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Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (Soluble NSF Attachment Protein Receptor) proteins involved in exocytosis, in fixed cells have resulted in images with high spatial resolution, at the expense of dynamical information. Here, we have imaged localized fluorescence bursts of DRONPA-fused SNAP-25 molecules in live chromaffin cells by Total Internal Reflection Fluorescence (TIRF) imaging. We find that this method allows tracking protein cluster dynamics over relatively long times (∼20 min.), partly due to the diffusion into the TIRF field of fresh molecules, making possible the simultaneous identification of cluster size, location and temporal evolution. The results indicate that the DRONPA-fused SNAP-25 clusters display rich dynamics, going from staying constant to disappearing and reappearing in specific cluster domains within minutes.

Diffraction-unlimited fluorescence imaging allows the visualization of intact, strongly heterogeneous systems at unprecedented levels of detail. Beyond the acquisition of detailed pictures, increasing efforts are now being focused on deriving quantitative insights from these techniques. In this work, we review the recent developments on sub-diffraction quantization that have arisen for the various techniques currently in use. We pay particular attention to the information that can be obtained but also the practical problems that can be faced, and provide suggestions for solutions or workarounds. We also show that these quantitative metrics not only provide a way to turn raw data into hard statistics but also help to understand the features and pitfalls associated with sub-diffraction imaging. Ultimately, these developments will lead to a highly standardized and easily applicable toolbox of techniques, which will find widespread application in the scientific community.

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Escherichia coli (E. coli) cells replicate their genome once per cell cycle to pass on genetic information to the daughter cells. The SeqA protein binds the origin of replication, oriC, after DNA replication initiation and sequesters it from new initiations in order to prevent overinitiation. Conventional fluorescence microscopy studies of SeqA localization in bacterial cells have shown that the protein is localized to discrete foci. In this study we have used photo-activated localization microscopy (PALM) to determine the localization of SeqA molecules, tagged with fluorescent proteins, with a localization precision of 20-30 nm with the aim to visualize the SeqA subcellular structures in more detail than previously possible. SeqA-PAmCherry was imaged in wild type E. coli, expressed from plasmid or genetically engineered into the bacterial genome, replacing the native seqA gene. Unsynchronized cells as well as cells with a synchronized cell cycle were imaged at various time points, in order to investigate the evolution of SeqA localization during the cell cycle. We found that SeqA indeed localized into discrete foci but these were not the only subcellular localizations of the protein. A significant amount of SeqA-PAmCherry molecules was localized outside the foci and in a fraction of cells we saw patterns indicating localization at the membrane. Using quantitative PALM, we counted protein copy numbers per cell, protein copy numbers per focus, the numbers of foci per cell and the sizes of the SeqA clusters. The data showed broad cell-to-cell variation and we did not observe a correlation between SeqA-PAmCherry protein numbers and the cell cycle under the experimental conditions of this study. The numbers of SeqA-PAmCherry molecules per focus as well as the foci sizes also showed broad distributions indicating that the foci are likely not characterized by a fixed number of molecules. We also imaged an E. coli strain devoid of the dam methylase (Δdam) and observed that SeqA-PAmCherry no longer formed foci, and was dispersed throughout the cell and localized to the plasma membrane more readily. We discuss our results in the context of the limitations of the technique.

The ultrafast excited state dynamics of the fluorescent protein Kaede has been investigated by employing time resolved fluorescence and transient absorption. Upon irradiation of its neutral state, the protein undergoes an efficient conversion to a state that fluoresces at longer wavelengths. The molecular basis of the photoconversion involves an expansion of the chromophore π-conjugation by formal β-elimination but details of the reaction pathway remain subject to debate. Based on the kinetics observed in experiments on the protein sample in both H2O and D2O buffers, we suggest that a light-initiated cleavage mechanism (20 ps) could take place, forming the neutral red state in which the red chromophore resides. Excitation of the neutral red form results in the formation of the red anionic species via two Förster resonance energy transfer (FRET) channels. FRET between red neutral and red anionic forms occurs within the tetramer with time constants of 13.4 ps and 210 ps. In contrast to literature proposals no ESPT was observed.

