High-Resolution Single-Molecule Fluorescence Imaging of Zeolite Aggregates within Real-Life Fluid Catalytic Cracking Particles**

Fluid catalytic cracking (FCC) is a major process in oil refineries to produce gasoline and base chemicals from crude oil fractions. The spatial distribution and acidity of zeolite aggregates embedded within the 50–150 μm-sized FCC spheres heavily influence their catalytic performance. Single-molecule fluorescence-based imaging methods, namely nanometer accuracy by stochastic chemical reactions (NASCA) and super-resolution optical fluctuation imaging (SOFI) were used to study the catalytic activity of sub-micrometer zeolite ZSM-5 domains within real-life FCC catalyst particles. The formation of fluorescent product molecules taking place at Brønsted acid sites was monitored with single turnover sensitivity and high spatiotemporal resolution, providing detailed insight in dispersion and catalytic activity of zeolite ZSM-5 aggregates. The results point towards substantial differences in turnover frequencies between the zeolite aggregates, revealing significant intraparticle heterogeneities in Brønsted reactivity.

Fluid catalytic cracking (FCC) is am ajor industrial process to convert crude oil into gasoline and valuable hydrocarbons, such as propylene. [1][2][3] In this catalytic process 50-150 mmsized spherical particles are used, which contain an acidic zeolite embedded in amatrix of clay,silica, and alumina. The zeolite components with acidic properties,b eing either zeolite YorZSM-5, play acrucial role in the overall catalytic cracking properties. [4][5][6] Thea cidity of zeolite domains changes during catalyst activation and aging, but ad etailed characterization of the acidity distribution within as ingle FCC catalyst particle has proven to be extremely difficult due to their intrinsic chemical and structural complexity.
More recently,F CC catalyst particles have been the subject of detailed studies at the single-particle level.
Confocal fluorescence microscopy (CFM) in combination with acid-catalyzed staining reactions was used to visualize the dispersion of zeolites Yand ZSM-5 domains within FCC particles. [7][8][9] By integrating afluorescence microscope within at ransmission electron microscope Karreman et al. have been able to correlate the changes in acidity as probed with fluorescence microscopy,w ith structural changes and damage. [10] Using the CFM approach, complemented by the results of X-ray microscopy techniques, [11] it is now possible to evaluate the aging process of the catalyst because of the metal deposition (poisoning) and steaming (dealumination). Unfortunately,C FM cannot resolve sub-micrometer zeolite domains and does not provide the quantitative information about catalytic activity of individual zeolite aggregates.
Single-molecule fluorescence microscopy has emerged as avery sensitive and informative technique in life sciences,and more recent efforts have directed to extend this powerful approach to the field of materials science,i ncluding but not limited to homogeneous and heterogeneous catalysis. [12][13][14][15][16][17] Thes ingle-molecule sensitivity of fluorescence detection is used to enhance the spatial resolution down to the nanometer scale,making it an ideal candidate for assessing the reactivity of catalytic solids.The technique has been used for studies on well-defined heterogeneous catalysts;f or example,s inglemolecule kinetics of nanoparticle catalysts, [18][19][20] and highresolution imaging of catalytic activity in porous heterogeneous catalysts. [21][22][23] Here we report the first application of single-molecule fluorescence microscopyand the required analysis methods to quantitatively study Brønsted-catalyzed reactivity of hierarchically structured, multi-component and industrially applied FCC catalyst particles,c ontaining zeolite ZSM-5 as . . the active cracking phase.T he presented approach can be widely applied to other complex catalyst materials like granulated particles that suffer from an elevated background luminescence.W ea lso anticipate that this approach will broaden the scope of fluorogenic reactions that can be used for quantitative reactivity mapping since it possesses less stringent demands on the fluorophore properties.S ince individual catalytic events no longer need to be isolated, measurements with higher catalytic turnover densities can be used for quantitative analysis leading to more information in the same experiment time.
