruction of a super - resolved image.table  1single - molecule localization microscopy techniquestechniquebasic principleexperimental detailsreferencespointillismfluorescence intermittencies of emitters such as triplet - state blinking can be used to temporally separate fluorescence emission , localize emitters , and reconstruct a structure of interest ( e.g. , the cytoskeleton ) by essentially painting them in a
 fashionblinking of randomly distributed quantum dots on a coverslip ( qd655 ) ; ~1.000 photons per on - event ; off - state lifetime too short to demonstrate usability to resolve cellular structurelidke et al .
( 2005)palm 
 photoactivated localization microscopy uses photoactivation or photoconversion of fluorescent proteins to generate a sparse subset of fluorescent molecules per framephotoactivatable and photoconvertible fluorescent proteins ( e.g. , pagfp , meos , and pamcherry ) ; no special buffer additions required ; typically , a few hundred photons per on - event ; ( betzig et al .
( 2007)storm 
 stochastic optical reconstruction microscopy uses pairs of fluorophores ( e.g. , cy3cy5 ) to photoswitch the reporter ( cy5 ) in the presence of thiols between a bright on- and a reduced off - state with long lifetime ( >  1  s ) ; the activator ( cy3 ) is used for efficient reactivation of the reporterstandard fluorophore dye pairs ( cy5/cy3 and alexa 647/cy3 ) ; addition of 10100  mm of thiols such as mercaptoethylamine ( mea ) ; oxygen depletion ; off - state lifetimes of seconds to minutes ; ~3.000 photons per on - event of cy5/alexa 647bates et al .
( 2006)fpalm 
 fluorescence photoactivation localization microscopy uses photoactivation or photoconversion of fluorescent proteins to generate a sparse subset of fluorescent molecules per framephotoactivatable fluorescent proteins ( e.g. , pagfp ) ; no special buffer additions required ; typically , a few hundred photons per on - statehess et al .
( 2006 , 2007)palmira 
 palm with independently running acquisition uses an asynchronous acquisition protocol and a fast camera that allows matching the camera frame time to the average on time of fluorophoresfluorescent protein ( rsfastlime ) and standard fluorophore ( cy5 ) ; embedding in poly ( vinyl - alcohol ) ( pva ) to prolong triplet - state lifetime ; ~200 photons per on - state of rsfastlime , ~700 photons per on - state of cy5bock et al .
( 2007 ) 
 dstorm 
 direct storm photoswitches standard fluorescent probes in the presence of thiols between a bright on- and a reduced off - state with long lifetime ( > 1  s)standard fluorophores ( cyanine , rhodamine , and oxazine dyes , e.g. , atto- and alexa dyes ) ; addition of 10100  mm of thiols such as mercaptoethylamine ( mea ) ; off - state lifetimes of seconds to minutes ; 5005.000 photons per on - event dependent on dyeheilemann et al .
/ spectral position determination microscopy uses  reversible photobleaching   of fluorophores and fluorescent proteins under high irradiation intensitiesstandard fluorophores and fluorescent proteins ( e.g. , yfp ) mounted in an antifade reagent ; irradiation intensities of 10  kw  cm1  mw  cm ; > 1.000 photons detected per on - event of yfp moleculesbaddeley et al . ( 2009 ) , gunkel et al .
( 2008)gsdim 
 ground state depletion microscopy followed by individual molecule return uses triplet and other metastable off - states of fluorophores for temporal separation of fluorescence emission of individual fluorophoresstandard fluorophores ( atto 532 , alexa 488 , texas red ) and fluorescent proteins ( e.g. , egfp and eyfp ) ; oxygen depletion or embedding in poly(vinyl - alcohol ) ( pva ) to prolong off - state lifetime to 10100  ms ; 8002.600 photons per on - eventflling et al .
( 2008 ) , hell ( 2009)blink microscopyengineering on- and off - states by controlling the photophysics of the fluorophores by electron transfer reactionsstandard fluorophores ( cy5 , atto 655 , atto 680 , atto 700 ) ; oxygen removal and addition of 100  m ascorbic acid to quench  the triplet state and generate reduced fluorophores with off - state lifetimes > 20  ms ; 70700 photons per on - eventsteinhauer et al . ( 2008 ) , vogelsang et al . ( 2009)most techniques work optionally with a second laser ( 405  nm ) to increase the reactivation efficiency of the on - state ( the on - switching probability ) .