Stochastic optical fluctuation imaging (SOFI) is a superresolution imaging technique that uses the flickering of fluorescent labels to generate a microscopic image with a resolution better than what the diffraction limit allows. Its adaptation towards fluorescent protein-labeled samples (called photoconversion SOFI or pcSOFI) allows for a straightforward and easily accessible way of generating superresolution images. In this protocol, we will discuss how so-called "smart labels," and specifically the reversibly switchable fluorescent proteins, have opened doors towards superresolution imaging in general and we provide a protocol on how to perform pcSOFI on HeLa cells expressing human β-actin labeled with the reversibly photoswitchable fluorescent protein Dronpa.

The key events in the light-induced switching mechanism of the photochromic green fluorescent protein Padron have been investigated by employing femtosecond fluorescence up-conversion, femtosecond transient absorption, and time-correlated single photon counting techniques. In contrast to Dronpa, excitation of protein's neutral state at 395 nm triggers an efficient and complex photoswitching to a dark state whereas irradiation with 495 nm light reverses the protein to its initial state restoring the bright fluorescence. On the basis of the kinetics observed upon irradiation of the chromophore in the protonated state, we suggest that the switching mechanism consists of a light-initiated excited state process (presumably ESPT) with a time constant of 1 ps producing an unstable intermediate state, tentatively assigned to the excited state of the cis-anionic form, that is followed by a cis- to trans- isomerization (14.5 ps) forming the trans-anionic state in which the dark chromophore resides. In the trans-state, the protonation equilibrium strongly favors the anionic form. Consequently, upon excitation of the formed anionic species a trans-cis isomerization of the chromophore was found to occur with a time constant as fast as 5.2 ps switching the chromophore quantitatively to the bright (anionic) state.

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.

The crystal structure of the on-state of PDM1-4, a single-mutation variant of the photochromic fluorescent protein Dronpa, is reported at 1.95 Å resolution. PDM1-4 is a Dronpa variant that possesses a slower off-switching rate than Dronpa and thus can effectively increase the image resolution in subdiffraction optical microscopy, although the precise molecular basis for this change has not been elucidated. This work shows that the Lys145Asn mutation in PDM1-4 stabilizes the interface available for dimerization, facilitating oligomerization of the protein. No significant changes were observed in the chromophore environment of PDM1-4 compared with Dronpa, and the ensemble absorption and emission properties of PDM1-4 were highly similar to those of Dronpa. It is proposed that the slower off-switching rate in PDM1-4 is caused by a decrease in the potential flexibility of certain β-strands caused by oligomerization along the AC interface.

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.

Venus is a recently developed, fast maturating, yellow fluorescent protein that has been used as a probe for in vivo applications. In the present work the photophysical characteristics of Venus were analyzed spectroscopically at the bulk and single molecule level. Through time-resolved single molecule measurements we found that single molecules of Venus display pronounced fluctuations in fluorescence emission, with clear fluorescence on- and off-times. These fluorescence intermittencies were found to occupy a broad range of time scales, ranging from milliseconds to several seconds. Such long off-times can complicate the analysis of single molecule counting experiments or single-molecule FRET experiments., (This journal is © The Royal Society of Chemistry and Owner Societies 2012)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

Photoswitchable fluorophores play an essential role in super-resolution fluorescence microscopy, including techniques such as photoactivated localization microscopy (PALM). A determining factor in the precision of the images generated by PALM measurements is the photon numbers that can be detected from the fluorophores. Dronpa is a reversibly photoswitchable fluorescent protein that has been successfully used in PALM experiments. The number of photons per switching cycle that can be acquired for Dronpa depends on its off-switching rate, limiting the number of photons that can be recorded. In this study we report our discovery that the tetrameric ancestor of Dronpa, 22G, shows slower switching, and develop a mutant that displays switching kinetics between those of Dronpa and 22G. We show that the kinetics of the photoswitching are strongly related to self-association of the protein, supporting our view of dynamic flexibility as determining in the photoswitching. Similarly we find that higher-resolution PALM images can be acquired with slower-switching proteins due to their higher number of emitted photons per switching cycle.

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.

