Steve Wolter

Direct stochastic optical reconstruction microscopy with standard fluorescent probes

Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ∼20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.

Live-cell dSTORM with SNAP-tag fusion proteins

To the Editor: Since the publication of our Correspondence1 and the reply of Joung et al.2, we improved zinc-finger nuclease (ZFN) modular assembly. ZFNs are artificial restriction enzymes3 composed of tailor-made zinc-finger DNA-binding arrays and the FokI nuclease domain, which can induce site-specific mutations4 and large chromosomal deletions5 in higher eukaryotic cells and organisms. To reduce the number of ZFNs that need to be synthesized to identify a functional enzyme, we previously compared zinc fingers with equivalent DNA-binding specificity and chose ones that are often found in functional ZFNs4. Based on that analysis, we recommended 37 zinc fingers for use in genome editing4. Here we tested 33 of these fingers, which collectively recognize 39 of 64 three–base-pair subsites (15 GNN subsites and 24 non-GNN subsites, where G is guanine and N is any base; Supplementary Table 1). We prepared a combinatorial library of two-finger modules (Supplementary Methods) consisting of 1,089 (33 × 33 zinc fingers) two-finger arrays, each linked to the FokI nuclease domain. This library allowed us to construct three-finger or four-finger ZFNs in a single subcloning step (Supplementary Fig. 1). Previously, assembling hundreds of ZFNs to target a single gene had taken at least several weeks because up to four repetitive

Real-Time Computation of Subdiffraction-Resolution Fluorescence Images

In the recent past, single‐molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction‐resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high‐precision fluorophore localization and subsequent visualization of found fluorophore positions in a super‐resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non‐maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real‐time data processing is possible and that super‐resolution imaging with organic fluorophores of cellular structures with ∼20 nm optical resolution can be completed in less than 10 s.

rapidSTORM: accurate, fast open-source software for localization microscopy

Besides being versatile and fast, rapidSTORM is easy to use, deploy, inspect and extend. It is open source and based on widespread, mature, portable and open technologies such as C++, the GNU tool chain and wxWidgets. A graphical user interface and user manual allow quick acquaintance. Automated prerelease validation and wide use on a range of biological targets ensure reliable results. rapidSTORM is regularly updated, and both source code and compiled packages are available from our website at http://www.superresolution.biozentrum.uni-wuerzburg.de/.

The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging

Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. Since most of these methods rely on the transfer of the majority of fluorophores to a non-fluorescent dark state and precise localization of separated fluorescent fluorophores, the photophysical properties of photoswitchable fluorophores have to be carefully controlled. The achievable resolution and herewith the ability to resolve a structural feature depends not only on the brightness of the fluorophores, but also on the labeling density and on the stability or lifetime of the non-fluorescent dark state. Here, we discuss how the ratio of off- and on-switching of a fluorophore affects resolution. We compare experimental data with theoretical simulations and present a strategy to customize photoswitching characteristics to achieve optimal optical resolution.

Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters.

Background: Syntaxin forms nano-sized clusters at the plasma membrane whose inner organization is unknown. Results: In the clusters, the density of proteins gradually decreases toward the periphery. Conclusion: Syntaxin reactivity is influenced by its location within the clusters. Significance: dSTORM imaging combined with cluster analysis significantly contributes to understanding membranal protein distribution and cluster organization. Key synaptic proteins from the soluble SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters, and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and nonclustered molecules at the membrane of PC12 cells with single-molecule precision. Direct stochastic optical reconstruction microscopy images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The nonclustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Although syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical, and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule-based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.

Measuring localization performance of super-resolution algorithms on very active samples.

Super-resolution fluorescence imaging based on single-molecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as three-dimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer.

Investigating Cellular Structures at the Nanoscale with Organic Fluorophores

Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.

Subdiffraction-Resolution Fluorescence Microscopy of Myosin–Actin Motility

Subdiffraction-resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of approximately 20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video-like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore-labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of approximately 100 Hz, only 100 consecutive frames are sufficient to generate a single high-resolution image of moving actin filaments with a lateral resolution of approximately 30 nm. A video-like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to approximately 0.18 microm s(-1), observed strong bending and disruption of filaments as well as locally immobile fragments.

Single-molecule Photoswitching and Localization

Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ∼20 nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.