in many cases , the reactivation efficiency of the readout laser is high enough to ensure a constant number of fluorophores residing in the on - state at any time of the experiment . with the exception of spdm ,
all other localization microscopy techniques apply excitation intensities of a few kw  cm ( typically 13  kw  cm ) for fluorescence readout and off - switching or photobleaching , respectively . the recovery or reactivation intensities are two to three orders of magnitude smaller single - molecule localization microscopy techniques most techniques work optionally with a second laser ( 405  nm ) to increase the reactivation efficiency of the on - state ( the on - switching probability ) . in many cases ,
the reactivation efficiency of the readout laser is high enough to ensure a constant number of fluorophores residing in the on - state at any time of the experiment . with the exception of spdm ,
all other localization microscopy techniques apply excitation intensities of a few kw  cm ( typically 13  kw  cm ) for fluorescence readout and off - switching or photobleaching , respectively . the recovery or reactivation intensities are two to three orders of magnitude smaller all stochastic single - molecule localization microscopy techniques ( table  1 ) share the same concept that only a sparse subset of fluorophores is allowed to stay in its fluorescent on - state at any time of the experiment
this is achieved by stochastic activation of individual fluorophores , single - molecule detection using a wide - field fluorescence microscope equipped with a sensitive charge - coupled device ( ccd ) camera , and precise position determination ( localization ) , i.e. , fitting of ideal point - spread functions ( psfs ) to the measured photon distributions .
as long as the distance between individual fluorophores residing simultaneously in their on - state ( i.e. , per frame ) is larger than the distance resolvable by the microscope ( > /2 on the ccd camera ) , individual fluorophores can be clearly localized .
since the localization precision ( i.e. , the accuracy of position determination of a single fluorophore ) depends mainly on the number of collected photons , n , and on the standard deviation ,  , of the psf , single fluorophores can be localized with an accuracy approximated by /n for negligible background ( mortensen et al .
while all single - molecule localization microscopy methods share the same concept of temporal separation of fluorescence emission , they use different concepts to accomplish this experimental requirement ( table  1 ) . to ensure that only a sparse subset of fluorophores resides in  the on - state at any time of the experiment and that each fluorophore defining the structure of interest is detected as individual emitter and thus precisely localized , the generation of stable off - states with lifetimes exceeding seconds is of outstanding importance . while this task is comparatively easy to accomplish using photoactivatable fluorescent proteins , which are non - fluorescent at the beginning of the experiment , the use of standard organic fluorophores requires the transfer of the majority of bright fluorophores to a long - lived , stable off - state .
this succeeds for most alexa and atto dyes  belonging to the cyanine , rhodamine , and oxazine dye classes  upon irradiation of an aqueous solution in the presence of millimolar concentrations of reducing thiols .
the dyes are cycled between the singlet ground and first excited singlet state until the dye enters the triplet state .
the triplet state can then be quenched to repopulate the singlet ground state either by oxygen or by the thiolat ( the actual reducing species ) to form a semi - reduced fluorophore radical anion , the fully reduced leuco form of the fluorophore , or a thiol adduct ( dempsey et al .
finally , the singlet ground state is recovered by the oxidation of the reduced off - state , e.g. , with molecular oxygen . because the off - state of most organic fluorophores absorbs at 400  nm
, photoinduced recovery can be applied to control the number of fluorophores residing in the on - state ( van de linde et al .
since dstorm is easy to perform , i.e. , it works with commercial available fluorescent standard probes in aqueous solvents and standard protocols for immunostaining ( heilemann et al .
2008 , 2009 , 2011b , 2012 , 2013 ) , it is currently the most applied single - molecule localization microscopy concept using organic fluorophores . the simple thiol - assisted photoswitching mechanism ( millimolar concentrations of thiols such as -mercaptoethylamine , cysteine , or glutathione have to be added to the imaging buffer ) and the availability of efficient open - source software for two - dimensional ( 2d ) and three - dimensional ( 3d ) multicolor localization microscopy ( e.g. , rapidstorm ) ( wolter et al .
2010 , 2012 ) facilitated the acceptance and broad applicability of dstorm for different super - resolution imaging studies ( dempsey et al .
, the feasibility and reliability of different localization microscopy techniques has to be judged by test samples with well - defined molecular structure and composition . among the various natural biological test samples , the nuclear pore complex ( npc ) with its well - defined molecular composition , subdiffraction dimension , and eightfold symmetry holds a special position ( adams and wente 2013 ) .
because the npc is composed of a defined number of proteins and exhibits a central channel with a diameter of ~40  nm , it is suited for testing of the achievable resolution of different super - resolution imaging methods and counting the number of molecules in a biological structure . the combination of raw dstorm localization data and particle averaging techniques as first demonstrated by lschberger et al .
