zeolites are microporous 
 aluminosilicates that are heavily used 
 as solid acid catalysts due to the unique combination of acidic and 
 shape - selective properties . in particular , zeolites 
 are of high importance in catalytic processes in the petrochemical 
 industry .
a challenge in the rational design of zeolite - based catalysts is 
 to control the number , distribution and nature of ( brnsted ) 
 acid sites .
significant progress has been made recently in the direction 
 of tailoring hierarchical zeolite catalysts with remarkably improved 
 catalytic activity , selectivity , and stability .
understanding the synergy between the numerous physicochemical 
 processes taking place in zeolite - based catalysts demands for fundamental 
 insights into the host  guest interactions , preferably at the 
 single particle  single molecule level .
porosity , crystalline 
 defects of the catalyst , guest mobility , as well as solvent effects , 
 can strongly interfere with brnsted acid - catalyzed processes 
 taking place within zeolite catalysts .
nowadays , there is a tendency 
 to perform catalytic reactions in the liquid phase leading to an abundance 
 of various organic guest molecules that influence the proton - transfer 
 processes taking place within the cages and channels of zeolites . 

finally , recent characterization studies at the single particle level 
 emphasize the importance of the underlying structural differences 
 between individual zeolite particles where surface diffusion barriers 
 and crystalline defects may play an important role .
it should be evident that the chemical nature of the above - described 
 processes at the solid  liquid interface of zeolites is poorly 
 studied at the single particle level and relevant time and length 
 scales , partially due to the insensitivity and ensemble averaging 
 nature of traditionally used spectroscopic methods .
consequently , 
 spatially resolved information about the accessibility of brnsted 
 acid sites and the real - time changes in reactivity of a single zeolite 
 particle can not be visualized by these bulk characterization methods . to overcome this limitation , several techniques based on optical 
 microscopy methods
have been developed in recent years for the study 
 of brnsted acid - catalyzed reactions at the single particle level .
hence , selective staining of zeolite acid sites is becoming a popular strategy to study brnsted reactivity 
 of zeolite particles where fluorescent species are generated after 
 an acid catalyzed conversion of molecules , such as furfuryl alcohol , thiophene , and styrene .
after the great success in life sciences , single molecule fluorescence microscopy has become a powerful 
 and 
 sensitive tool to study chemistry , physics and materials science at 
 the molecular scale .
the localization of fluorescent emitters leads 
 to the spatiotemporal resolutions in the orders of 10 nm and 10 ms , 
 provided that the fluorescent species possess sufficient brightness 
 and photostability .
several excellent reviews highlight the high relevance 
 of the single molecule fluorescence approach in many fields of chemistry .
the broad scope of single molecule fluorescence microscopy includes 
 studies of kinetics and mechanisms of stoichiometric chemical reactions , collisions of conjugated polymers in liquid phase , distribution and transport of charge carriers in semiconductors , diffusion in porous materials , localization 
 of plasmonic hotspots , and single - turnover kinetics 
 of solid catalysts . among the above - mentioned fluorogenic reactions ,
the brnsted - acid - catalyzed 
 oligomerization of styrene - based derivatives has received high interest 
 due to the existence of several carbocationic species that are considered 
 as frequently encountered reaction intermediates in zeolite chemistry .
importantly , styrene and its derivatives fit 
 inside the pores of medium and large pore zeolites and oligomerize 
 further to generate carbocationic species that absorb and emit light 
 in the visible region . by changing the substituents on the styrene 
 moiety , it is possible to probe the acidic and shape - selective properties 
 of zeolites .
the oligomerization reactions 
 using 14 different styrene substituents were initially performed on 
 large zeolite h - zsm-5 crystals as model systems and further extended to industrially relevant 
 zeolite - based catalysts , such as fluid catalytic cracking ( fcc ) particles 
 and catalyst bodies based on h - zsm-5 and h - y . in this 
 work ,
we apply for the first time the brnsted - acid - catalyzed 
 oligomerization of styrene derivatives to study the influence of solvent 
 polarity and defect density on the reactivity of zeolite h - zsm-5 at 
 the single particle  single molecule level . using 4-methoxystyrene 
 and 4-fluorostyrene as selective and versatile probes we showcase 
 the importance of pore accessibility and defect chemistry for the 
 selective formation of different reaction intermediates . to accomplish 
 this ,
we have studied two types of zeolite crystals , namely , highly 
 microporous parent zeolite h - zsm-5 crystals and defect - rich , mildly 
 steamed zeolite h - zsm-5 crystals with induced mesoporosity .
based on this 
 study , a spectroscopic marker for nanoscale quality control of zeolite - based 
 materials could be identified .
furthermore , we show that the single 
 molecule oligomerization kinetics and stability of fluorescent carbocations 
 can be further controlled by the polarity of solvent molecules .
large zeolite h - zsm-5 crystals ( dimensions : 
 100  20  20 m ) were used as provided 
 by exxonmobil ( machelen , belgium ) .
tetrapropylammonium as template 
 was removed by calcination at 823 k for 8 h ( 1 k / min ) .
the crystals 
 were subsequently converted into their acidic form by a triple ion 
 exchange with 10 wt % ammonium nitrate ( 99+% acros organics ) at 353 
 k , followed by 6 h calcination at 773 k. the steamed zeolite h - zsm-5 
 was treated at 773 k in a water - saturated n2 flow ( 150 
 ml / min ) for 5 h. the resulting crystals were previously characterized 
 in detail by aramburo et al . an inverted 
 epifluorescence wide - field microscope ( olympus ix-71 ) was used for 
 the single molecule experiments .
both a 532 nm excitation ( with a 
 545 nm long - pass filter ) and a 561 nm excitation ( a 575 nm long - pass 
 filter ) were used .
the excitation light was focused by a 100 
 oil immersion objective lens ( 1.4 na ) and the emitted fluorescence 
 light was detected by a highly sensitive electron multiplying charge - coupled 
 device ( emccd ) camera ( imagem enhanced c9100 - 23b , hamamatsu ) .
the 
 fluorescence microscopy movies were recorded with acquisition times 
 of 30 and 100 ms per frame .
the recorded single 
 molecule fluorescence 
 microscopy movies were analyzed with the localizer software developed by dedecker et al .
the localization of fluorescent events was done by 
 independent segmentation of each frame into emissive spots and background 
 as described by serg et al .
a 
 list of initial emitter positions was determined with subdiffraction 
 limited resolution by fitting a 2-d gaussian using the levenberg 
the emitter - tracking algorithm , as implemented in the localizer , has been used for analysis of the single emitter survival .
h - zsm-5 crystals were 
 loaded on a cover glass that was previously attached in a reactor 
 designed for liquid - phase single molecule microscopy experiments . 
 typically , 1 ml of the solution containing 4-methoxystyrene or 4-fluorostyrene 
 dissolved in either heptane ( spectroscopic grade ) or butanol ( spectroscopic 
 grade , vacuum distilled ) was subsequently added .
the reaction was 
 then monitored by focusing at the surface of the bottom facet or by 
 moving the focus to any provisional focal depth in the axial z - direction up to z = 20  0.2 m . 

