Eagle eyes: dSTORM uses conventional photoswitchable fluorescent dyes that can be reversibly cycled between a fluorescent and a dark state by irradiation with light of different wavelengths (see picture). This elegant approach can visualize cellular structures with a resolution of approximately 20 nm, far beyond the diffraction limit of light, without the need of an activator molecule.

Optical microscopy based on waveguide chips significantly reduces the complexity of the entire optical setup, enabling miniaturization by completely removing the excitation light path from the microscope. Instead, waveguides which tightly confine the guided light by total internal reflection due to a high refractive index contrast (HIC) to the surrounding media such as water and cells are used to deliver the illumination light to the sample. The evanescent field on top of the waveguide can be utilized for total internal reflection fluorescence (TIRF) excitation over an almost arbitrarily wide FOV that is intrinsically independent of the detection objective lens and in principle only limited by the waveguide design. Evanescent field excitation using waveguides was first introduced by Grandin et al. 17 , where a slab waveguide was used to generate an evanescent field over the large stretch of the waveguide chip. Slab waveguides (Fig. 1a) Here, we demonstrate waveguide chip-based super-resolution fluorescence imaging by two complementary approaches using ESI and dSTORM. The high intensity in the evanescent field generated by the HIC waveguide material is used for optical switching of fluorophores as required by dSTORM. In addition, the intrinsically multi-mode interference pattern within the waveguide is used to generate fluctuating intensity patterns for ESI. To demonstrate the applicability of waveguide chip-based super-resolution microscopy we visualize the connection of the actin cytoskeleton and plasma membrane fenestrations in liver sinusoidal endothelial cells (LSECs). RESULTS Chip-based single molecule localization microscopyThe performance of chip-based dSTORM is shown by imaging immunostained microtubules in rat LSECs 26 plated directly on the waveguide (Fig. 2a). Measuring the lateral profile along one straight microtubule filament reveals a hollow structure 27 which has been used earlier in localization microscopy as a benchmark sample [28][29][30] , discussed in detail in 31 . This shows a resolution of better than 50 nm ( Fig. 2b), confirmed by full-width-at-half-maximum (FWHM) values, localization precision 32 , and Fourier ring correlation 33,34 (FRC) calculations ( Supplementary Fig. 1). The resolution capability was further investigated by using DNA origami nanorulers that provide markers at (50 ± 5) nm distance as references. These can be clearly resolved in chip-based dSTORM (Fig. 2c,d, Supplementary Fig. 2) which shows a comparable performance to the widely used architecture of an inverted TIRF dSTORM setup (Fig. 2c,d, Supplementary Fig. 3).As an advantage over conventional setups, waveguide chip-based nanoscopy greatly benefits from the fact that the fluorescence excitation is independent of the detection objective lens. As fluorescence is excited by the evanescent field of the waveguide, the technique provides optical sectioning and excellent signal to background ratios at penetration depths below 200 nm ( Supplementary Fig. 4, Supplementary Fig. 5, Video 1), similar to objective-based TIRF...

SummaryIn 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.

We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as beta-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quantification of proteins in the inner mitochondrial membrane with approximately 20 nm optical resolution.

Adlerauge: dSTORM nutzt das reversible Schalten von konventionellen Fluoreszenzfarbstoffen bei der Bestrahlung mit Licht zweier Wellenlängen, um hochaufgelöste Bilder zu erhalten. Die elegante und einfache Methode kann zelluläre Strukturen mit einer Aufösung von ca. 20 nm ohne Aktivatorfarbstoff deutlich unterhalb der Auflösungsgrenze sichtbar machen.

Many commercial as well as custom-built fluorescence microscopes use scientific-grade cameras that represent a substantial share of the instrument’s cost. This holds particularly true for super-resolution localization microscopy where high demands are placed especially on the detector with respect to sensitivity, noise, and also image acquisition speed. Here, we present and carefully characterize an industry-grade CMOS camera as a cost-efficient alternative to commonly used scientific cameras. Direct experimental comparison of these two detector types shows widely similar performance for imaging by single molecule localization microscopy (SMLM). Furthermore, high image acquisition speeds are demonstrated for the CMOS detector by ultra-fast SMLM imaging.

The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.

In order to preserve genomic stability, cells rely on various repair pathways for removing DNA damage. The mechanisms how enzymes scan DNA and recognize their target sites are incompletely understood. Here, by using high-localization precision microscopy along with 133 Hz high sampling rate, we have recorded EndoV and OGG1 interacting with 12-kbp elongated λ-DNA in an optical trap. EndoV switches between three distinct scanning modes, each with a clear range of activation energy barriers. These results concur with average diffusion rate and occupancy of states determined by a hidden Markov model, allowing us to infer that EndoV confinement occurs when the intercalating wedge motif is involved in rigorous probing of the DNA, while highly mobile EndoV may disengage from a strictly 1D helical diffusion mode and hop along the DNA. This makes EndoV the first example of a monomeric, single-conformation and single-binding-site protein demonstrating the ability to switch between three scanning modes.