Peter Dedecker

Super‐Resolution Reactivity Mapping of Nanostructured Catalyst Particles

For almost a century, heterogeneous catalysts have been at the heart of countless industrial chemical processes, but their operation at the molecular level is generally much less understood than that of homogeneous catalysts or enzymes. The principal reason is that despite the macroscopic dimensions of solid catalyst particles, their activity seems to be governed by compositional heterogeneities and structural features at the nanoscale. Progress in understanding heterogeneous catalysis thus requires that the nanoscale compositional and structural data be linked with local catalytic activity data, recorded in the same small spatial domains and under in situ reaction conditions. Light microscopy is a recent addition to the toolbox for in situ study of solid catalytic materials. It combines high temporal resolution and sensitivity with considerable specificity in distinguishing reaction products from reagents. However, lens-based microscopes are subjected to light diffraction which limits the optical resolution to 250 nm in the image plane. This resolution is far too limited to resolve the nanosized domains on solid catalysts. Nanometer-accurate localization of single emitters can be achieved by fitting a Gaussian distribution function to the intensity of the observed fluorescence spot (point-spread function, PSF). This method has been used to map out diffusion pathways in mesoporous or clay materials under highly dilute conditions. However, for more concentrated systems, several molecules simultaneously located within a diffraction-limited area cannot be distinguished. Separating the emission of the different fluorescent labels in time, for example by selective photoactivation, solves the problem for imaging of static systems, 13–18] but not when looking at the dynamics of a working catalyst. Herein, we used single catalytic conversions of small fluorogenic reactants, which occurred stochastically on the densely packed active sites of the catalyst, to reconstruct diffraction-unlimited reactivity maps of catalyst particles. As successive catalytic reactions do not overlap in time, one can precisely determine the location of reaction sites that show turnovers at different moments in time, even if the distance between them is only 10 nm (or less, depending on the signal-to-noise ratio), and reconstruct images of catalytically active zones with super-resolution. Although fluorogenic substrates are widely used in singlemolecule enzymology, so far only a few studies have reported single-turnover counting using fluorescence microscopy on solid chemocatalysts. 24, 25] Such studies typically use large polycyclic substrates, which cannot enter the micropores of many heterogeneous catalysts. Hence, similar experiments on microporous materials critically depend on identifying a small reagent that is converted to a product detectable at the single-molecule level. Surprisingly, furfuryl alcohol is such a reagent, and it appears that after acid-catalyzed reaction (see the Supporting Information), the pore-entrapped products are sufficiently fluorescent to be individually observed using a standard microscope equipped with a single excitation source (532 nm diode laser) and sensitive CCD camera (for experimental details, see the Supporting Information). We refer to this novel high-resolution reconstruction method based on catalytic conversion of fluorogenic substrates as NASCA microscopy, or nanometer accuracy by stochastic catalytic reactions microscopy. Figure 1a and b show the concept of NASCA microscopy and a 2D fluorescence intensity image of individual product molecules formed by an acid zeolite crystal, respectively. The fluorescence intensity plot of Figure 1c proves how well the intensity of the individual product molecules allows them to be distinguished from background signals, caused by scatter[*] Dr. M. B. J. Roeffaers, Dr. P. Dedecker, Prof. Dr. J. Hofkens Department of Chemistry, Katholieke Universiteit Leuven Celestijnenlaan 200F, 3001 Heverlee (Belgium) Fax: (+ 32)163-2799 E-mail: johan.hofkens@chem.kuleuven.be

Widely accessible method for superresolution fluorescence imaging of living systems

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule–based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging.

High-Resolution Single-Turnover Mapping Reveals Intraparticle Diffusion Limitation in Ti-MCM-41-Catalyzed Epoxidation†