( 2012 ) can also be used to reconstruct average protein positions in multiprotein complexes such as the npc with nanometer resolution ( szymborska et al .
it neglects , however , structural heterogeneity of the sample by averaging individual signals  the strength of all single - molecule methods  and does not enhance the optical resolution .
whereas super - resolution imaging or localization of fluorophore - labeled target molecules does not rely on the number of fluorophores attached to the molecule per se , the labeling density determines the achievable resolution of complex biological structures . according to the information theory
, the required density of fluorescent probes must be high enough to satisfy the nyquist 
in essence , this theorem states that the average distance between neighboring fluorophores ( the sampling interval ) must be at least two times smaller than the desired spatial resolution .
for example , to resolve structural features with a spatial resolution of 20  nm , fluorophores must be placed and localized every 10  nm . for a 2d structure ,
a labeling density of approximately 10 fluorophores m or approximately 600 fluorophores within a circular diffraction - limited region is required ( sauer 2013 ) .
thus , independent of the technique used , efficient and specific labeling with fluorescent probes is a decisive aspect of super - resolution imaging .
however , they also exhibit a non - negligible size ( a cylinder with a length of 4.2  nm and diameter of 2.4  nm ) ( tsien 1998 ) .
organic fluorophores such as rhodamine or carbocyanine dyes are substantially smaller ( ~1  nm ) but require a chemical labeling procedure such as standard immunofluorescence using primary and secondary igg antibodies with a size of ~10  nm ( weber et al .
1978 ) . alternatively , small labeled camelid antibodies ( nanobodies ) directed against green fluorescent protein ( gfp ) ( ries et al .
2012 ) with a smaller size ( 1.5  nm    2.5  nm ) can be used for efficient labeling .
one of the most promising future labeling methods for high - density labeling is the 1,2,3-triazole linkage ( click chemistry ) between a fluorophore modified as an azide and a reaction partner ( e.g. , protein ) modified as an alkyne , or vice versa ( laughlin et al .
this method , however , requires further improvements concerning specific labeling of the protein of interest in living and fixed cells .
photoswitchable or photoactivatable fluorescent proteins ( pa - fps ) were the first choice for super - resolution imaging in living cells , as they enable stoichiometric labeling and non - pertubative imaging of the sample ( lippincott - schwartz and patterson 2003 ) .
several studies have been conducted using pa - fps to unravel sub - diffractive morphologies and dynamics of cellular components ( table  2).table  2fluorophores used in live - cell single - molecule localization microscopy studiesfluorophoreabs/em ( nm)photons / on - eventtagadditivescamera frame rate ( hz)temporal resolution ( s)spatial resolutionreferencesoregon greenrhodamine490/514900snapnone100150303d : 50  nm xy ; 90  nm z 
 jones et al .
( 2012)tmrrhodamine554/5801,100snapnone10015030  3d : 45  nm xy ; 82  nm z 
 jones et al .
( 2008)tdeos 
 fp569/5811,200fusion proteinnone100150  3d : 30  3d : 40  nm xy ; 80  nm z 
 jones et al .
( 2011)eosfp 
 fp571/581n / afusion proteinnone20  500  25  nmmanley et al .
( 2008)meos2 
 fp573/5841,200fusion proteinnone100150  3d : 303d : 40  nm xy ; 80  nm z 
 jones et al .
( 2011)alexa 568rhodamine572/6001,700directly labeled-mercaptoethanol , oxygen scavenger500  3d : 303d : 40  nm xy ; 70  nm z 
 jones et al .
( 2012)mitotracker redcationic rosamine581/644790noneoxygen scavenger500900  210  3040  nmshim et al . ( 2012)er - tracker redbodipy587/615820noneoxygen scavenger500  10  35  nmshim et al . (
2012)alexa 647cyanine649/6703,500directly labeled , snap-mercaptoethanol , oxygen scavenger500  2d : 0.5  3d : 12  2d : 25  nm3d : 30  nm xy ; 50  nm z 
 jones et al .
( 2010)atto 655oxazine663/6841,200snapnone100150  30  3d : 40  nm xy ; 80  nm z 
 jones et al .