prior to measurements , the zeolite crystals were activated at 773 
 k ( 1 k / min ) for 24 h in static air to avoid residual fluorescence .
previous studies of the 
 styrene oligomerization reaction suggested a reaction mechanism for 
 the formation of fluorescent carbocationic species , as outlined in scheme 1 . upon protonation 
 of the neutral styrene molecule ( 1 )
further dimerization leads 
 to the formation of the linear dimeric 1,3-bis(phenyl)-1-butylium 
 cation ( 3 ) , that can transform into neutral diphenyl 
 alkene upon proton transfer to the zeolite framework ( 4 ) , or undergo cyclization to the cyclic dimeric 3-methyl-1,4-phenylindanyl 
 ( indanyl ) carbocation ( 7 ) .
the formation of the linear 
 allylic carbocation ( 5 ) has been the subject of particular 
 interest due to its photoproperties . according to cozens et al . and buurmans et al . , molecules ( 3 ) and ( 4 ) can undergo bimolecular 
 hydride transfer to form an allylic carbocation ( 5 ) . 

the conjugated carbocations ( 5 ) and ( 7 ) 
 are reported in the literature as the stable carbocations that can 
 absorb visible light .
the absorption originating from the trimeric 
 species ( 6 ) was previously proposed , although its precise molecular structure is still unknown .
the absorption maxima 
 for 
 the species ( 57 ) represent the experimentally observed absorption 
 bands shown in supporting information figure 
 s1 .
the absorption and emission properties 
 of the 4-methoxystyrene 
 and 4-fluorostyrene carbocations ( 57 ) under study were determined in a series of uv  vis and confocal 
 fluorescence microscopy experiments , which are summarized in the supporting information figures s1 and s2 .
the 
 oligomerization of both probe molecules resulted in several absorption 
 bands indicated in scheme 1 .
the fluorescence spectra of h - zsm-5 crystals exposed to 
 4-methoxystyrene exhibit two main emission bands at 600 and 
 650 nm ( supporting information figure s2 ) 
 that are assigned to the linear dimeric and trimeric species , respectively . 