Microand mesoporous materials offer unique opportunities for catalysis thanks to their large surface area. By introducing active elements inside the pore walls of such materials, a wide range of acid–base or redox catalysts has been developed. For example, incorporation of Ti sites in silicalite resulted in the TS-1 catalyst, which is known for its high performance in the selective oxidation and epoxidation of hydrocarbons. However, the small (0.55 nm) micropores of this catalyst hinder the uptake of larger olefins as substrates for the epoxidation. To circumvent this limitation of TS-1, titanosilicates with larger pores, such as Ti-Beta and Ti-MWW zeolites, have been synthesized. Even mesoporous titanosilicates such as Ti-MCM-41 were developed with the aim of faster diffusion of more bulky substrates towards the inner active sites. 8] MCM-41 materials are characterized by a hexagonal array of pores with a uniform diameter that can be tuned between 1.5 and 10 nm. Despite the relatively large pore size, maximal utilization of the Ti sites in diffusion unlimited conditions remains a major challenge. Typically, TiMCM-41 is prepared in the form of particles with sizes of a few micrometers. It was recently demonstrated that a decrease in particle size to about 100 nm was accompanied with a relevant increase in selectivity and reaction rate for the epoxidation of cyclohexene and cholesterol. 11] It was reasoned that intraparticle diffusion limitations in the mesopores of the large particles hindered an optimal use of the active titanium sites, similarly as previously described for the microporous TS-1 catalyst. The kinetics of a catalytic process are often governed by the interplay between diffusion and reaction. Such insights are classically gathered by macroscopic kinetic experiments, for example, by comparing reaction rates using crystals with different sizes, by varying space velocities of the feed, or by measuring apparent activation energies. Pulsed-field gradient NMR spectroscopy has been used to determine intraparticle diffusion coefficients during catalysis, but this technique is restricted to extremely large particles (> 10 mm) and only yields ensemble-averaged results. Recent technological evolutions in optical microscopy now offer the opportunity to confront these insights with in situ observations for single catalyst particles. The high spatiotemporal resolution (submicrometer and milliseconds) of (single-molecule) fluorescence microscopy has proven to be extremely useful to study catalysis at the level of individual particles or even at the level of individual reaction events, as well as to investigate diffusion processes in mesoporous materials. However, so far these two phenomena, catalytic conversion and diffusion in porous materials, were treated separately in single-molecule studies; no direct information on the interplay between these two processes has been obtained. Moreover optical microscopy is subject to the laws of diffraction, limiting the spatial resolution to a few hundred nanometers, whereas the interesting processes related to catalysis within porous particles typically occur on smaller length scales. The present contribution circumvents the resolution discrepancy by applying a single-turnover-based strategy in fluorescence microscopy to provide diffractionunlimited resolution. This approach allows mapping the catalytic activity with nanometer-scale spatial resolution, that is, in the order of 10 to 30 nm, which is competitive with the most recent, but more complex nanoscopy tools such as PALM, STORM, STED, and related techniques. The high spatial resolution provides the direct visualization, and thus the immediate localization of active sites within individual particles, while recording the catalytic process under realistic conditions. By exploiting the milliseconds time resolution of the technique, the direct evaluation and quantification of the kinetics is within reach with a very limited number of experiments, as will be demonstrated below for epoxidation over Ti-MCM-41. Typical parameters such as the Thiele modulus and the related effectiveness [*] G. De Cremer, E. Bartholomeeusen, Dr. K. Lin, Prof. Dr. P. P. Pescarmona, Prof. Dr. P. A. Jacobs, Prof. Dr. D. E. De Vos, Prof. Dr. B. F. Sels Department of Microbial and Molecular Systems Katholieke Universiteit Leuven Kasteelpark Arenberg 23, 3001 Heverlee (Belgium) Fax: (+ 32)16-321-998 E-mail: bert.sels@biw.kuleuven.be

Localizer: fast, accurate, open-source, and modular software package for superresolution microscopy

Abstract. We present Localizer, a freely available and open source software package that implements the computational data processing inherent to several types of superresolution fluorescence imaging, such as localization (PALM/STORM/GSDIM) and fluctuation imaging (SOFI/pcSOFI). Localizer delivers high accuracy and performance and comes with a fully featured and easy-to-use graphical user interface but is also designed to be integrated in higher-level analysis environments. Due to its modular design, Localizer can be readily extended with new algorithms as they become available, while maintaining the same interface and performance. We provide front-ends for running Localizer from Igor Pro, Matlab, or as a stand-alone program. We show that Localizer performs favorably when compared with two existing superresolution packages, and to our knowledge is the only freely available implementation of SOFI/pcSOFI microscopy. By dramatically improving the analysis performance and ensuring the easy addition of current and future enhancements, Localizer strongly improves the usability of superresolution imaging in a variety of biomedical studies.