( 2012 ) 
 fp fluorescent protein 
 converted / activated form fluorophores used in live - cell single - molecule localization microscopy studies 
 fp fluorescent protein 
 converted / activated form one of the first realizations of live - cell super - resolution imaging investigated the structure of the actin protein mreb labeled with eyfp by palm ( biteen et al .
furthermore , they introduced time - lapse palm to increase the effective labeling concentration and achieve a sub-40  nm spatial resolution .
images were obtained with cycles of reactivation at 407  nm and imaging of eyfp at 514  nm with a frame rate of 10  hz and 12  min temporal resolution .
palm was also successfully implemented to investigate the nanoscale dynamics of adhesion complexes by imaging paxillin fused to tdeosfp ( shroff et al .
since the dynamics of these complexes occurs at a timescale of minutes , a series of palm images , acquired over a time > 20  min , with a temporal resolution of 2560  s was sufficient to study morphological changes .
palm imaging revealed sub - diffractive structures and different dynamics within an adhesion complex and between complexes at a spatial resolution of about 60  nm .
assembly and disassembly of adhesions in different cell lines were also studied . with fpalm imaging of pa - gfp ,
the motion of hemagglutinin ( ha ) clusters in the cell membrane of fibroblasts with a localization precision of ~40  nm was investigated ( hess et al . 2007 ) by calculating the distance distribution of ha molecules in consecutive frames with a time resolution of 100300  s. thus , an effective diffusion coefficient could be determined . a combination of single - particle tracking and palm imaging ( sptpalm )
was introduced to study the mobility and distribution of two membrane proteins ( vsvg and gag ) by manley et al .
the experiments gave single - molecule trajectories at a high density ( 50  m ) and the mapping of single - molecule diffusion coefficients of vsvg and gag evidenced that vsvg proteins exhibit a larger mobile fraction than gag proteins .
due to the comparatively low fluorescence quantum yield and photostability of fps , ( f)palm in living cells is restricted to slower cellular processes .
imaging faster dynamic processes in cells requires higher temporal resolution , ideally without decreasing the spatial resolution . in this
therefore , they are well suited for improving the spatiotemporal resolution in live - cell localization microscopy experiments
2008 ; miller et al . 2005 ) offer an elegant method for specific labeling of proteins with organic dyes in living cells ( table  2 ) . here , a polypeptide tag , which is genetically fused to the target protein , binds to a fluorescently labeled substrate . on the other hand , live - cell labeling with
chemical tags is also challenging because the fluorescently labeled substrates have to exhibit high membrane permeability and negligible non - specific binding in living cells .
thus , the labeling procedure has to be optimized for each protein and fluorescent substrate separately concerning substrate concentration and incubation time .
furthermore , the amount of non - specific binding of fluorescently substrates to cellular organelles has to be carefully checked .
nonetheless , some fluorescently labeled substrates are very well suited for specific labeling of proteins in living cells .
the first demonstration of live - cell single - molecule localization microscopy with organic dyes was performed by dstorm employing the trimethoprim ( tmp ) tag ( wombacher et al .
the cell - permeable dye atto 655 was used to label the edhfr tagged core histone protein h2b . since the cell provides its own reductive environment , mostly due to the presence of millimolar concentration of the thiol glutathione ( van de linde et al .
2012 ) , atto 655 molecules switch between a bright on- and a non - fluorescent off - state upon irradiation under physiological conditions . applying a sliding window analysis on the localization data , the dynamic movement of histone proteins could be observed at a temporal resolution of 10  s. slightly later , the snap - tag technology was also successfully applied for live - cell dstorm in various cell lines using the two rhodamine - based substrates snap - cell tmr - star and snap - cell 505 ( fig .  1 ) ( klein et al .
the results demonstrated the formation of long - lived fluorophore off - states under physiological conditions , which can be effectively and repeatedly reactivated upon irradiation at 405  nm ( klein et al .
because rhodamine dyes tend to bind non - specifically on glass surfaces , coverslips were coated with 2  m glycine.fig . 
1live - cell dstorm with snap tags . a fluorescence image of histone h2b proteins in a cos-7 cell stained with snap - cell tmr - star ( 1  m ) .
b fluorescence image of the same cell but with 532  nm excitation of ~1  kw  cm , which induced photoswitching . c 
 dstorm image reconstructed from 10,000 images ( acquired at 50  hz ) . adapted from ( klein et al .
2011 ) , with permission live - cell dstorm with snap tags . a fluorescence image of histone h2b proteins in a cos-7 cell stained with snap - cell tmr - star ( 1  m ) .
b fluorescence image of the same cell but with 532  nm excitation of ~1  kw  cm , which induced photoswitching . c 
 dstorm image reconstructed from 10,000 images ( acquired at 50  hz ) . adapted from ( klein et al .
2011 ) , with permission the photoswitchability of some organic fluorophores in living cells was also used to study the organization of intracellular microcompartments ( appelhans et al .