similar conclusions can be made with respect to the origin of the 
 fluorescence bands observed for the 4-fluorostyrene oligomers . in 
 this case , the corresponding emission bands were observed at 580 
 and 640 nm . in
what follows , we will use these spectroscopic assignments 
 to explain the observations from our single molecule fluorescence 
 microscopy measurements . under optimal conditions 
 for single molecule imaging , the speed of fluorescent product disappearance 
 due to photobleaching or migration out of focus should match the formation 
 of novel fluorescent oligomers .
the room temperature reaction in pure 
 4-methoxystyrene resulted in a very fast buildup of the fluorescence 
 signal that could not be matched by the photobleaching induced by 
 the excitation laser . under such conditions , for the large majority 
 of the crystals , the fast accumulation of overall fluorescence hampers 
 the continuous detection of single fluorescent reaction products being 
 formed . in this case , the reaction rate needs to be controlled by 
 introducing a solvent , which will be discussed below .
notably , single 
 molecule sensitivity could be reached in solvent - free conditions by 
 studying h - zsm-5 crystals with low brnsted acidity , making use 
 of the large interparticle differences in reactivity ( supporting information figure s3 ) .
the 
 localization of fluorescent reaction products that appeared as the 
 result of stochastic catalytic turnovers was achieved by the nanometer 
 accuracy by stochastic chemical reactions ( nasca ) super - resolution 
 fluorescence method .
the method enables following real - time catalytic 
 events taking place within a catalytic solid . by using nasca approach ,
the 4-methoxystyrene oligomerization was 
 followed with a 30100 ms temporal resolution and individual 
 product molecules were localized using 2-d gaussian fitting procedure 
 with an estimated lateral resolution of about 20 nm ( supporting information figure s4 ) .
this is more than a 10-fold 
 improvement of the diffraction - limited resolution and a remarkable 
 improvement in sensitivity as compared to the uv  vis and fluorescence 
 microscopy studies reported previously .
figure 1 summarizes 
 the results for a parent zeolite h - zsm-5 crystal recorded in solvent - free 
 conditions and at two different focal depths  at the zeolite 
 surface ( figure 1a , z = 0  0.2 m ) and in the middle of the zeolite 
 crystal ( figure 1b , 
 z = 8 m ) .
the reconstructed high - resolution maps in figure 1a , b present the total 
 number of detected single emitting molecules , accumulated independently 
 over 5000 frames (  500 s ) . at the surface , fluorescent reaction 
 product molecules cluster in certain regions ( figure 1a ) , whereas at the middle of the crystal 
 ( z = 8 m ) an active region with more evenly distributed fluorescence 
 events is present ( figure 1b ) .
the scatter plots of individual localizations support 
 this conclusion ( supporting information figure s4 ) .
the low surface reactivity of the studied parent h - zsm-5 
 crystal ( as compared to the more reactive crystals ) points to the 
 acid site inaccessibility due to the extremely high and inhomogeneous 
 surface diffusion resistance caused by zeolite pore blockage .
high - resolution 
 single molecule fluorescence microscopy imaging 
 of fluorescent reaction products recorded after 1 h during 500 
 s of the solvent - free oligomerization of 4-methoxystyrene on a parent 
 zeolite h - zsm-5 crystal ; the excitation wavelength was 561 nm .
( a , b ) 
 accumulated maps of the total number of detected fluorescent events 
 counted separately in each of 5000 frames ( color bar : number of events 
 per 200  200 nm presented on the logarithmic scale , 
 noncorrected for repeated detection of single product molecules ) , 
 ( a ) recorded in the near - surface region of the crystal and ( b ) recorded 
 at z = 8 m from the surface and toward the 
 middle of the crystal .
the edges are not fluorescent due to the orientation 
 of the product molecules with respect to the laser beam and light 
 polarization .
( c ) normalized occurrence of emitters with different 
 survival times , calculated for the surface ( n1(t ) , red ) and z = 8 m 
 ( n2(t ) , blue ) .
( d ) the 
 difference in normalized occurrences for the distributions in ( c ) .
an important observation from figure 1a is that a high 
 number of fluorescent events 
 can be detected in the yellow / red color - coded crystalline domains . 

inspection of the recorded fluorescent movie ( supporting information movie s1 ) reveals high fluorescence 
 movie activity in these regions .
highly photostable fluorescent emitters 
 were found to contribute about 25% to the observed fluorescence activity . 
 to confirm this observation
a particle - tracking 
 algorithm was applied to account for reappearing individual fluorescent 
 molecules within certain spatial and temporal constrains . based on the analysis described in supporting information figure s5
, we considered 
 that the consecutive localizations within 60 nm distance originate 
 from the same molecule ; additionally , the blinking dark states ( off 
 times ) in duration shorter than 2 s were considered to originate from 
 the same molecule .
these values were found to represent well the reappearance 
 of the highly photostable emitters . by using determined localization 
 values
, we have examined the survival 
 times of fluorescent 4-methoxystyrene oligomers for both studied cases 
 in figure 1a and b , 
 respectively .
figure 1c summarizes the normalized distributions of survival times for the 
 fluorescent molecules at the surface ( n1(t ) ) and at z = 8 m ( n2(t ) ) .
we detect a broad range 
 of survival times for fluorescent emitters , covering 3 orders of magnitude 
 from 100 ms to 50 s. the difference (  ) between the normalized 
 distributions n2(t ) 
the result indicates that 
 the fraction of molecules with the lower survival times ( 700 
 ms ) is higher for the movie recorded at z = 8 m , 
 whereas the movie recorded at the surface of the parent zeolite h - zsm-5 
 crystal has a higher contribution of the highly photostable emitters 
 ( survival times > 700 ms ) , supporting information movie s1 .
the measured differences in the survival times of 
 fluorescent products 
 suggest the formation of fluorescent species with different photostability . 

the linear dimeric carbocations ( 5 ) form easily in the 
 zeolite micropores and under highly diluted conditions they should 
 be the predominant species . the trimeric ( 6 ) and cyclic 
 dimeric ( 7 ) carbocations are likely to be formed at more 
 accessible acid sites close to the zeolite outer surface .
defects 
 in the crystalline structure may facilitate further oligomerizations 
 in larger voids where the local porous environment does not induce 
 severe shape constraints .
the formation of the trimeric carbocation species ( 6 ) seems to be kinetically controlled and follows the formation of 
 the linear dimeric carbocation species ( 5 ) , as evidenced from scheme 1 and the uv  vis spectroscopy experiments 
 ( supporting information figure s3 ) . in 
 the single molecule fluorescence microscopy experiments we have used 
 532 and 561 nm nm laser excitation , meaning that we simultaneously 
 excite both the linear dimeric ( 5 ) ( emission at 600 nm ) 
 and the trimeric species ( 6 ) ( emission at 650 nm ) . therefore , 
 the formation of the photostable trimeric fluorescent products close 
 to the surface could explain the observed differences in survival 
 times of individual emitters .
the high stability of the trimeric species 
 is most probably related to their low reactivity , as further oligomerization 
 is unlikely to happen . after applying the correction for the 
 reappearance of individual 
 molecules , it was possible to construct super - resolution nasca reactivity 
 maps by binning individual catalytic turnovers in 200  200 nm areas . to distinguish between dimeric and trimeric species , 