To selectively study the zeolite domains within the FCC catalyst particles we have used the oligomerization of furfuryl alcohol as ap robe reaction. This reaction is catalyzed by Brønsted acid sites present within zeolites or metal-organic frameworks and can be used for catalytic reactivity mapping with single turnover sensitivity. [22,24,25] As chematic of the method is shown in Figure 1a.T he 532 nm laser light can efficiently excite fluorescent oligomers that are catalytically formed from non-fluorescent furfuryl alcohol molecules ( Figure 1b;f or the experimental details and the mechanism of the probe reaction see sections S1-3 in the Supporting Information).
As the fluorescent products are formed exclusively on Brønsted acid sites,t heir fluorescence can be used for the 3D localization of zeolite ZSM-5 domains embedded within the matrix material of asingle FCC particle ( Figure 1c). The individual fluorescent reaction products are detected with an EM-CCD camera (Figure 1d). At ypical fluorescence intensity trajectory of an individual hotspot is shown in Figure 1e. It was found that the characteristic survival time of fluorescent products before photobleaching is typically smaller than 0.3 s ( Figure 1f). Therefore,w em ay conclude that fluctuations in the fluorescence intensity happening at specific locations at the second time scale are mostly caused by the formation of new fluorescent product molecules on acid sites of individual zeolite domains. Figure 2a illustrates four isolated fluorescent product molecules localized by fitting their point spread functions (PSF) with a2 DGaussian (see section S4 in the Supporting Information). This method of localizing stochastic catalytic turnovers in heterogeneous catalysis is commonly known as NASCAm icroscopy,that is,nanometer accuracyb ys tochastic chemical reactions microscopy. [22] Recording afluorescence movie (see Movie S1 in the Supporting Information) allows reconstructing of ah igh-resolution NASCAi mage based on the precise localization of individual reaction events,a si llustrated in Figure 2b.H owever, NASCAm easurements typically require wellcontrolled reaction conditions with ag ood signal-to-noise ratio and therefore are challenging for industrial catalysts with high structural complexity and intrinsic background fluorescence.I deally,acomplementary method that can operate under less stringent conditions and in aw ider range of concentrations is necessary to support the results of the NASCAanalysis.
In order to complement the results of the NASCAm ethod we opted to use the superresolution optical fluctuation imaging (SOFI) analysis-a method that is developed recently for imaging of cellular structures in experiments with low signal-to-noise ratio. [26][27][28][29] This method relies on statistical analysis of temporal fluctuations in consecutive fluorescence images to provide essentially background-free, contrast-enhanced images with improved resolution in all three dimensions. [26] In our experiments,i ndependent stochastic fluctuations of fluorescent emitters appear as aresult of the constant formation, diffusion and photobleaching of fluorophores taking place at zeolite domains.T he recorded signal in the SOFI images is not trivially related to the recorded fluorescence intensity. [26,28] However, it is proportional to the local concentration of fluorophores,p rovided that they exhibit similar emission properties and fast fluctuations of the fluorescence signal-conditions that are met in our experiment. As an example,F igure 2c shows an accumulated SOFI image

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Chemie reconstructed based on an identical movie as the NASCA image in Figure 2b.A no verlay of the images based on the two methods in Figure 2d indicates that the brightest regions in the SOFI image are indeed the ones where most of the fluorescent events and thus catalytic turnovers are recorded.
