Real-Time analysis and visualization for single-molecule based super-resolution microscopy

Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy

Quantum yield optimized fluorophores for site-specific labeling and super-resolution imaging

Single molecule applications, saturated pattern excitation microscopy, or stimulated emission depletion (STED) microscopy demand for bright and highly stable fluorescent dyes1,2. Despite of intensive research the choice of fluorphores is still very limited. Typically a stable fluorescent dyes is covalently attached to the target. This methodology brings forward a number of limitations, in particular, in case of protein labeling. First of all the fluorescent probes need to be attached selectively and site-specifically to prevent unspecific background. This often requires single cysteine mutations for covalent protein modification. Employing quantum dots allows overcoming problems of photo-bleaching3-6. However, the downsides are their large size, rendering the probe inaccessible to spatially confined architectures, issues in biocompatibility due to proper particle coating, and cellular toxicity6-8. Here we propose a new method to overcome the above outlined problems

A variational analysis of Einstein-scalar field Lichnerowicz equations on compact Riemannian manifolds

We establish new existence and non-existence results for positive solutions of the Einstein-scalar field Lichnerowicz equation on compact manifolds. This equation arises from the Hamiltonian constraint equation for the Einstein-scalar field system in general relativity. Our analysis introduces variational techniques, in the form of the mountain pass lemma, to the analysis of the Hamiltonian constraint equation, which has been previously studied by other methods.Comment: 15 page

The constraint equations for the Einstein-scalar field system on compact manifolds

We study the constraint equations for the Einstein-scalar field system on compact manifolds. Using the conformal method we reformulate these equations as a determined system of nonlinear partial differential equations. By introducing a new conformal invariant, which is sensitive to the presence of the initial data for the scalar field, we are able to divide the set of free conformal data into subclasses depending on the possible signs for the coefficients of terms in the resulting Einstein-scalar field Lichnerowicz equation. For many of these subclasses we determine whether or not a solution exists. In contrast to other well studied field theories, there are certain cases, depending on the mean curvature and the potential of the scalar field, for which we are unable to resolve the question of existence of a solution. We consider this system in such generality so as to include the vacuum constraint equations with an arbitrary cosmological constant, the Yamabe equation and even (all cases of) the prescribed scalar curvature problem as special cases.Comment: Minor changes, final version. To appear: Classical and Quantum Gravit

Multiple Routes for Glutamate Receptor Trafficking: Surface Diffusion and Membrane Traffic Cooperate to Bring Receptors to Synapses

Trafficking of glutamate receptors into and out of synapses is critically involved in the plasticity of excitatory synaptic transmission. Endocytosis and exocytosis of receptors have initially been thought to account alone for this trafficking. However, membrane proteins also traffic through surface lateral diffusion in the plasma membrane. We describe developments in electrophysiological and optical approaches that have allowed for the real time measurement of glutamate receptor surface trafficking in live neurons. These include (i) specific imaging of surface receptors using a pH sensitive fluorescent protein, (ii) design of a photoactivable drug to inactivate locally surface receptors and monitor electrophysiologically their recovery, and (iii)application of single molecule fluorescence microscopy to directly track the movement of individual surface receptors with nanometer resolution inside and outside synapses. Altogether, these approaches have demonstrated that glutamate receptors diffuse at high rates in the neuronal membrane and suggest a key role for surface diffusion in the regulation of receptor numbers at synapses

Monitoring the emergence of antibiotic resistance using the technology ot the DebugIT platform in the HEGP context

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A Septin-Dependent Diffusion Barrier at Dendritic Spine Necks

Excitatory glutamatergic synapses at dendritic spines exchange and modulate their receptor content via lateral membrane diffusion. Several studies have shown that the thin spine neck impedes the access of membrane and solute molecules to the spine head. However, it is unclear whether the spine neck geometry alone restricts access to dendritic spines or if a physical barrier to the diffusion of molecules exists. Here, we investigated whether a complex of septin cytoskeletal GTPases localized at the base of the spine neck regulates diffusion across the spine neck. We found that, during development, a marker of the septin complex, Septin7 (Sept7), becomes localized to the spine neck where it forms a stable structure underneath the plasma membrane. We show that diffusion of receptors and bulk membrane, but not cytoplasmic proteins, is slower in spines bearing Sept7 at their neck. Finally, when Sept7 expression was suppressed by RNA interference, membrane molecules explored larger membrane areas. Our findings indicate that Sept7 regulates membrane protein access to spines