we have chosen a threshold value for survival times of 0.7 s. note 
 that this arbitrary threshold value , as determined from figure 1d , will be only used to illustrate 
 the concept of distinguishing fluorescent species with different lifetimes . 
 in figure 2 , we have 
 spatially resolved the appearance of trimeric fluorescent molecules 
 ( survival times > 0.7 s ) and dimeric fluorescent molecules ( survival 
 times  0.7 s ) .
the dimeric species are formed both at the 
 surface and in the middle of a zeolite crystal ( figure 2a , c ) , whereas the trimeric species were predominantly 
 detected close to the outer surface of the zeolite h - zsm-5 crystal 
 ( figure 2b , d ) .
high - resolution 
 single molecule fluorescence microscopy maps of 
 the short - lived ( < 0.7s ; dimeric ) and long - lived ( > 0.7s ; trimeric ) 
 species , reconstructed based on the survival times of fluorescent 
 molecules shown in figure 1a , b . ( a  d ) accumulated maps of ( a ) dimeric products 
 at the outer surface of the crystal ,
( c ) dimeric products at z = 8 m , and ( d ) trimeric products at z = 
 8 m .
color 
 scale bars denote a total number of dimeric and trimeric emitters 
 per 200  200 nm detected during 500 s. our interpretation can be further supported by 
 inspecting the nanoscopic 
 regions of interest , such as the region highlighted in figure 2a , b and presented as a scatter 
 plot of fluorescent events in figure 3a .
we represent the 
 dimeric and trimeric fluorescent species by the blue and red circles , 
 respectively .
qualitatively , the highly photostable events ( in red ) 
 seem to appear in agglomerates and in close proximity to each other . 
 the less photostable dimeric species ( in blue )
a similar conclusion can be 
 made for the entire maps of dimeric and trimeric species presented 
 in figure 2a , b .
this 
 is a clear indication that the two types of fluorescent reaction products 
 are formed in conditions of different accessibility of acid sites . 

consequently , the local reactivity rates and the kinetics of dimer / trimer 
 formation are largely determined by the accessible brnsted acid 
 sites .
nearest neighbor ( nn ) analysis for the formation of the dimeric 
 ( blue ) and trimeric fluorescent species ( red ) .
( a ) scatter plot of 
 the fluorescent products . ( b ) frequency of the nn distances for the 
 dimeric ( blue ) and trimeric carbocations ( red ) calculated for the 
 entire data set presented in figure 2 .
the spatial arrangement 
 of the dimeric and trimeric fluorescent 
 species can be quantified by calculating the nearest neighbor distances 
 for both types of the formed products .
the results are shown in figure 3b for both short - lived 
 ( dimeric ) and long - lived ( trimeric ) species .
both distributions 
 show a maximum number of counts for the very short nn distances ( < 25 
 nm ) .
a close inspection of the scatter plot in figure 3a confirms that these localizations predominantly 
 originate from clustered events , where the formation of both dimeric 
 and trimeric species is facilitated .
it is not possible to make a 
 clear distinction between both reaction products at distances that 
 are below the resolution limit as the optimized parameters of localization 
 are arbitrary and can not exclude the blinking of individual molecules 
 and statistical distribution in survival times of fluorescent products . 

therefore , within this resolution limit , the measured numbers should 
 be seen as a semiquantitative description of the nn distances .
however , 
 the dimeric species show also a considerable fraction of nn distances 
 that are in the range of 100300 nm ( figure 3b ) , values that are well above the resolution 
 limit .
this is expected from the initial assumption that the dimeric 
 species are more evenly formed throughout the microporous crystalline 
 domains , as qualitatively presented in figure 2 .
the presented analytical approach 
 offers a possibility to study 
 defect - induced chemistry of zeolite zsm-5 by following two distinct 
 types of fluorescent species .
the working hypothesis was further tested 
 by deliberately inducing structural defects into the zeolite framework 
 by dealumination of the parent zeolite h - zsm-5 crystals via mild steaming 
 at 773 k. 4-methoxystyrene appeared to be very sensitive to the induced 
 crystalline defects .
the reactivity of parent and steamed zeolite 
 h - zsm-5 crystals were compared in a series of concentration experiments 
 with a 4-methoxystyrene solution in n - heptane . as discussed earlier , the exposure of the parent h - zsm-5 crystals 
 to liquid ( nondiluted ) 4-methoxystyrene leads to the fast accumulation 
 of fluorescence where single fluorescent reaction products can not 
 be resolved anymore , as illustrated in figure 4a .
significant dilution of 4-methoxystyrene 
 in heptane ( 7.4 mm ) was necessary to observe well - separated and localized 
 fluorescent reaction products in such active parent h - zsm-5 crystals 
 ( figure 4b ) . reducing 
 the concentration of 4-methoxystyrene also slows down the intracrystalline 
 diffusion with fluorescent oligomers predominantly detected near the 
 zeolite surface ( 0.5 m ) . as expected , the reactivity 
 decreased for more than 1 order of magnitude when the crystals were 
 exposed to a 1.5 mm 4-methoxystyrene solution ( figure 4c ) . in both cases ,
the individual single 
 molecule products could be clearly resolved by the localization algorithm 
 ( red circles in figure 4 ) . however , when the steamed h - zsm-5 crystals were exposed to a 1.5 
 mm 4-methoxystyrene solution , an overwhelming amount of fluorescent 
 carbocation products was detected making it impossible to efficiently 
 resolve individual molecules ( figure 4e ) . reducing
the concentration 10-fold did not resolve 
 this problem ( figure 4f ) ; only further 50-fold dilution of 4-methoxystyrene to 30 m 
 made it possible to observe isolated fluorescent species ( figure 4 g ) .
probing the accessibility 
 of parent ( top ) and steamed zeolite h - zsm-5 
 crystals ( bottom ) .
wide - field fluorescence micrographs of the brnsted 
 acid - catalyzed oligomerization of 4-methoxystyrene at different molar 
 concentrations in heptane ( bottom right ) ; images are recorded at the 
 surface of the parent ( a  c ) and steamed ( e  g ) zeolite 
 h - zsm-5 crystals by using a 532 nm excitation ; ( a ) was measured in 
 solvent - free conditions ( 7.4 m ) .
the red circles indicate the efficiency 
 of the localization algorithm to detect individual fluorescent events . 