Using SOFI images as ar eference for the stochastic fluctuations of fluorescence signal, we applied ab inary thresholding procedure in order to statistically analyze the size of the catalytically most active zeolite domains (Figure 2e). Thep rocedure separates the fluorescence domains with high intensity in SOFI images from the ones with low signal by setting at hreshold value based on as ystematic analysis of domain brightness and size (see section S5). The reactivity of an individual FCC catalyst particle is monitored for three different focal depths,c lose to the surface (Z = 0 AE 0.3 mm), for Z = 1 mm, and Z = 2 mmb elow the surface (Figure 3). Reconstructed SOFI images are presented in Figure 3. Thea nalysis of the SOFI signal points towards notable attenuation of both the excitation and emission by matrix additives of the FCC particle (section S6). Therefore, in our further analysis only the outer regions of similar brightness are investigated for reliable comparison and subsequent thresholding analysis.A fter the thresholding procedure,t he binary images of highly active fluorescent domains (Figure 3, thresholding) are segmented further to account for pixel artifacts and attenuation of the fluorescence in the inner parts (Figure 3, segmentation). Figure 3shows the obtained histograms of the size distribution of zeolite domains.M ost of the zeolite domains are well-dispersed in the analyzed 3D volume and within the 2D projection size of 0.2 mm 2 ,w hich corresponds to spherical particles of about 500 nm in diameter.Asimilar distribution of zeolite particle sizes was recently reported in aC FM study,s upporting the correctness of our approach. [9] However,t he analysis of smaller zeolite domains is hardly possible with CFM because of its intrinsic limitations in resolution and sensitivity (section S7). This observation highlights the real strength of the fluorescence-based single molecule approach reported in this work. TheN ASCAm ethod can typically localize single catalytic turnovers with 20 nm resolution, while the more broadly applicable SOFI method routinely achieves aspatial resolution of 120 nm.
Theo btained NASCAa nd SOFI maps of reactivity indicate that zeolite ZSM-5 domains embedded within as ingle FCC particle differ in their overall fluorescence activity,which suggests potential differences in their catalytic reactivity.T he illustration of this observation is presented in Figure 4a.T he fluorescence intensity trajectories in these regions confirm dependence of SOFI intensity from the catalytic activity of individual domains,such as the regions of high (Figure 4b), medium (Figure 4c), and low catalytic activity (Figure 4d). To support the quantitative aspect of the SOFI analysis we have analyzed 65 zeolite domains localized within a66mm 2 region of interest from the SOFI image in Figure 3a and attempted to correlate their averaged SOFI intensities with corresponding catalytic turnover frequencies.T urnover frequencies of zeolite domains were calculated based on two separate quantification methods. Thef irst method is based on the previously described Thec omparison of both methods is presented in Figure 4e.T he average brightness of the domains in aS OFI image seems to be in ag ood approximation proportional to the corresponding numbers of detected catalytic turnovers. Thed eviation from the linear trend is ac onsequence of inherent properties of the applied methods.F or instance, brighter emitters will have ah igher contribution to the intensity of aS OFI image. [26] Figure 4e suggests that the zeolite domains may differ significantly in their SOFI brightness,thus catalytic reactivity.The average turnover frequency of highly active zeolite domains is calculated to be around five events per second per square micrometer at the studied . .

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Communications experimental conditions.T his is approximately an order of magnitude difference in activity when compared to the less reactive zeolite ZSM-5 domains.Most probably,the origin of this difference in reactivity is related to differences in framework aluminium content of zeolite domains or local accessibility differences.
In conclusion, single-molecule fluorescence methods proved to be avery sensitive tool to localize with high spatial resolution and single turnover sensitivity the activity of acidic zeolite domains within ah ierarchically structured, industrially used FCC catalyst particle.Inaddition, the explored SOFI analysis emerged as ap ractical method that can bridge the inherent deficiencies of confocal and single-molecule localization methods,e specially in experiments with pronounced intraparticle differences in reactivity and low signal-to-noise ratio. Using the developed analysis approach one could imagine aw ide range of in-depth characterization studies of various complex multi-component catalytic materials.   Figure 4a.e)Catalytic turnover rate as af unction of average brightness in the SOFI image, calculated for 65 individual zeolite domains. White circles:v alues calculated based on fluorescence intensity trajectories;black line is the best linear fit (R 2 = 0.88, k = 0.038 AE 0.002). Red circles:v alues calculated based on Gaussian fitting procedure;r ed line is the best linear fit (R 2 = 0.69, k = 0.040 AE 0.004).