Comparison between SOFI and STORM

A straightforward method to achieve super-resolution consists of taking an image sequence of stochastically blinking emitters using a standard wide-field fluorescence microscope. Densely packed single molecules can be distinguished sequentially in time using high-precision localization algorithms (e.g., PALM and STORM) or by analyzing the statistics of the temporal fluctuations (SOFI). In a face-to-face comparison of the two post-processing algorithms, we show that localization-based super-resolution can deliver higher resolution enhancements but imposes significant constraints on the blinking behavior of the probes, which limits its applicability for live-cell imaging. SOFI, on the other hand, works more consistently over different photo-switching kinetics and also delivers information about the specific blinking statistics. Its suitability for low SNR acquisition reveals SOFI's potential as a high-speed super-resolution imaging technique

Radar-based assessment of hail frequency in Europe

In this study we present a unique 10 year climatology of severe convective storm tracks for a large European area covering Germany, France, Belgium and Luxembourg. For the period 2005–2014, a high-resolution hail potential composite of 1×1 km$^{2}$ is produced from two-dimensional radar reflectivity and lightning data. Individual hailstorm tracks as well as their physical properties, such as radar reflectivity along the tracks, were reconstructed for the entire time period using the Convective Cell Tracking Algorithm (CCTA2D). A sea-to-continent gradient in the number of hail days per year is found to be present over the whole domain. In addition, the highest number of severe storms is found on the leeward side of low mountain ranges such as the Massif Central in France and the Swabian Jura in southwest Germany. A latitude shift in the hail peak month is observed between the northern part of Germany, where hail occurs most frequently in August, and southern France, where the maximum amount of hail is 2 months earlier. The longest footprints with high reflectivity values occurred on 9 June 2014 and on 28 July 2013 with lengths reaching up to 500 km. Both events were associated with hailstones measuring up to 10 cm diameter, which caused damage in excess of EUR 2 billion

Multiplane 3D superresolution optical fluctuation imaging

By switching fluorophores on and off in either a deterministic or a stochastic manner, superresolution microscopy has enabled the imaging of biological structures at resolutions well beyond the diffraction limit. Superresolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a conventional widefield microscope. So far, three-dimensional (3D) SOFI has only been demonstrated by sequential imaging of multiple depth positions. Here we introduce a versatile imaging scheme which allows for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. Consequently, the simultaneous acquisition of multiple focal planes reduces the acquisition time and hence the photo-bleaching of fluorescent markers. We demonstrate multiplane 3D SOFI by imaging the mitochondria network in fixed C2C12 cells over a total volume of $65\times65\times3.5 \mu\textrm{m}^3$ without depth scanning.Comment: 7 pages, 3 figure

Spectral Cross-Cumulants for Multicolor Super-resolved SOFI Imaging

Super-resolution optical fluctuation imaging (SOFI) provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Using nth order spatio-temporal cross-cumulants the spatial resolution as well as the sampling can be increased up to n-fold in all three spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a novel multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increase the spectral sampling. The number of discriminable fluorophore species is thus not limited to the number of physical detection channels. Using two color channels, we demonstrate spectral unmixing of three fluorophore species in simulations and multiple experiments with different cellular structures, fluorophores and filter sets. Based on an eigenvalue/ vector analysis we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a novel route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property. This makes simultaneous multiplexed super-resolution fluorescence imaging widely accessible to the life science community interested to probe colocalization between two or more molecular species.Comment: main: 21 pages & 4 figures, supplementary 20 pages & 16 figure

Mapping molecular statistics with balanced super-resolution optical fluctuation imaging (bSOFI)

