Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation.
Stimulated emission depletion (STED) microscopy usually employs a scanning excitation beam that is superimposed by a donut-shaped STED beam for keeping the fluorophores at the periphery of the excitation spot dark. Here, we introduce a simple birefringent device that produces a donut-shaped focal spot with suitable polarization for STED, while leaving the excitation spot virtually intact. The device instantly converts a scanning (confocal) microscope with a co-aligned STED beam into a full-blown STED microscope. The donut can be adapted to reveal, through the resulting fluorescence image, the orientation of fluorophores in the sample, thus directly providing subdiffraction resolution images of molecular orientation.
Reuss
Matthias
M
German Cancer Research Center (DKFZ), Optical Nanoscopy Division, Heidelberg, Germany.
Engelhardt
Johann
J
Hell
Stefan W
SW
D001704:Biopolymers / Q000032:analysis / Q000737:chemistry*; D001718:Birefringence; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation*; D018613:Microscopy, Confocal / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D008968:Molecular Conformation; D012031:Refractometry / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001704:Biopolymers