( d , h ) cartoons illustrating the formation of dimeric and trimeric 
 fluorescent species in parent ( d ) and steamed ( h ) zeolite h - zsm-5 
 crystals .
the light blue dots indicate the formation of linear dimeric 
 carbocations , while the red dots represent the bulkier trimeric carbocations , 
 formed close to defects / mesopores .
our previous work has shown that the measured turnover frequencies 
 of the parent and mildly steamed h - zsm-5 crystals differ by a factor 
 of 25 .
the variation in fluorescence 
 response with styrene derivatives is hence probably related to the 
 formation of different , more photostable oligomers at the crystal 
 defects giving rise to a pronounced increase in background fluorescence , 
 as postulated earlier in the text .
two distinct 
 absorption bands initially appear for both types of zeolite h - zsm-5 
 crystals at 565 and 590 nm ( supporting information figure s3 ) .
we attribute these absorption bands 
 to the existence of two isomers of the allylic dimeric carbocation 
 ( 5 ) .
the absorption bands at 490 , 530 , and 640 nm appear 
 later in the spectrum , but faster on steamed zeolite h - zsm-5 crystals 
 and parent crystals with visible crystalline defects ( supporting information figure s3 ) .
the absorption band at 640 nm has been attributed 
 to the trimeric carbocationic species ( 6 ) . despite the lack of evidence for their precise molecular structure(s ) , 
 the extent of conjugation and late appearance of these species supports 
 their assignment to the trimeric carbocationic species . the additional 
 nonradiative relaxation pathways and energy transfer between the different 
 fluorescent species
are expected to be minimized in highly diluted 
 solutions of the reactant and large product - to - product distances 
the observed behavior underlines 
 the importance of pore accessibility 
 and the shape - selective role of framework in the formation of bulkier 
 reaction products .
for instance , the highly crystalline parent zeolite 
 h - zsm-5 crystals typically have a si - rich layer of up to 100 nm in 
 thickness , that is characterized by a very low acid site density ( si / al 
 ratio of 160 ) .
this layer also seems to act 
 as a shape - selective surface diffusion barrier hindering the formation 
 of the bulkier fluorescent products at the very surface , as depicted 
 in figure 4d .
the local 
 diffusion properties are drastically changed with the formation of 
 larger mesoporous defects , which can 
 facilitate the formation of bulkier oligomers that are apparently 
 more photostable ( red dots depicted in figure 4h ) .
the low concentrations of 4-methoxystyrene 
 that are used in the 
 experiments ( < 1 mm ) favor the predominant formation of the linear 
 dimeric carbocations in the micropores . considering the high density 
 of brnsted acid sites
, the formation of the highly photostable 
 trimeric carbocationic species can be used to selectively probe more 
 accessible brnsted acid sites at the crystalline defects .
a 
 significant fraction of the parent zeolite crystals appeared to have 
 intracrystalline defects ( supporting information figure s6 ) , such as cracks and mesoporous defects , which are inherent 
 to the sample preparation , such as synthesis , activation and calcination . 

notably , the important role of crystalline defects in base - leaching 
 of zeolite h - zsm-5 was reported recently on nanosized zeolite h - zsm-5 
 crystals , making the single molecule visualization of the defects 
 a very relevant topic for zeolite chemistry . in a similar fashion
, the developed method can be used to qualitatively 
 visualize interparticle differences in the uptake of probe molecules . 