Super-resolution optical fluctuation imaging (SOFI) achieves 3D super-resolution by computing temporal cumulants or spatio-temporal cross-cumulants of stochastically blinking fluorophores. In contrast to localization microscopy, SOFI is compatible with weakly emitting fluorophores and a wide range of blinking conditions. The main drawback of SOFI is the nonlinear response to brightness and blinking heterogeneities in the sample, which limits the use of higher cumulant orders for improving the resolution. Balanced super-resolution optical fluctuation imaging (bSOFI) analyses several cumulant orders for extracting molecular parameter maps, such as molecular state lifetimes, concentration and brightness distributions of fluorophores within biological samples. Moreover, the estimated blinking statistics are used to balance the image contrast, i.e. linearize the brightness and blinking response and to obtain a resolution improving linearly with the cumulant order. Using a widefield total-internal-reflection (TIR) fluorescence microscope, we acquired image sequences of fluorescently labelled microtubules in fixed HeLa cells. We demonstrate an up to five-fold resolution improvement as compared to the diffraction-limited image, despite low single-frame signal-to-noise ratios. Due to the TIR illumination, the intensity profile in the sample decreases exponentially along the optical axis, which is reported by the estimated spatial distributions of the molecular brightness as well as the blinking on-ratio. Therefore, TIR-bSOFI also encodes depth information through these parameter maps. bSOFI is an extended version of SOFI that cancels the nonlinear response to brightness and blinking heterogeneities. The obtained balanced image contrast significantly enhances the visual perception of super-resolution based on higher-order cumulants and thereby facilitates the access to higher resolutions. Furthermore, bSOFI provides microenvironment-related molecular parameter maps and paves the way for functional super-resolution microscopy based on stochastic switching

SOFI Simulation Tool: A Software Package for Simulating and Testing Super-Resolution Optical Fluctuation Imaging

Super-resolution optical fluctuation imaging (SOFI) allows one to perform sub-diffraction fluorescence microscopy of living cells. By analyzing the acquired image sequence with an advanced correlation method, i.e. a high-order cross-cumulant analysis, super-resolution in all three spatial dimensions can be achieved. Here we introduce a software tool for a simple qualitative comparison of SOFI images under simulated conditions considering parameters of the microscope setup and essential properties of the biological sample. This tool incorporates SOFI and STORM algorithms, displays and describes the SOFI image processing steps in a tutorial-like fashion. Fast testing of various parameters simplifies the parameter optimization prior to experimental work. The performance of the simulation tool is demonstrated by comparing simulated results with experimentally acquired data

Nonlinear Correlation Spectroscopy (NLCS) Theory

These theory files (Mathematica Notebooks) are a complement to the Thesis of M. Geissbuehler. The derivation is too lengthy to be included in this form in the thesis. For further explenations of the derivation please refer to the thesis of M. Geissbuehler "Novel Concepts for Functional High Resolution Microscopy" Ecole Polytechnique Fédérale de Lausanne (EPFL), 2011. http://library.epfl.ch/theses/ The authors keep full copyright of this derivation

Cumulant microscopy

The invention describes a method and a microscopy system for imaging and analysing stochastically and independently blinking point-like emitters. A multiple-order cumulants analysis in conjunction with an established blinking model enables the extraction of super-resolved environment-related parameter maps, such as molecular state lifetimes, concentration and brightness distributions of the emitter. In addition, such parameter maps can be used to compensate for the non-linear brightness and blinking response of higher-order cumulant images - used for example in Super-resolution Optical Fluctuation Imaging (SOFI) - to generate a balanced image contrast. Structures that otherwise would be masked by brighter regions in the conventional cumulant image become visible in the balanced cumulant image. The invention furthermore provides a method for the spectral unmixing of multi- colour samples using spectral cross-cumulants

Optical prism for creating multiple views of an image

Optical prism comprising two external parallel and reflecting surfaces, one internal interface, parallel to said surfaces and acting as light splitter; said prism furthermore comprising an entrance side forming an angle β with respect to said interface and at least two exit sides forming an angle γ with respect to said interface, said external surfaces being located at different distances from said interface, the lengths and the thicknesses of the prism are chosen such that at least one beam entering through said entrance side may cross said interface at least twice and exits through said exit sides

Nonlinear Correlation Spectroscopy (NLCS)

We present a novel concept for optical spectroscopy called nonlinear correlation spectroscopy (NLCS). NLCS analyses coherent field fluctuations of the second and third harmonic light generated by diffusing nanoparticles. Particles based on noncentrosymmetric nonlinear materials such as KNbO3 show a strong second as well as third harmonic response. The method and the theory are introduced and experimental NLCS results in fetal calf serum are presented showing the promising selectivity of this technique for measurement in complex biological environments