The reversibly photoactivatable green fluorescent protein analog Dronpa holds great promise as a marker for various new cellular imaging applications. Using a replica exchange method which combines both Hamiltonian and temperature exchanges, the ground-state dynamics of Dronpa and two mutants with increased switching kinetics, Val157Gly and Met159Thr, were compared. The dominant chromophore state was found to be the cis isomer in all three proteins. The simulation data suggest that both mutations strongly increase the chromophore flexibility and cis-trans isomerization rate. We identify three key amino acids, Val157, Met159, and Phe173, which are able to impede the bottom hula-twist transition path, depending on their position and rotameric state. We believe our insights will help to understand the switching process and provide useful information for the design of new variants with improved fluorescence properties.

By using single molecule fluorescence spectroscopy we have investigated the excitation energy migration processes occurring in a series of cyclic porphyrin arrays bearing a close proximity in overall architectures to the LH2 complexes in purple bacterial photosynthetic systems. We have revealed that the conformational heterogeneity induced by the structural flexibility in large cyclic porphyrin arrays, which provides the nonradiative deactivation channels as an energy sink or trap, reduces significantly the energy migration efficiency. Our study provides detailed information on the energy migration efficiency of the artificial light-harvesting arrays at the single molecule level, which will be a guideline for future applications in single molecular photonic devices in the solid state.

The fast and reversible on/off switching of the fluorescence emission of the GFP-like fluorescent protein Dronpa has attracted considerable interest for applications in subdiffraction imaging. In this paper we study the use of a donut-mode beam in combination with two more overlapping laser beams to increase the imaging resolution through selective switching to the nonfluorescent photoswitched state. We devise and run a series of numerical simulations to determine suitable photophysical parameters of prospective, thermally stable photoswitchable molecules, in terms of photoswitching quantum yields, fatigue resistance, and possible presence of transient nonfluorescent states. Many of our findings are applicable to other measurements that make use of donut beams, and these guidelines can be used in the synthesis and screening of novel photoswitchable compounds. We experimentally demonstrate the possibility of obtaining increased resolution by making use of the efficient and thermally stable Dronpa photoswitching, using equipment that is commonly available.

The photophysical properties and photoswitching scheme of the reversible photoswitchable green fluorescent protein-like fluorescent proteins Dronpa-2 and Dronpa-3 were investigated by means of ensemble and single-molecule fluorescence spectroscopy and compared to those of the precursor protein Dronpa. The faster response to light and the faster dark recovery of the new mutants observed in bulk also hold at the single-molecule level. Analysis of the single-molecule traces allows us to extract the efficiencies and rate constants of the pathways involved in the forward and backward switching, and we find important differences when comparing the mutants to Dronpa. We rationalize our results in terms of a higher conformational freedom of the chromophore in the protein environment provided by the beta-can. This thorough understanding of the photophysical parameters has allowed us to optimize the acquisition parameters for camera-based sub-diffraction-limit imaging with these photochromic proteins. We show that Dronpa and its mutants are useful for fast photoactivation-localization microscopy (PALM) using common wide-field microscopy equipment, as individual fluorescent proteins can be localized several times. We provide a new approach to achieve fast PALM by introducing simultaneous two-color stroboscopic illumination.

The single-molecule fluorescence blinking behavior of the organic dye Atto647N in various polymer matrixes such as Zeonex, PVK, and PVA as well as aqueous media was investigated. Fluorescence blinking with off-times in the millisecond to second time range is assigned to dye radical ions formed by photoinduced electron transfer reactions from or to the environment. In Zeonex and PVK, the measured off-time distributions show power law dependence, whereas, in PVA, no such dependence is observed. Rather, in this polymer, off-time distributions can be best fitted to monoexponential or stretched exponential functions. Furthermore, treatment of PVA samples to mild heating and low pressure greatly reduces the frequency of blinking events. We tentatively ascribe this to the removal of water pockets within the polymer film itself. Measurements of the dye immobilized in water in the presence of methylviologen, a strongly oxidizing agent, reveal simple exponential on- and off-time distributions. Thus, our data suggest that the blinking behavior of single organic molecules is sensitive to their immediate environment and, moreover, that fluorescence blinking on- and off-time distributions do not inherently and uniquely obey a power law.