these become obvious for the steamed zeolite h - zsm-5 crystals where 
 different crystals can have strikingly different reactivity ( supporting information figure s6 ) .
we speculate 
 that the interparticle differences arise due to structural heterogeneities , 
 such as amorphous deposition or framework distortions that may strongly 
 influence local surface mass transport and reactivity .
the selective formation of trimeric carbocations 
 can be maximized 
 in the conditions of extremely low concentration of the selected styrene 
 derivatives .
the high - resolution maps of a steamed h - zsm-5 crystal , 
 measured at four different focal depths , are shown in figure 5a .
the majority of the photostable 
 reaction products were formed in a 23 m thick surface 
 layer of the zeolite crystal ( supporting information movie s2 ) .
previous high - resolution sem studies of steamed zeolite 
 h - zsm-5 crystals detected near - surface mesopores with diameters of 
 550 nm . however , almost no damage of the crystalline structure 
 ( i.e. , mesoporosity ) was observed in h - zsm-5 crystals further away 
 from the outer surface .
this observation is 
 in accordance with the 3-d reactivity profiles of furfuryl alcohol 
 in the steamed zeolite h - zsm-5 .
therefore , 
 it is reasonable to suggest that the photostable emitters in figure 5 reveal the locations 
 of the accessible mesopores . even though the concentration of 4-methoxystyrene 
 in this experiment ( 30 m ) corresponds to only 17 molecules 
 per 100 nm of solvent , the kinetics of the product formation 
 is elevated in mesopores by specific local adsorption and diffusion 
 pathways . at z = 2 m below the zeolite surface , 

a higher number of photostable and highly localized fluorescent oligomers 
 was detected ( figure 5b ) .
the detected emitters did not show significant diffusion and 
 consecutive localizations are within the localization precision of 
 our method ( 20 nm ) , as shown in figure 5c .
the recorded fluorescent reaction products 
 show high photostability with occasional blinking , as evidenced from 
 their fluorescence intensity trajectories ( figure 5d ) . by using single molecule fluorescence 
 microscopy with 30 ms acquisition time it is possible to track the 
 temporal changes in fluorescence for every individual fluorescent 
 product .
high - resolution fluorescence microscopy imaging of defect - induced 
 reactivity of 4-methoxystyrene in a steamed h - zsm-5 crystal , recorded 
 in 30 m solution in heptane by using a 532 nm excitation with 
 30 ms acquisition time .
( a ) accumulated images of photoactivity for 
 different focal depths denoted in the top left corner , recorded after 
 1 h from the start of the reaction .
the color scale bar represents 
 the number of detected fluorescent styrene oligomers per 200  
 200 nm during 150 s of the recording time .
( b ) high resolution 
 scatter plot indicating the location of fluorescent products formed 
 in a steamed zeolite h - zsm-5 crystal at z = 2  
 0.3 m .
( c , d ) corresponding ( c ) scatter plot and ( d ) fluorescence 
 intensity trajectory of the single molecule indicated with the red 
 arrow in ( b ) . the background intensity in ( d )
is around 10k . in attempt to optimize the 
 reaction rates for the highly reactive 
 4-methoxystyrene
, we have tested the oligomerization reaction in 1-butanol . 
 whereas 4-methoxystyrene reacted readily at the brnsted acid 
 sites in a 1.5 mm ( 0.02 vol % ) solution in n - heptane
, 
 hardly any fluorescent events were observed in a highly concentrated 
 2.5 m ( 33.3 vol % ) solution in 1-butanol .
the spatial reactivity distribution 
 was clearly different with respect to n - heptane , 
 with extremely low reactivity mostly observed at the edges of the 
 h - zsm-5 crystals ( figure 6a ) .
oligomerization of 4-methoxystyrene ( 2.5 m in 1-butanol ) at parent 
 zeolite h - zsm-5 crystals .
( a  c ) wide - field fluorescence micrographs 
 recorded 30 min after start of the reaction by using a 561 nm excitation . 

the sequence illustrates the effect of photobleaching , ( a ) at the 
 beginning of recording , ( b ) after 20 s , and ( c ) after 130 s ; ( d ) recorded 
 after 24 h , at z = 4 m .
( e , f ) accumulated 
 high - resolution reactivity maps recorded at ( e ) the middle of the 
 zeolite crystal , ( f ) the edge of the zeolite crystal . (
g ) integrated 
 total fluorescence background intensity and the number of detected 
 fluorescent events , as measured for the crystal in ( f ) .
color scale 
 bars denote the total number of turnovers per 200  200 nm detected during 500 s. acquisition time in ( a  d ) was 
 100 ms .
scale bars are 5 m . the initial fluorescence signal , recorded 30 min after initiating 
 the reaction , vanished after 130 s of continuous illumination due 
 to fast photobleaching of individual fluorescent molecules ( figure 6a  c ) .
after 
 24 h , we observed significant penetration and accumulation of the 
 fluorescent oligomers along the sinusoidal pores of the zeolite h - zsm-5 
 crystal tips , also in the deeper regions of the crystals ( figure 6d ) . the observed 
 reactivity and directionality of the fluorescent products in 1-butanol 
 is a result of anisotropic diffusivity and reactivity of 4-methoxystyrene 
 along the straight and sinusoidal channels .
reported higher 
 accessibility of the tip regions via the straight pores ( running along 
 the b lattice parameter ) open to the surface , as 
 illustrated in supporting information figure 
 s2 .
the fluorescence response can be 
 described in terms of low single molecule reactivity presented in figure 6e and f ; the analysis 
 of the average fluorescence background signal and the number of detected 
 fluorescent events showed that both quantities decayed fast in time 
 ( figure 6 g ) .
we concluded 
 that the rate of the oligomerization reaction in 1-butanol was very 
 low and that only the initially formed fluorescent products were photobleached .
the change in the reaction medium affects not only the reactivity , 
 but also the survival times of fluorescent products .
the analysis 
 of the survival times of the 4-methoxystyrene - derived carbocationic 
 species in n - heptane and 1-butanol is shown in figure 7a .
although the total 
 number of detected fluorescent events is 2 orders of magnitude higher 
 for the fluorescent reaction products in n - heptane , 
 the measured exponential decays of the survival time frequencies clearly 
 indicate a lower photostability for the fluorescent reaction products 
 in 1-butanol ; all exponential time constants appeared to be smaller 
 in the latter case .
( a ) survival times of fluorescent reaction products during 
 the 
 oligomerization of 4-methoxystyrene with parent zeolite zsm-5 crystals 
 in 1-butanol ( 2.5 m , red circles ) and n - heptane ( 7.4 
 mm , blue circles ) .
exponential time constants for the fits are 0.055 
  0.001 s ( 27% contribution ) , 0.22  0.01 s ( 45% ) , and 1.1 
  0.1 s ( 28% ) for the emitters in n - heptane 
 ( blue fit ) and 0.015  0.05 s ( 18% ) , 0.13  0.01 s ( 64% ) 
 and 0.8  0.2 s ( 18% ) for the emitters in 1-butanol ( black fit ) . 