Synthesis, Spectroscopy, Crystal Structure, Electrochemistry, and Quantum Chemical and Molecular Dynamics Calculations of a 3-Anilino Difluoroboron Dipyrromethene Dye

An asymmetrically substituted fluorescent difluoroboron dipyrromethene (BODIPY) dye, with a phenylamino group at the 3-position of the BODIPY chromophore, has been synthesized by nucleophilic substitution of 3,5-dichloro-8-(4-tolyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. The solvent-dependent spectroscopic and photophysical properties have been investigated by means of UV-vis spectrophotometry and steady-state and time-resolved fluorometry and reflect the large effect of the anilino substituent on the fluorescence characteristics. The compound has a low fluorescence quantum yield in all but the apolar solvents cyclohexane, toluene, and chloroform. Its emission maxima in a series of solvents from cyclohexane to methanol are red-shifted by approximately 50 nm in comparison to classic BODIPY derivatives. Its oxidation potential in dichloromethane is at ca. 1.14 V versus Ag/AgCl. The absorption bandwidths and Stokes shifts are much larger than those of typical, symmetric difluoroboron dipyrromethene dyes. The values of the fluorescence rate constant are in the (1.4-1.7) x 10(8) s(-1) range and do not vary much between the solvents studied. X-ray diffraction analysis shows that the BODIPY core is planar. Molecular dynamics simulations show that there is no clear indication for aggregates in solution.

Rational Design of Photoconvertible and Biphotochromic Fluorescent Proteins for Advanced Microscopy Applications

Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. IrisFP was recently reported as the first fluorescent protein combining these two types of phototransformations. The introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging. However, the spectroscopic properties of IrisFP are far from being optimal and its tetrameric organization complicates its use as a fusion tag. Here, we demonstrate how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins and develop and characterize a new set of such enhanced optical highlighters derived from mEosFP and Dendra2. We present in particular NijiFP, a promising new fluorescent protein with photoconvertible and biphotochromic properties that make it ideal for advanced fluorescence-based imaging applications.

Spectroscopic Rationale for Efficient Stimulated-Emission Depletion Microscopy Fluorophores

We report a rationale for identifying superior dyes for stimulated-emission depletion (STED) microscopy. We compared the dyes pPDI and pTDI, which displayed excellent photostability in single-molecule spectroscopy. Surprisingly, their photostability and performance in STED microscopy differed significantly. While single pTDI molecules could be visualized with excellent resolution (35 nm), pPDI molecules bleached rapidly under similar conditions. Femtosecond transient absorption measurements proved that the overlap between the stimulated-emission band and the excited-state absorption band is the main reason for the observed difference. Thus, assessment of the excited-state absorption band provides a rational means of dye selection and determination of the optimal wavelength for STED.

DNA fluorocode: A single molecule, optical map of DNA with nanometre resolution

We present a new method for single-molecule optical DNA mapping using an exceptionally dense, yet sequence-specific coverage of DNA with a fluorescent probe. The method employs a DNA methyltransferase enzyme to direct the DNA labelling, followed by molecular combing of the DNA onto a polymer-coated surface and subsequent sub-diffraction limit localization of the fluorophores. The result is a ‘DNA fluorocode’; a simple description of the DNA sequence, with a maximum achievable resolution of less than 20 bases, which can be read and analyzed like a barcode. We demonstrate the generation of a fluorocode for genomic DNA from the lambda bacteriophage using a DNA methyltransferase, M.HhaI, to direct fluorescent labels to four-base sequences reading 5′-GCGC-3′. A consensus fluorocode that allows the study of the DNA sequence at the level of an individual labelling site can be generated from a handful of molecules.

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.

Green-to-red photoconvertible Dronpa mutant for multimodal super-resolution fluorescence microscopy

Advanced imaging techniques crucially depend on the labels used. In this work, we present the structure-guided design of a fluorescent protein that displays both reversibly photochromic and green-to-red photoconversion behavior. We first designed ffDronpa, a mutant of the photochromic fluorescent protein Dronpa that matures up to three times faster while retaining its interesting photochromic features. Using a combined evolutionary and structure-driven rational design strategy, we developed a green-to-red photoconvertible ffDronpa mutant, called pcDronpa, and explored different optimization strategies that resulted in its improved version, pcDronpa2. This fluorescent probe combines a high brightness with low photobleaching and photoblinking. We herein show that, despite its tetrameric nature, pcDronpa2 allows for multimodal subdiffraction imaging by sequentially imaging a given sample using both super-resolution fluctuation imaging and localization microscopy.