Zdeněk Švindrych

ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging

Summary: ThunderSTORM is an open-source, interactive and modular plug-in for ImageJ designed for automated processing, analysis and visualization of data acquired by single-molecule localization microscopy methods such as photo-activated localization microscopy and stochastic optical reconstruction microscopy. ThunderSTORM offers an extensive collection of processing and post-processing methods so that users can easily adapt the process of analysis to their data. ThunderSTORM also offers a set of tools for creation of simulated data and quantitative performance evaluation of localization algorithms using Monte Carlo simulations. Availability and implementation: ThunderSTORM and the online documentation are both freely accessible at https://code.google.com/p/thunder-storm/ Contact: guy.hagen@lf1.cuni.cz Supplementary information: Supplementary data are available at Bioinformatics online.

Three-dimensional super-resolution structured illumination microscopy with maximum a posteriori probability image estimation

We introduce and demonstrate a new high performance image reconstruction method for super-resolution structured illumination microscopy based on maximum a posteriori probability estimation (MAP-SIM). Imaging performance is demonstrated on a variety of fluorescent samples of different thickness, labeling density and noise levels. The method provides good suppression of out of focus light, improves spatial resolution, and allows reconstruction of both 2D and 3D images of cells even in the case of weak signals. The method can be used to process both optical sectioning and super-resolution structured illumination microscopy data to create high quality super-resolution images.

Comparison of image reconstruction methods for structured illumination microscopy

Structured illumination microscopy (SIM) is a recent microscopy technique that enables one to go beyond the diffraction limit using patterned illumination. The high frequency information is encoded through aliasing into the observed image. By acquiring multiple images with different illumination patterns aliased components can be separated and a highresolution image reconstructed. Here we investigate image processing methods that perform the task of high-resolution image reconstruction, namely square-law detection, scaled subtraction, super-resolution SIM (SR-SIM), and Bayesian estimation. The optical sectioning and lateral resolution improvement abilities of these algorithms were tested under various noise level conditions on simulated data and on fluorescence microscopy images of a pollen grain test sample and of a cultured cell stained for the actin cytoskeleton. In order to compare the performance of the algorithms, the following objective criteria were evaluated: Signal to Noise Ratio (SNR), Signal to Background Ratio (SBR), circular average of the power spectral density and the S3 sharpness index. The results show that SR-SIM and Bayesian estimation combine illumination patterned images more effectively and provide better lateral resolution in exchange for more complex image processing. SR-SIM requires one to precisely shift the separated spectral components to their proper positions in reciprocal space. High noise levels in the raw data can cause inaccuracies in the shifts of the spectral components which degrade the super-resolved image. Bayesian estimation has proven to be more robust to changes in noise level and illumination pattern frequency.

Analysis of magnetic response of critical state in second-generation high temperature superconductor YBa2Cu3Ox wire

We have measured magnetic response of second-generation high temperature superconductor YBa2Cu3Ox wire in perpendicular ac field as a function of temperature. We compare complex ac susceptibility to the calculated susceptibility based on the model of Bean’s critical-state response in two-dimensional (2D) disk in perpendicular field and infinitely long cylinder in parallel field. We present a method of linking model and experimental susceptibility. Good agreement of experimental susceptibility with model susceptibility of 2D disk allows noncontact estimation of critical depinning current density and its temperature dependence.

High density 3D localization microscopy using sparse support recovery

Single-molecule localization microscopy methods offer high spatial resolution, but they are not always suitable for live cell imaging due to limited temporal resolution. One strategy is to increase the density of photoactivated molecules present in each image, however suitable analysis algorithms for such data are still lacking. We present 3denseSTORM, a new algorithm for localization microscopy which is able to recover 2D or 3D super-resolution images from a sequence of diffraction limited images with high densities of photoactivated molecules. The algorithm is based on sparse support recovery and uses a Poisson noise model, which becomes critical in low-light conditions. For 3D data reconstruction we use the astigmatism and biplane imaging methods. We derive the theoretical resolution limits of the method and show examples of image reconstructions in simulations and in real 2D and 3D biological samples. The method is suitable for fast image acquisition in densely labeled samples and helps facilitate live cell studies with single molecule localization microscopy.