The interactions between single molecules and three-dimensional donut modes in fluorescence microscopy are discussed based on the vector diffraction theory of light.We find that the use of donut modes generated from a linearly polarized laser beam can yield information about the orientation of immobilized single molecules, allowing for their use in orientational imaging. While fairly insensitive over a range of orientations, this technique is seen to be very sensitive for the subset of orientations where the transition dipole of the molecule is oriented close to the optical axis of the microscope and perpendicular to the input polarization. In a second part of the paper we discuss the impact of the molecular orientation on the resolution improvement in STED microscopy. We find that, even for circularly polarized excitation light, the expected resolution improvement depends on the orientation of the molecule relative to the optical axis of the microscope.

Reversible photoswitching of individual molecules has been demonstrated for a number of mutants of the green fluorescent protein (GFP). To date, however, a limited number of switching events with slow response to light have been achieved at the single-molecule level. Here, we report reversible photoswitching characteristics observed in individual molecules of Dronpa, a mutant of a GFP-like fluorescent protein that was cloned from a coral Pectiniidae. Ensemble spectroscopy shows that intense irradiation at 488 nm changes Dronpa to a dim protonated form, but even weak irradiation at 405 nm restores it to the bright deprotonated form. Although Dronpa exists in an acid-base equilibrium, only the photoinduced protonated form shows the switching behavior. At the single-molecule level, 488- and 405-nm lights can be used to drive the molecule back and forth between the bright and dim states. Such reversible photoswitching could be repeated >100 times. The response speed to irradiation depends almost linearly on the irradiation power, with the response time being in the order of milliseconds. The perfect reversibility of the Dronpa photoswitching allows us to propose a detailed model, which quantitatively describes interconversion among the various states. The fast response of Dronpa to light holds great promise for following fast diffusion or transport of signaling molecules in live cells.

The percutaneous access device (PAD) is used to connect an external drive unit to the Kantrowitz CardioVad (KCV), a cardiac assist device for the treatment of chronic heart failure. The PAD conveys pneumatic power from a drive unit to the implanted blood pump and an electrocardiogram signal from the myocardium to the drive unit. The device-tissue interface of the PAD is precoated with autologous fibroblasts cultured from a skin sample of the intended recipient. This preparation demonstrated long-term stability in animals and was adopted for use in patients receiving the KCV. The KCV is activated intermittently, and the drive unit may be connected and disconnected by the patient, which subjects the PAD to frequent manipulation. To date, the PAD has been implanted in nine patients ranging in age from 41 to 74 years. Implant times ranged from 2 to 458 days, for a total of 1082 days, of which 557 days were in an outpatient setting. Two patients experienced episodes of infection that did not originate from the PAD-tissue interface. This feasibility study demonstrated that (1) the PAD is stable and infection resistant in long-term ambulatory patients, (2) the PAD withstood the challenge of daily manipulation (the KCV is turned on and off electively), and (3) PADs can be replaced, if necessary.

The mechanical auxiliary ventricle (MAV) is an avalvular, inseries left ventricular assist device (LVAD) comprised of a 60 cc Biomer blood pump implanted in the descending thoracic aorta, a percutaneous access device (PAD), and an external pneumatic drive. In four calves the MAV was implanted and activated intermittently for 192, 249, 423, and 785 days, respectively; no anticoagulants were administered. When the animals were killed, the implants were retrieved, and autopsies, including gross, light microscopic, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) examinations, were performed. The MAV was securely attached to the aorta in every animal. A thin, even smooth, pseudoneointimal layer (PNI) contiguous with the aorta at the level of the suture line covered the MAV's blood-contacting surface. Areas of calcification beneath the PNI, expected in growing animals with implanted pumping devices, were noted. At autopsy there was no evidence of thrombosis or infection in the regions of the blood pump or the PAD. These findings confirm the calves' benign histologic response to the MAV, and together with the results of hematologic and biochemiccal studies, and bench tests of system components being reported elsewhere, indicate the system's readiness for limited clinical investigation in selected patients with chronic congestive failure.