( b , c ) examples 
 of the fluorescence intensity trajectories of individual fluorescent 
 molecules in ( b ) n - heptane and ( c ) 1-butanol .
( d , e ) 
 schematic of the styrene oligomerization reaction in ( d ) n - heptane and ( e ) 1-butanol ; the interaction of a solvent molecule 
 with a brnsted acid site is highlighted . by inspecting the fluorescence behavior of individual emitters , 
 it is possible to give a more qualitative description of their interactions 
 with the zeolite framework . in a parent zeolite crystal ,
the fluorescent 
 reaction products are confined within the micropores and the measured 
 survival times provide information about framework - emitter interactions . 

manual inspection of a large number of individual fluorescence trajectories 
 confirmed the results presented in figure 7a .
the fluorescent reaction products in n - heptane on average have longer survival times , as illustrated 
 for the fluorescence intensity trajectory of an emitter in figure 7b . in stark contrast , 
 fluorescent reaction products in 1-butanol predominantly show short 
 survival times , often within the acquisition time of 100 ms .
this 
 is illustrated in figure 7c , where one fluorescence trajectory consists of several short 
 intensity bursts possibly originating from one fluorescent molecule .
the experimentally observed results can be explained by the competing 
 role of solvent molecules as they may adsorb on the brnsted 
 acid sites , as depicted in figure 7d , e .
the significant drop in the reaction rates and 
 the photostability of individual fluorescent reaction products are 
 due to the strong chemisorption of 1-butanol at brnsted acid 
 sites .
this solvent is known to occupy 96% of the zeolite volume with 
 about 2 molecules per acid site ( measured for a si / al ratio of 72 ) .
the close packing of 1-butanol molecules is 
 due to their strong interaction with brnsted acid sites , but 
 also due to favorable interactions of the hydrocarbon chain with the 
 zeolite wall ( figure 7e ) . as a result ,
the formation and thermodynamic 
 stability of the benzylic and dimeric carbocationic species is severely 
 inhibited by the presence of the polar solvent molecules .
heptane , 
 on the other hand , has a similar pore filling as 1-butanol for the 
 same reasons of c  h / zeolite wall interactions , but does not interact strongly with the brnsted acid 
 sites , which can additionally promote the formation of carbocationic 
 species ( figure 7d ) . 

similar tests in 1,4-dioxane did not yield any visible photoproducts . 
 in line with our results ,
yoon et al . have reported in their bulk 
 experiments a full conversion of styrene in dichloromethane over zeolite 
 h  y powder , and no evidence of reaction in tetra - hydrofuran 
 or acetonitrile .
the presented single molecule 
 fluorescence microscopy approach 
 can be successfully extended to other substituted styrene probe molecules . 
 in this way , a broad range of reaction conditions can be achieved 
 considering the large difference in reactivity between differently 
 substituted derivatives . the trends in reactivity , as determined by 
 the stabilization of the initial benzylic carbocation , can be predicted 
 by hammett parameters . in a comparative study ,
stavitski et al . have shown 
 that the presence of different functional groups may affect the net 
 stabilization of the benzylic species .
for example , fluoro- substituent has less stabilizing effect than 
 methoxy- group in the para position , resulting in 
 the much lower reactivity of 4-fluorostyrene at room temperature . 

however , it should be noted that the reactivity trends in zeolites 
 may strongly deviate from the hammett equation , as the confinement 
 effects may play important role .
the 
 fluorescent reaction products for 4-fluorostyrene can be detected 
 at room temperature by using single molecule fluorescence microscopy . 
 using this less - reactive probe molecule similar solvent effect
is 
 observed , whereas imaging of the crystalline interior is readily possible 
 in solvent - free conditions ( supporting information figure s8 ) .
single molecule fluorescence microscopy can be considered 
 as a quantitative 
 technique provided that conditions for efficient localization of fluorescent 
 molecules are met . by quantifying the individual fluorescent reaction 
 products it was possible to estimate the averaged turnover frequencies 
 recorded in the studied oligomerization reactions . for simplicity 