, mitochondrial proteins of the outer and inner membrane were labeled with tetramethylrhodamine ( tmr ) via the halotag .
single - molecule tracking and localization of individual protein complexes showed protein - specific diffusion behavior within both membranes and their substructures like cristae , indicating mitochondrial compartmentalization .
as the different chemical tags can be used orthogonally , multi - color super - resolution imaging is likewise possible . for single - molecule tracking of two different plasma membrane receptor proteins ,
a multi - color approach employing snap- and halotag has been demonstrated ( benke et al .
the fluorophores used ( dy-547 for snap and alexa 488 for halo ) are spectrally well separated and were imaged sequentially .
in addition , the study demonstrated that tracking is not restricted to membrane proteins , but is also applicable to intracellular proteins like h2b .
using a snap- and clip tag in combination with the two dyes alexa 647 and tmr , dual - color live - cell dstorm of the beta-2-adrenergic receptor and h2b histone proteins has been demonstrated ( klein et al .
the membrane receptor was labeled with the cell - impermeable dye alexa 647 and the nucleus with tmr . to induce photoswitching of alexa 647 ,
the medium was supplemented with 100  mm glutathione and an oxygen scavenger system . however , in all live - cell experiments using organic fluorophores , the generation of reactive oxygen species ( ros ) has to be considered ( van de linde et al .
ros may already perturb the intracellular environment during the experiment and thus can be expected to be a serious source of cell damage . the possibility to combine palm and dstorm for two - color super - resolution imaging
for live - cell imaging , the combination of pa - fps and chemical tags enabled also three - color palm / dstorm of the spatiotemporal organization of membrane receptors ( wilmes et al .
fusion proteins of the type i interferon receptors ( ifnar1 and ifnar2 ) with pa - gfp and patagrfp were coexpressed with the actin - binding lifeact peptide ( riedl et al .
three - color super - resolution imaging was performed through cycles of photoactivation at 405  nm and sequential excitation at 488  nm for pa - gfp , 568  nm for patagrfp , and 647  nm for atto 655 .
the reconstructed images showed a cytoskeletal association of the two receptors , indicating an interaction with actin or actin - bound adaptor proteins ( wilmes et al .
they bind directly to the membrane of mitochondria , endoplasmic reticulum , lysosomes , or the plasma membrane .
these commercially available probes proved to be photoswitchable in living cells upon irradiation with the respective wavelength and reactivation at 405  nm ( shim et al .
due to the high labeling density , the ultrastructural dynamics of organelles could be resolved with a spatial resolution of ~40  nm and temporal resolution of 115  s using multi - emitter fitting algorithms .
time - lapse imaging of mitochondria with mitotracker red , for example , revealed thin tubular intermediates between mitochondria at fission and fusion events , which could not be resolved in conventional diffraction - limited imaging ( shim et al . 2012 ) .
three - dimensional super - resolution imaging in live cells has also been demonstrated ( jones et al .
clathrin - coated pits and their cargo transferrin were labeled with different fluorophores using snap - tag fusion proteins or direct labeling .
due to high laser intensities used of up to 15  kw  cm and the resulting fast switching kinetics of alexa 647 , a time resolution of 0.5  s and 25  nm spatial resolution was achieved in 2d and 2  s and 50  nm ( axial ) in 3d , respectively .
only with alexa 647 , which was delivered into the cell by electroporation , the cup - like morphology of ccps could be resolved .
very recently , a new near - infrared fluorophore has been introduced for live - cell imaging ( lukinavicius et al . 2013 ) . this new silicon 
rhodamine ( sir ) is highly cell - permeable and shows a fivefold increase in fluorescence upon binding to its target protein .
the fluorogenic character results from the formation of the non - fluorescent spirolactone if the dye aggregates or binds unspecifically to hydrophobic structures , whereas it becomes fluorescent upon reaction with the respective protein tag .
single - molecule localization microscopy is also feasible , as sir exhibits switching behavior under physiological conditions , which was shown for cells expressing h2b  snap ( lukinavicius et al .
in order to enable three - dimensional ( 3d ) super - resolution imaging , the axial symmetry of the psf has to be broken , that is , in conventional microscopy ( in the case of zero aberrations ) , the psf of a fluorophore slightly above and below the image plane appears equally blurred preventing the extraction of the accurate axial fluorophore position . to overcome
the obstacle methods have been introduced that tweak the signal in a way that the psf above and below the image plane looks different .
some of the ideas have already been successfully used in single - particle tracking , and with the ability to image only a few fluorophores defining a densely labeled structure at a time due to temporal separation of fluorophore emission , it was straightforward to use these established methods also for localization microscopy .
methods used to infer information about the axial origin of a signal can roughly be split into four groups : ( 1 ) astigmatism , ( 2 ) biplane , ( 3 ) psf splitting into lobes , and ( 4 ) interferometric approaches ( table  3).table  3three - dimensional single - molecule localization microscopy techniquestechniquelateral / axial resolution ( fwhm ) ( nm)axial capture rangereferencesastigmatism ( polynomial)2030/60700.6  m ; with stacking up to 3  mhuang et al .
al . ( 2009)prilm3847/822  mbaddeley et al . ( 2011)ipalm22.8/9.80.225  mshtengel et al . (
2009 ) 
 z - position determination via sigma difference lookup table 
 direct z - position determination by comparison with experimental psf three - dimensional single - molecule localization microscopy techniques 
 z - position determination via sigma difference lookup table 
 direct z - position determination by comparison with experimental psf the astigmatism approach introduces a cylindrical lens into the detection path of the microscope system .
this leads to a stretching of the psf because only one spatial direction is tightly focused while the other is defocused ( fig . 
