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Methods that fuse multiple localization microscopy images of a single structure can improve signal-to-noise ratio and resolution, but they generally suffer from template bias or sensitivity to registration errors. We present a template-free particle-fusion approach based on an all-to-all registration that provides robustness against individual misregistrations and underlabeling. We achieved 3.3-nm Fourier ring correlation (FRC) image resolution by fusing 383 DNA origami nanostructures with 80% labeling density, and 5.0-nm resolution for structures with 30% labeling density., Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., ImPhys/Quantitative Imaging, ImPhys/Imaging Physics

ImPhys/Imaging Physics, Applied Sciences

Quantification in localization microscopy with reversibly switchable fluorophores is severely hampered by the unknown number of switching cycles a fluorophore undergoes and the unknown stoichiometry of fluorophores on a marker such as an antibody. We overcome this problem by measuring the average number of localizations per fluorophore, or generally per fluorescently labeled site from the build-up of spatial image correlation during acquisition. To this end we employ a model for the interplay between the statistics of activation, bleaching, and labeling stoichiometry. We validated our method using single fluorophore labeled DNA oligomers and multiple-labeled neutravidin tetramers where we find a counting error of less than 17% without any calibration of transition rates. Furthermore, we demonstrated our quantification method on nanobody- and antibody-labeled biological specimens., ImPhys/Imaging Physics, Applied Sciences

Contains fulltext : 127296.pdf (Publisher’s version ) (Open Access), Podosomes are multimolecular mechanosensory assemblies that coordinate mesenchymal migration of tissue-resident dendritic cells. They have a protrusive actin core and an adhesive ring of integrins and adaptor proteins, such as talin and vinculin. We recently demonstrated that core actin oscillations correlate with intensity fluctuations of vinculin but not talin, suggesting different molecular rearrangements for these components. Detailed information on the mutual localization of core and ring components at the nanoscale is lacking. By dual-color direct stochastic optical reconstruction microscopy, we for the first time determined the nanoscale organization of individual podosomes and their spatial arrangement within large clusters formed at the cell-substrate interface. Superresolution imaging of three ring components with respect to actin revealed that the cores are interconnected and linked to the ventral membrane by radiating actin filaments. In core-free areas, alphaMbeta2 integrin and talin islets are homogeneously distributed, whereas vinculin preferentially localizes proximal to the core and along the radiating actin filaments. Podosome clusters appear as self-organized contact areas, where mechanical cues might be efficiently transduced and redistributed. Our findings call for a reevaluation of the current "core-ring" model and provide a novel structural framework for further understanding the collective behavior of podosome clusters.

We report on a new generation, commercial prototype of a programmable array optical sectioning fluorescence microscope (PAM) for rapid, light efficient 3D imaging of living specimens. The stand-alone module, including light source(s) and detector(s), features an innovative optical design and a ferroelectric liquid-crystal-on-silicon (LCoS) spatial light modulator (SLM) instead of the DMD used in the original PAM design. The LCoS PAM (developed in collaboration with Cairn Research, Ltd.) can be attached to a port of a(ny) unmodified fluorescence microscope. The prototype system currently operated at the Max Planck Institute incorporates a 6-position high-intensity LED illuminator, modulated laser and lamp light sources, and an Andor iXon emCCD camera. The module is mounted on an Olympus IX71 inverted microscope with 60-150X objectives with a Prior Scientific x,y, and z high resolution scanning stages. Further enhancements recently include: (i) point- and line-wise spectral resolution and (ii) lifetime imaging (FLIM) in the frequency domain. Multiphoton operation and other nonlinear techniques should be feasible. The capabilities of the PAM are illustrated by several examples demonstrating single molecule as well as lifetime imaging in live cells, and the unique capability to perform photoconversion with arbitrary patterns and high spatial resolution. Using quantum dot coupled ligands we show real-time binding and subsequent trafficking of individual ligand-growth factor receptor complexes on and in live cells with a temporal resolution and sensitivity exceeding those of conventional CLSM systems. The combined use of a blue laser and parallel LED or visible laser sources permits photoactivation and rapid kinetic analysis of cellular processes probed by photoswitchable visible fluorescent proteins such as DRONPA., Quantitative Imaging Group, Electrical Engineering, Mathematics and Computer Science