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Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high-resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high-resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD-TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD-TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup.

Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has been considered as an actin bundling protein because its N-terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross-linked actin filaments, here we analyzed if full length-ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F-actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C-terminal ABD, we exclude that ITPKA cross-links actin filaments by simultaneous F-actin binding with two different ABDs. Instead, stimulated-emission-depletion-microscopy and measurement of InsP3Kinase activity give evidence that that N-terminal ABD-homodimers of ITPKA bind to F-actin while the monomeric C-termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3Kinase activity is doubled and the product of InsP3Kinase activity, Ins(1,3,4,5)P4, inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3Kinase domain substantially contributes to this process. © 2015 Wiley Periodicals, Inc.

The capture of circulating tumor cells (CTCs) is still a challenging application for microfluidic chips, as these cells are rare and hidden in a huge background of blood cells. Here, different microfluidic ceiling designs in regard to their capture efficiency for CTCs in model experiments and more realistic conditions of blood samples spiked with a clinically relevant amount of tumor cells are evaluated. An optimized design for the capture platform that allows highly efficient recovery of CTCs from size-based pre-enriched samples under realistic conditions is obtained. Furthermore, the viability of captured tumor cells as well as single cell recovery for downstream genomic analysis is demonstrated. Additionally, the authors' findings underline the importance of evaluating rational design rules for microfluidic devices based on theoretical models by application-specific experiments.

Higher order laser modes, mainly called doughnut modes (DMs) have use in many different branches of research, such as, bio-imaging, material science, single-molecule microscopy, and spectroscopy. The main reason of their increasing importance is that recently, the techniques to generate well-defined DMs have been refined or rediscovered. Although their potential is still not fully utilized, their specifically polarized field distribution gives rise to a wide field of applications. They are contributing to complete our fundamental knowledge of the optical properties of single emitting species, such as molecules, nanoparticles, or quantum dots, offering insight into the three-dimensional dipole or particle orientation in space. The perfect zero intensity in the focus center qualifies some DMs for stimulated emission depletion (STED) microscopy. For the same reason, they have been suggested for trapping and tweezing applications.

Lasermoden mit ringformigem Strahlprofil, die zumeist als Doughnut-Moden (DMs) bezeichnet werden, finden Anwendung in unterschiedlichen Forschungsbereichen wie den Bio- und Materialwissenschaften oder der Einzelmolekulmikroskopie und -spektroskopie. Der Hauptgrund fur die wachsende Bedeutung der DMs liegt darin, dass in letzter Zeit die Techniken zur Herstellung dieser Moden mit hoher Prazision verfeinert oder wiederentdeckt wurden. Obwohl ihr Potential bisher noch nicht vollkommen ausgeschopft wird, ermoglichen die speziellen Polarisationseigenschaften der DMs eine Vielzahl von Anwendungen. So tragen sie dazu bei, unser grundlegendes Verstandnis der optischen Eigenschaften einzelner emittierender Spezies wie Molekule, Nanopartikel oder Quantenpunkte zu vervollstandigen und erlauben Einblick in die dreidimensionale Dipol- oder Partikelorientierung im Raum. Durch ihre vollstandig verschwindende Intensitat im Zentrum des Fokus eignen sich einige DMs auch fur die stimulierte Emissionsloschung in der Fluoreszenzmikroskopie. Aus demselben Grund hat man sie auch fur die Verwendung in optischen Fallen und Pinzetten vorgeschlagen.

Summary We demonstrate a novel optical method for characterizing single Au nanoparticles by acquiring their scattering patterns. This technique combines confocal microscopy and higher-order laser modes for detecting the light scattered by sub-wavelength–sized nanoobjects. The optical patterns are generated by the coherent superposition of the field scattered by individual metallic particles and the excitation field reflected at the cover slide–air interface and provide information about the particles' position, orientation, size and shape. Detectable changes in the full width at half maximum (FWHM) of the signal intensity permit to distinguish between 20- and 60-nm diameter Au spheres. The confocal images are also very sensitive to the particle's geometry and polarizability, that is, Au nanospheres, Au nanorods and triangular Au nanoplates give different characteristic patterns if the excitation wavelength is varied.