Claus Seidel

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Papers by Claus Seidel

Nucleobase-Specific Quenching of Fluorescent Dyes. 1. Nucleobase One-Electron Redox Potentials and Their Correlation with Static and Dynamic Quenching Efficiencies

The Journal of Physical Chemistry, 1996

Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives ... more

Analyzing Aβ Aggregates with High Resolution Microscopy

Biophysical Journal, 2011

In a world where more people grow older aging-related neurodegeneration like Alzheimer's disease ... more In a world where more people grow older aging-related neurodegeneration like Alzheimer's disease (AD) affects more and more people. Today, AD can be diagnosed with certainty only post mortem, detecting insoluble b-amyloid peptide (Ab) aggregates and neurofibrillary tangles in the patient's brain tissue. Aggregates consisting of Ab are a fundamental pathologic feature of AD. Today in many studies, concentrations of monomeric Ab in body fluids are investigated, especially for diagnostic purposes. Nevertheless, for the detection, quantitation and qualification of aggregated pathologic Ab forms, also in the course of aging, a highly sensitive detection assay system for aggregated Ab species is necessary. We developed an ultra-sensitive assay for the detection of aggregated protein species out of body fluids. This highly specific and sensitive assay uses confocal fluorescence spectroscopy methods and is sensitive enough to detect single aggregates. For the procedure, pathologic aggregates out of body fluids are immobilized on a glass chip, subsequently fluorescence labeled and detected via confocal spectroscopy. Actually, we are optimizing the assay in concerns of instrumentation (imaging) and microscopy high-resolution and even super-resolution methods. We are developing methods to analyze aggregates via super-resolution microscopy. Setups like PAINT (Point Accumulation for Imaging in Nanoscale Topography) or STORM (Stochastic Optical Reconstruction Microscopy) allow resolutions in nanometer-range. PAINT is based on replacing the point-spreadfunction (PSF) of a fluorophore by a point in the middle of a 2D gaussian fit. First measurements show resolutions of 30 nm. STORM is based on highaccuracy localization of photoswitchable fluorophores. During one imaging cycle, only a small part of the fluorophores is turned on. This allows a high accuracy in determining the fluorophore position by replacing the PSF. The fluorophore positions obtained from a series of imaging cycles can be used to reconstruct the whole image. Spectral imaging techniques are important for many biological experiments, particularly live-cell quantitative imaging of multiple fluorescence probes. Existing hyperspectral imaging systems require sequential techniques, limiting the data acquisition rate. A newly-developed snapshot device, the Image Mapping Spectrometer (IMS), acquires full spectral information simultaneously from every pixel in the field with image acquisition rates up to 10 frames/second. The IMS maps adjacent pixels from the object to create space between them in the image, and then uses a grating to spread wavelength content from each pixel into this space. Direct image re-mapping provides the final 3D (x, y, lambda) data cube. Fluorescent protein (FP)-based biosensors are increasingly valuable tools for identifying subcellular dynamic processes in live cells. Many biosensors are based on FP-FRET, and measurements of the resultant small changes in FRET require high quality data. Tracking intracellular free Ca 2þ levels is also crucial to elucidating signaling events, but the best Ca 2þ indicator dyes overlap in the spectral emission range of the common FP-FRET biosensors. We have used the IMS system to simultaneously image multicolored FPs (i.e., CFP, GFP, YFP) in combination with extrinsic indicator dyes, such as Fluo-4. To demonstrate the speed and resolution of the IMS approach, we monitored both intracellular Ca 2þ oscillations and caspase-3 activity during hydrogen peroxide-induced apoptosis. Ca 2þ activity was measured with Fluo-4 (emission peak at 517 nm) and caspase-3 activity was measured with SCAT3.1, a FRET biosensor based on the ECFP (emission peak at 484 nm) and EYFP (emission peak at 527 nm) pair. Using the IMS, the three fluorophores were imaged with sub-second temporal resolution and spectrally unmixed in real-time. This permits direct correlation of Ca 2þ activity with other apoptotic signaling events and demonstrates the power of the IMS for measuring dynamic physiological processes. Diabetes is a disease resulting from changes in pancreatic islets, which are insulin secreting micro-organs within the pancreas. With increased blood glucose, insulin is secreted from beta cells in the islets in a coordinated pulsatile manner. At the same time alpha cell glucagon secretion is inhibited. Mechanisms controlling these processes at the intercellular and at the inter-islet level remain unclear. We suggest that the three-dimensional organization of islet cells and the dynamics of islet blood flow have a role in regulating insulin and glucagon secretion. This is suggested by observation that the density of blood vessels within islets is much greater than in surrounding pancreatic tissue, and that most individual islet cells are adjacent to a blood vessel. As an initial test of our hypothesis, we have developed a high-speed in vivo fluorescence imaging method to track pancreatic blood flow in a living mouse. We are also developing methods necessary to analyze the large amounts of data generated. Using high speed line scan confocal microscopy the method has full frame sub-micron spatial and less than 10 ms temporal resolution. Islets are located within the pancreas by using mice with GFP-labeled beta cells. Blood plasma is labeled with a fluorescent dextran, allowing mapping of vascular dimensions and pathway. Individual blood cells are fluorescently labeled by osmotic shock loading with an Alexa dye, which allows tracking of the blood flow. We present current results for blood flow under different levels of blood glucose in clamping experiments. Our previous qualitative results have suggested that there are differences in blood flow parameters at different glucose levels. Here, a more quantitative analysis of blood flow velocity, any observed changes in vessel dimensions, and changes in blood flow coverage inside and outside islets is presented. To study the structural organization of mitochondrial proteins, we applied Stimulated Emission Depletion (STED) microscopy in isolated mitochondria. In STED microscopy, two laser beams are used: one for excitation of fluorophores and the other, with doughnut shape, to deplete them in order to allow fluorescence emission only from the excited volume located at the doughnut's center. With STED a lateral resolution of~45 nm was achieved in images of isolated mitochondria. We investigated the localization pattern and distribution of MaxiKa, COX4 and VDAC1. After a combined analysis of classical confocal and STED images, we found distinct distributions for VDAC1, MaxiKa and COX4. COX4 distribution was consistent with localization in the cristae. We established that there are 7-15 clusters of Max-iKa, 10-15 clusters of VDAC1, and 15-20 clusters of COX4 per mitochondria. We have demonstrated that protein clusters in the mitochondria can be resolved with a separation power of~45 nm, and that it is possible to retrieve quantitative information about the number of clusters and density of proteins in mitochondria. This approach can be extended to eins in mitochondria and subcellular organelles. Supported by NIH.