Analysis of Critical State Response in Thin Films by AC Susceptibility Measurements

Low frequency AC magnetic response of the Nb thin film in perpendicular oscillating applied fields is detected by continuously reading SQUID magnetometer. Harmonic analysis of the temperature dependence of the nonlinear AC susceptibility gives excellent agreement with the susceptibility calculated on basis of model of the hysteretic critical state in 2D disk. This complete analytical model relates to Beans critical state described by the critical depinning current density. We map the experimental and model data and we trace the temperature dependence of the critical current density. The model fits up to temperature above which nonlinear response turns to linear one. This technique is also suitable for characterization of HTS thin films.

Giardia intestinalis mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum

BackgroundMitochondria of opisthokonts undergo permanent fission and fusion throughout the cell cycle. Here, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, in the anaerobic protist parasite Giardia intestinalis, a member of the Excavata supergroup of eukaryotes. The mitosomes have abandoned typical mitochondrial traits such as the mitochondrial genome and aerobic respiration and their single role known to date is the formation of iron–sulfur clusters.ResultsIn live experiments, no fusion events were observed between the mitosomes in G. intestinalis. Moreover, the organelles were highly prone to becoming heterogeneous. This suggests that fusion is either much less frequent or even absent in mitosome dynamics. Unlike in mitochondria, division of the mitosomes was absolutely synchronized and limited to mitosis. The association of the nuclear and the mitosomal division persisted during the encystation of the parasite. During the segregation of the divided mitosomes, the subset of the organelles between two G. intestinalis nuclei had a prominent role. Surprisingly, the sole dynamin-related protein of the parasite seemed not to be involved in mitosomal division. However, throughout the cell cycle, mitosomes associated with the endoplasmic reticulum (ER), although none of the known ER-tethering complexes was present. Instead, the ER–mitosome interface was occupied by the lipid metabolism enzyme long-chain acyl-CoA synthetase 4.ConclusionsThis study provides the first report on the dynamics of mitosomes. We show that together with the loss of metabolic complexity of mitochondria, mitosomes of G. intestinalis have uniquely streamlined their dynamics by harmonizing their division with mitosis. We propose that this might be a strategy of G. intestinalis to maintain a stable number of organelles during cell propagation. The lack of mitosomal fusion may also be related to the secondary reduction of the organelles. However, as there are currently no reports on mitochondrial fusion in the whole Excavata supergroup, it is possible that the absence of mitochondrial fusion is an ancestral trait common to all excavates.

Ecology of Testate Amoebae in the Komořany Ponds in the Vltava Basin

Testate amoeba (TA) assemblages were collected in 2005 from four ponds in Komořany (Prague, Czech Republic). An analysis of seasonal taxonomic variability of TA populations and its correlation with the limnological characteristics of the area (temperature, pH, total organic carbon, nitrogen, phosphorus, heavy metals, etc.) was performed. The predominant genera were Difflugia, Arcella, and Centropyxis. The most significant changes in the TA community occurred between March and July. Arcella genus dominated in March and April; in May, Arcella and Centropyxis genera were present in the same amount; in June, Arcella genus disappeared, and Difflugia genus started to dominate the community. A multivariate redundancy analysis showed statistically significant correlations between the environmental parameters and the composition of the TA community. The results indicate a negative correlation between TA quantities and Ni, Cd, PAH, Mn, As, and Pb. TA were also affected by concentrations of NH4+, NO3−, and P, as well as by temperature variations. The observed correlations between the species composition and environmental parameters can be used in paleoecological interpretations of fossil TA communities. Our results also prove the suitability of TA as water quality indicators in urban areas.

Live Cell Imaging With Spatial Light Modulator-based Optical Sectioning Structured Illumination Microscopy

Structured illumination microscopy (SIM) is a method in fluorescence microscopy which works by acquiring a set of images using widefield detection. Each image in the set is made with a different position of an illumination mask, but with no mask in the detection path [1]. Subsequent image processing is used to produce an optically sectioned image (OS-SIM) [2 4], or an image with resolution beyond the diffraction limit (super-resolution SIM or SR-SIM) [5,6].

Cheese Matrix Microstructure Studied by Advanced Microscopic Techniques

1 . Teagasc Food Research Centre, Moorepark, Fermoy, Co Cork, Republic of Ireland 2. Third Faculty of Medicine, Charles University, Prague, Czech Republic 3. First Faculty of Medicine, Charles University, Prague, Czech Republic 4. Leica Microsystems CMS GmbH, Mannhein, Germany 5. Institute of Scientific Instrument ASCR, Department of Electron Microscopy, Brno, Czech Republic 6. Brno University of Technology, Institute of Physical Engineering, Brno, Czech Republic