, there is one point of equal psf widths ( which can be set to be z  =  0  nm ) , and psfs originating from above or below are spread to a horizontal or vertical line , respectively .
the rough axial position can be extracted from the orientation of the psf and the widths in x and y can be calibrated to yield exact z - coordinates .
after the initial use in single - particle tracking ( holtzer et al . 2007 ;
kao and verkman 1994 ) , the method has been broadly applied to single - molecule localization microscopy ( dani et al .
2012 ) . for calibration of the defocusing behavior , usually single fluorophores , quantum dots or small fluorescent beads
the obtained psf widths in x and y are fitted with a polynomial of second order , which represents a physically derived model ( holtzer et al .
2007 ) or a fourth - order polynomial to account for imperfections in the optical system ( huang et al .
2008a ) . to avoid fitting of a more or less physically derived function to the calibration data , a lookup table can be created for the extraction of the actual axial position . in the open - source quickpalm plugin for imagej ( henriques et al .
2010 ) , the standard deviations of the calibration psf in x and y are determined and the known z - position is plotted against x    y . the obtained straight line serves to look up the z - coordinates during the course of a measurement.fig .  2 
 a nucleus of a xenopus laevis a6 cell stained against the nuclear pore complex protein gp210 with pale white bar indicating the area where the x  z - cross section b is taken ;
c and d show the respective x  y- and y  z - views of the distal appendage protein cep152 of centrioles from a u2os cell ; e represents another pair of centrioles in a cos-7 cell .
scale bar 
 a , b 1  m ; c , d 200  nm ; e 500  nm ; color - code ( blue to red ) a , b 04.6  m ; c , d 0400  nm 
 a nucleus of a xenopus laevis a6 cell stained against the nuclear pore complex protein gp210 with pale white bar indicating the area where the x  z - cross section b is taken ; c and d show the respective x  y- and y  z - views of the distal appendage protein cep152 of centrioles from a u2os cell ; e represents another pair of centrioles in a cos-7 cell .
scale bar 
 a , b 1  m ; c , d 200  nm ; e 500  nm ; color - code ( blue to red ) a , b 04.6  m ; c , d 0400  nm to use biplane imaging , the detected signal has to be split into two parts with an optical delay added to one of the two signals .
both signal paths can be either imaged on the same camera ( juette et al .
2008 ) , or if a second camera is at hand , the two signals can be detected separately . in the second scheme ,
a relative offset in the axial position between both image planes has to be realized .
the psf widths detected on detector a and b encode the z - coordinate just as the ratio between the x- and y - psf widths using the astigmatism approach .
the third family of 3d localization microscopy techniques uses additional optics to split the psf of a fluorophore into two lobes that rotate around each other with respect to the axial position of the emitter .
it was first proposed to engineer the psf to a double - helix by the introduction of a phase mask , which is created by a polarization - sensitive spatial light modulator ( slm ) ( pavani et al .
the two resulting lobes rotate around each other in a double - helix ( dh - psf ) manner , which baptized the technique .
it was later proposed to use a simplified approach adding a phase ramp in the objective aperture to half of the signal ( baddeley et al .
termed phase ramp imaging localization microscopy ( prilm ) , in contrast to dh - psf , one lobe remains fixed while the other rotates around it .
both techniques have in common that the available axial range is significantly larger than in biplane- or astigmatic imaging ( baddeley et al .
