Dresden 2020 – wissenschaftliches Programm
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BP 35.2: Vortrag
Donnerstag, 19. März 2020, 15:15–15:30, HÜL 386
Confocal single molecule localisation microscopy for super-resolved fluorescence lifetime imaging — •Jan Christoph Thiele, Eugenia Butkevich, Oleksii Nevskyi, and Jörg Enderlein — Third Institute of Physics - Biophysics, Georg August University, Göttingen, Germany
Localisation based super-resolution microscopy techniques like dSTORM, PALM and PAINT usually rely on wide field or TIRF illumination and wide field detection. This allows for simultaneous acquisition of the whole field of view but comes with the limitations of a camera based detection. Instead, we use a confocal setup with a pulsed excitation, single photon detection and a fast laser scanner. We evaluate different dyes and conditions to achieve slow blinking kinetics and a high number of photons per switching event. Individual switching events could be localised utilising our confocal scanning approach and the corresponding super-resolved image could be reconstructed. The huge advantage of a single photon detection is that each localisation contains information about the fluorescence lifetime. This enables us to combine dSTORM with metal induced energy transfer (MIET), a distance dependant modulation of the lifetime of a fluorophore by a thin metal film. MIET enables axial localising single fluorophores with a precision below 5 nm. Our goal is to achieve a high, isotropic 3D-localisation accuracy by combining the high lateral precision of dSTORM with the high axial precision of MIET.