Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS). 2012

Marcel Leutenegger, and Christian Ringemann, and Theo Lasser, and Stefan W Hell, and Christian Eggeling
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.

We characterize a novel fluorescence microscope which combines the high spatial discrimination of a total internal reflection epi-fluorescence (epi-TIRF) microscope with that of stimulated emission depletion (STED) nanoscopy. This combination of high axial confinement and dynamic-active lateral spatial discrimination of the detected fluorescence emission promises imaging and spectroscopy of the structure and function of cell membranes at the macro-molecular scale. Following a full theoretical description of the sampling volume and the recording of images of fluorescent beads, we exemplify the performance and limitations of the TIRF-STED nanoscope with particular attention to the polarization state of the laser excitation light. We demonstrate fluorescence correlation spectroscopy (FCS) with the TIRF-STED nanoscope by observing the diffusion of dye molecules in aqueous solutions and of fluorescent lipid analogs in supported lipid bilayers in the presence of background signal. The nanoscope reduced the out-of-focus background signal. A lateral resolution down to 40-50 nm was attained which was ultimately limited by the low lateral signal-to-background ratio inherent to the confocal epi-TIRF scheme. Together with the estimated axial confinement of about 55 nm, our TIRF-STED nanoscope achieved an almost isotropic and less than 1 attoliter small all-optically induced measurement volume.

UI MeSH Term Description Entries
D007089 Image Enhancement Improvement of the quality of a picture by various techniques, including computer processing, digital filtering, echocardiographic techniques, light and ultrastructural MICROSCOPY, fluorescence spectrometry and microscopy, scintigraphy, and in vitro image processing at the molecular level. Image Quality Enhancement,Enhancement, Image,Enhancement, Image Quality,Enhancements, Image,Enhancements, Image Quality,Image Enhancements,Image Quality Enhancements,Quality Enhancement, Image,Quality Enhancements, Image
D008856 Microscopy, Fluorescence Microscopy of specimens stained with fluorescent dye (usually fluorescein isothiocyanate) or of naturally fluorescent materials, which emit light when exposed to ultraviolet or blue light. Immunofluorescence microscopy utilizes antibodies that are labeled with fluorescent dye. Fluorescence Microscopy,Immunofluorescence Microscopy,Microscopy, Immunofluorescence,Fluorescence Microscopies,Immunofluorescence Microscopies,Microscopies, Fluorescence,Microscopies, Immunofluorescence
D004867 Equipment Design Methods and patterns of fabricating machines and related hardware. Design, Equipment,Device Design,Medical Device Design,Design, Medical Device,Designs, Medical Device,Device Design, Medical,Device Designs, Medical,Medical Device Designs,Design, Device,Designs, Device,Designs, Equipment,Device Designs,Equipment Designs
D013050 Spectrometry, Fluorescence Measurement of the intensity and quality of fluorescence. Fluorescence Spectrophotometry,Fluorescence Spectroscopy,Spectrofluorometry,Fluorescence Spectrometry,Spectrophotometry, Fluorescence,Spectroscopy, Fluorescence
D019544 Equipment Failure Analysis The evaluation of incidents involving the loss of function of a device. These evaluations are used for a variety of purposes such as to determine the failure rates, the causes of failures, costs of failures, and the reliability and maintainability of devices. Materials Failure Analysis,Prosthesis Failure Analysis,Analysis, Equipment Failure,Analysis, Materials Failure,Analysis, Prosthesis Failure,Analyses, Equipment Failure,Analyses, Materials Failure,Analyses, Prosthesis Failure,Equipment Failure Analyses,Failure Analyses, Equipment,Failure Analyses, Materials,Failure Analyses, Prosthesis,Failure Analysis, Equipment,Failure Analysis, Materials,Failure Analysis, Prosthesis,Materials Failure Analyses,Prosthesis Failure Analyses
D036103 Nanotechnology The development and use of techniques to study physical phenomena and construct structures in the nanoscale size range or smaller. Nanotechnologies

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