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Outcome of the First wwPDB Hybrid/Integrative Methods Task Force Workshop

Structure, 2015

Structures of biomolecular systems are increasingly computed by integrative modeling that relies ... more Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models?

Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy

by Claus Seidel and Linnea Olofsson

Nature communications, 2014

Efficient cell-to-cell communication relies on the accurate signalling of cell surface receptors.... more Efficient cell-to-cell communication relies on the accurate signalling of cell surface receptors. Understanding the molecular bases of their activation requires the characterization of the dynamic equilibrium between active and resting states. Here, we monitor, using single-molecule Förster resonance energy transfer, the kinetics of the reorientation of the extracellular ligand-binding domain of the metabotropic glutamate receptor (mGluR), a class C G-protein-coupled receptor. We demonstrate that most receptors oscillate between a resting- and an active-conformation on a sub-millisecond timescale. Interestingly, we demonstrate that differences in agonist efficacies stem from differing abilities to shift the conformational equilibrium towards the fully active state, rather than from the stabilization of alternative static conformations, which further highlights the dynamic nature of mGluRs and revises our understanding of receptor activation and allosteric modulation.

Dye-Exchange Dynamics in Micellar Solutions Studied by Fluorescence Correlation Spectroscopy

by Wajih al-Soufi and Claus Seidel

The Journal of Physical Chemistry B, 2007

We investigated the dye-exchange dynamics between rhodamine 123 (R123), a mitochondrial fluoresce... more We investigated the dye-exchange dynamics between rhodamine 123 (R123), a mitochondrial fluorescent dye, and micelles as membrane mimetic systems. In the presence of neutral micelles (Triton X-100 and Brij 35) R123 partitions between the aqueous solution and the micellar pseudo-phase, undergoing red shift of the absorption and the emission spectra. Fluorescence correlation spectroscopy (FCS) was used to study the dynamics of these systems over an extremely wide time range and at the single-molecule level, yielding information in one and the same experiment about the diffusional dynamics of free and bound rhodamine and about the dye-exchange dynamics as well as several photophysical properties of the rhodamine bound to the micelles. It was found that the entry rate constants are diffusion-controlled, indicating that there are no geometric or orientational requirements for the association of the dye with the micelle. With respect to the dye-exchange dynamics, micelles are found to behave as soft supramolecular cages in contrast to other rigid supramolecular cavities, such as cyclodextrins. The exit rate constants depend on the surfactant and determine the stability of the binding. Single-molecule multiparameter fluorescence detection (MFD) was used to examine the fluorescence properties of individual molecules in comparison to ensembles of molecules. The MFD histograms confirm the fast dye-exchange dynamics observed by FCS and yield mean values of fluorescence lifetimes and anisotropies in agreement with those obtained in bulk measurements.