the results are summarized in figure 8 . a concentration range for successful single molecule 
 fluorescence imaging of zeolites will depend on the reactivity of 
 a probe molecule ( i.e , the substituent effect ) and the polarity of 
 a solvent ( i.e. , the solvent effect ) . in both cases the oligomerization 
 rate
is determined by the tendency of the reactant to form the initial 
 benzylic and dimeric carbocations . the polarity of a solvent and its 
 interaction with brnsted acid sites plays a crucial role in 
 the carbocation formation , as illustrated for the reactions in n - heptane and 1-butanol .
the oligomerization of both reactants 
 is significantly inhibited when n - heptane is replaced 
 with 1-butanol ( figure 8a ) .
( a ) reactivity of 4-methoxystyrene ( in red ) and 4-fluorostyrene 
 ( in blue ) as probe molecules for single molecule chemical imaging 
 in n - heptane ( circles ) and 1-butanol ( rhombi ) .
the 
 squares represent 99% pure 4-fluorostyrene . averaged turnover frequencies 
 of the parent and steamed zeolite h - zsm-5 crystals ( denoted as  s  ) 
 were recorded close to the outer surface of the single crystals ; x - axis denotes the concentration of the probe molecules 
 in the solvents .
the blue and red lines indicate the lowest and highest 
 turnover rates that can be imaged by the presented single molecule 
 approach . the linearity shown is provided only as a guidance for the 
 observed trends in reactivity .
( b ) frames indicating the density of 
 the individual emitters and an approximate level of reactivity for 
 corresponding turnover frequencies .
the oligomerization of 4-methoxystyrene in zeolite h - zsm-5 
 proceeds 
 much faster than in the case of 4-fluorostyrene , as visible from the 
 comparison of their reactivity in heptane ( figure 8a ) .
a successful single molecule fluorescence 
 microscopy imaging of zeolite h - zsm-5 crystals reacting with 4-methoxystyrene 
 can be performed at a high dilution of the reactant ( 1010 m in n - heptane ) . 

in contrast , the experiments with 4-fluorostyrene can routinely be 
 performed with the pure reagent ( 8.3 m ) for at least 30 min , even 
 for the highly acidic zeolite h - zsm-5 crystals .
the maximum 
 recorded rate of fluorescent reaction product formation 
 is in the order 10 events per m s. if all al atoms are considered catalytically equally active , this 
 number approximately translates into the turnover frequencies in the 
 order of 10 s ( red line in figure 8a ) .
the low turnover 
 numbers of 10 s ( blue line figure 8a ) indicate the detection 
 rate of < 0.01 events m s. based on the presented results , it is clear that a subset of the 
 acid sites are probed with our approach and this number is determined 
 by the frequency and density of detected fluorescent events ( figure 8b ) .
the approach 
 does not determine the total concentration of acid sites , but rather 
 relies on the accurate comparison of reaction rates recorded in a 
 certain time frame , which ultimately determine the rate constants 
 in the low - concentration regimes .
by 
 using single molecule fluorescence microscopy in combination 
 with the selective oligomerization of styrene derivatives , we were 
 able to visualize for the first time host- and guest - dependent proton 
 transfer processes in zeolite zsm-5 crystals at the single particle 
the formation of linear dimeric and trimeric styrene 
 carbocations , characterized by different photostabilities , can be 
 used to selectively probe highly accessible acid sites at crystalline 
 defects .
interestingly , the oligomerization rate was found to be very 
 sensitive to changes in the polarity of the solvent medium and chemical 
 nature of the para - substituent of the styrene moiety . 

both changes lead to several orders of magnitude difference in reactivity 
 of zeolite crystals . with this approach in hand
, we have established 
 a probe concept that is very sensitive to chemical changes in the 
 nature of the zeolite host as well as of the guest species and that 
 is capable of resolving nanoscopic details in the reactivity of zeolite 
 crystals with the ultimate single molecule sensitivity .
 visualizing proton - transfer 
 processes at the nanoscale is essential 
 for understanding the reactivity of zeolite - based catalyst materials . 
 in this work , 
 the brnsted - acid - catalyzed oligomerization of 
 styrene derivatives was used for the first time as a single molecule 
 probe reaction to study the reactivity of individual zeolite h - zsm-5 
 crystals in different zeolite framework , reactant and solvent environments . 
 
 this was accomplished via the formation of distinct dimeric and trimeric 
 fluorescent carbocations , characterized by their different photostability , 
 as detected by single molecule fluorescence microscopy . 
 the oligomerization 
 kinetics turned out to be very sensitive to the reaction conditions 
 and the presence of the local structural defects in zeolite h - zsm-5 
 crystals . 
 the remarkably photostable trimeric carbocations were found 
 to be formed predominantly near defect - rich crystalline regions . 
 this 
 spectroscopic marker offers clear prospects for nanoscale quality 
 control of zeolite - based materials . 
 interestingly , replacing n - heptane with 1-butanol as a solvent led to a reactivity 
 decrease of several orders and shorter survival times of fluorescent 
 products due to the strong chemisorption of 1-butanol onto the brnsted 
 acid sites . 
 a similar effect was achieved by changing the electrophilic 
 character of the para - substituent of the styrene 
 moiety . based on the measured turnover rates we have established a 
 quantitative , single turnover approach to evaluate substituent and 
 solvent effects on the reactivity of individual zeolite h - zsm-5 crystals . 
Introduction
Experimental Section
Results and Discussion
Conclusions