, the sample is placed between two objectives , and thus , the signal of a single fluorophore travels along two different light paths .
exploiting the wave  particle duality , upon superposition of the respective signals with a three - way beam splitter , every single photon will interfere with itself . depending on the axial position ,
i.e. , whether it is closer to objective a or b , the fluorophore will show distinct interference that gives information about the z - coordinate ( kanchanawong et al .
interferometry so far yields the best axial localization precision but is limited in axial range to about half the emitted wavelength ( table  3 ) .
most 3d localization microscopy approaches use high na oil - immersion objectives for surface confined total internal reflection fluorescence ( tirf ) irradiation .
another very popular irradiation mode for intracellular imaging only available with oil immersion is highly inclined and laminated optical sheet ( hilo ) microscopy ( tokunaga et al . 2008 ) . in a sense , hilo is similar to tirf , where illuminating light is approaching the sample at a critical angle , so that only molecules very close to the coverslip are illuminated by the evanescent wave .
however , instead of illuminating only the molecules in close proximity to the coverslip as in tirf , hilo illuminates by a highly inclined and thin beam under a particular angle allowing high irradiation intensities combined with high signal - to - noise ratio even inside of cells .
unfortunately , using an oil - immersion lens for imaging while measuring in aqueous buffer induces aberrations due to the refractive - index mismatch at the coverslip  buffer interface .
thus , the volume image of a specimen will appear stretched along the optical axis , an effect well described for both confocal ( hell et al .
furthermore , the refractive - index mismatch leads to spherical aberration along the z - axis that is difficult to correct ( deng and shaevitz 2009 ) . by localizing a single fluorescent particle placed in various but well - controlled depths by an optical tweezer
, it was shown that the determined z - position could be up to 30  % erroneous .
this issue is especially tricky , as the amount of false localization is not uniform along the z - axis , that is , if single fluorophores adsorbed on a bare glass surface were used for calibration and the subsequent measurement is taken slightly above the surface , the localization determination between coverslip and focal plane will perform precisely , while a systematic false coordinate determination will occur for fluorophores above the focal plane .
both sorts of aberrations can be avoided by either adjusting the refractive index of the imaging buffer ( huang et al .
2008a ) or by using objectives that use immersion media with matching refractive index ( glycerol immersion for tissue imaging or water immersion for adherent cells ) . however , the resulting epifluorescence illumination geometry results in lower excitation intensities , higher background , and increased photodamage rendering live - cell super - resolution imaging experiments more difficult .
in addition , when working with glycerol and water objectives , it is crucially important to ensure that the sample is aligned perfectly horizontally .
it was shown that even small tilt angles of less than 1 deteriorate the concentric pattern of the psf ( arimoto and murray 2004 ) .
the center of a psf above or below the focal plane will thus appear to be shifted to the same side , which is a nice indication for coverslip tilt if encountered .
thus , it has to be affirmed that the stage is perfectly leveled before the immersion medium is changed . finally , it has to be considered that the time needed to fully image a large 3d volume is significantly higher compared to one single 2d snapshot because several layers have to be imaged
. unfortunately , with longer imaging times , setups are more prone to drift , which causes severe difficulties for later image processing .
drift compensation is usually performed by fluorescence tracking of non - blinking fiducials . using a hydrogel with a refractive index close to water
, 100  nm fiducials can be stably immobilized and homogeneously distributed in 3d even in close vicinity of cells ( zessin et al .
in order to enable super - resolution imaging of whole living organisms , novel long - term continuous imaging methods with high spatial resolution and minimal photodamage such as selective plane illumination microscopy ( spim ) ( huisken et al .
2004 ) or bessel beam plane illumination ( gao et al . 2012 ; planchon et al . 2011 ) are urgently needed .
ideally , these methods are combined with the enhanced penetration depth and axial sectioning capabilities of two - photon microscopy .
first , realizations of super - resolution imaging with confined two - photon activation ( york et al . 2011 ) ( fig .  3 ) and spim ( cella zanacchi et al . 2011 , 2013 ) have recently been demonstrated . besides the use of photoactivatable fluorescent proteins , so - called caged , non - fluorescent organic dyes that can be activated upon irradiation with uv or ir light , such as rhodamine nn dyes ( belov et al .
a 
 xy ( top ) and xz ( bottom ) maximum - intensity projections of pa - mcherry1vimentin . about 1 million unlinked localizations
insets show further magnification of white rectangles in xy ( lines in xz ) maximum - intensity projection , highlighting individual vimentin fibrils in 60-nm - thick z - slices ( localizations are linked ) .
arrow region of fibril with apparent width < 100  nm . b axial extent of vimentin network with z - location indicated as a color map . for clarity , localizations corresponding to 01.5  m ( top ) and 1.53  m ( bottom ) are shown separately .
arrowheads in a and b indicate a fibril that persists over > 2  m axially .
scale bars 3  m ( a ) , 600  nm ( insets ) , 3  m ( b ) . reproduced from york et al .
