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For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of(More)
In microscopy, single fluorescence point sources can be localized with a precision several times greater than the resolution limit of the microscope. We show that the intermittent fluorescence or 'blinking' of quantum dots can analyzed by an Independent Component Analysis so as to identify the light emitted by each individual nanoparticle, localize it(More)
In the plasma membrane, syntaxin 1 and syntaxin 4 clusters define sites at which secretory granules and caveolae fuse, respectively. It is widely believed that lipid phases are mandatory for cluster formation, as cluster integrity depends on cholesterol. Here we report that the native lipid environment is not sufficient for correct syntaxin 1 clustering and(More)
We describe a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and xenon arc lamp illumination. Our Bayesian analysis of the blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores that may or may not be(More)
Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by(More)
Neuronal exocytosis is driven by the formation of SNARE complexes between synaptobrevin 2 on synaptic vesicles and SNAP-25/syntaxin 1 on the plasma membrane. It has remained controversial, however, whether SNAREs are constitutively active or whether they are down-regulated until fusion is triggered. We now show that synaptobrevin in proteoliposomes as well(More)
We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of(More)
Subtractive imaging in confocal fluorescence light microscopy is based on the subtraction of a suitably weighted widefield image from a confocal image. An approximation to a widefield image can be obtained by detection with an opened confocal pinhole. The subtraction of images enhances the resolution in-plane as well as along the optic axis. Due to the(More)
The techniques of patterned excitation microscopy (PEM, also referred to in the literature as structured illumination, harmonic excitation light microscopy, or laterally modulated excitation microscopy), has recently been extended to the non-linear regime, permitting a further increase in resolution breaking the Abbe diffraction limit (saturated PEM,(More)
By physical rotation of the sample, axial tomography enables the acquisition of otherwise inaccessible spatial information from an object. In combination with confocal microscopy, the method can fundamentally improve the effective three-dimensional (3D) resolution. In this report we present a novel method for high resolution reconstruction of confocal axial(More)