( 2011 ) , with permission three - dimensional palm imaging of a vimentin network .
a 
 xy ( top ) and xz ( bottom ) maximum - intensity projections of pa - mcherry1vimentin . about 1 million unlinked localizations was rendered in each view .
insets show further magnification of white rectangles in xy ( lines in xz ) maximum - intensity projection , highlighting individual vimentin fibrils in 60-nm - thick z - slices ( localizations are linked ) .
arrow region of fibril with apparent width < 100  nm . b axial extent of vimentin network with z - location indicated as a color map . for clarity ,
localizations corresponding to 01.5  m ( top ) and 1.53  m ( bottom ) are shown separately .
arrowheads in a and b indicate a fibril that persists over > 2  m axially .
scale bars 3  m ( a ) , 600  nm ( insets ) , 3  m ( b ) . reproduced from york et al .
( 2011 ) , with permission alternatively , photoswitchable fluorescent proteins and superimposed orthogonal standing light waves generating thousands of doughnuts can be used for live - cell super - resolution imaging according to the resolft principle ( standing for reversible saturable optical fluorescence transitions ) .
relying on the use of fluorescent proteins , which can be photoswitched between a stable on- and off - state under substantially lower light intensities than sted microscopy , the method enables super - resolution imaging of large field of views ( > 100  m    100  m ) in less than 1  s with subdiffraction spatial resolution ( chmyrov et al .
for sensitive fluorescence imaging of fixed whole organs , new sample preparation methods such as clarity ( chung and deisseroth 2013 ) and 3disco ( ertrk et al .
2012 ) have recently been developed that make organs transparent to light while keeping them intact , providing a detailed glimpse of their inner structure .
for example , clarity works by removing the fatty tissue that surrounds cells and makes them opaque , while preserving the tissue s structure .
importantly , the tissue can be labeled with different fluorescent probes after a washing step .
the potential of the technique has been demonstrated by imaging the brain of an adult mouse allowing the visualization of neuronal connections and local circuitry deep inside the brain on a cellular level ( chung and deisseroth 2013 ) .
congruously , the next step will be the visualization of neuronal connections inside the brain with molecular resolution applying the above - mentioned method .
single - molecule localization microscopy techniques have the potential to substantially improve our understanding of how cellular structure and function is organized .
currently , they can not compete with deterministic super - resolution imaging techniques such as sted and sim regarding imaging speed and temporal resolution in live - cell experiments , but they can provide single - molecule information about molecular distributions , even giving absolute numbers of proteins present in subcellular compartments .
this ability provides the fascinating possibility to study encoding of cellular function at the molecular level . to produce faster image readout speeds over large fields of view , huang et al .
used next - generation scientific cmos camera technology in combination with localization microscopy ( huang et al .
this enabled an imaging speed of up to 32 frames per second applying an efficient noise correction algorithm .
however , with increasing imaging speed , the photon output of fluorophores also has to be improved .
therefore , new methods have to be developed that increase the localization precision by collecting more fluorescence photons per on - event .
this can be achieved for example using two opposing objective lenses ( ram et al .
alternatively , the photoswitching rates can be decelerated by chemical or physical means , allowing the detection of more fluorescence photons per switching event ( sauer 2013 ) .
for example , triplet - state quenchers such as cyclooctatetraene ( cot ) ( altman et al . 2012 ) can be used to increase the number of fluorescence photons emitted per switching event by optimizing the cycling between the singlet and triplet states before the fluorophore eventually enters a long - lived off - state ( olivier et al .
for some red - absorbing dyes , the fluorescence quantum yield and lifetime could be substantially increased exchanging water by heavy water ( d2o ) ( lee et al .
the magnitude of this effect scales with the emission wavelength , reaching a particularly high value of 2.63-fold for the cyanine dye cy7 ( klehs et al . 2013 ) .
alternatively , the already mentioned caged non - fluorescent organic dyes that can be activated upon irradiation with uv or ir light ( belov et al . 2010 ; grimm et al . 2013 ) could be used in combination with a reducing and oxidizing system ( roxs ) , e.g. , of 1  mm ascorbic acid and 1  mm methyl viologen under oxygen depletion ( vogelsang et al .
roxs increases the photostability of fluorophores by efficient depopulation of the triplet state and recovery of the singlet state ( vogelsang et al .
   super - resolution imaging by single - molecule localization ( localization microscopy ) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution . 
 constant improvements in fluorescent probes , efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy . 
 today , it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes . 
 thus , it exhibits potential to address fundamental questions of cell and developmental biology . here 
 , we briefly introduce the history , basic principles , and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy  ( dstorm ) and summarize key developments and examples of two- and three - dimensional localization microscopy of the last 8  years . 
Introduction
Single-molecule localization microscopy with synthetic fluorophores
Live-cell localization microscopy
3D super-resolution imaging
Technical challenges of 3D localization microscopy
Super-resolution imaging of tissue and organs
Conclusion and perspective