Abstract
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.
Highlights
Fluorescence microscopy is one of the most extensively used tools for the structural and functional investigation of the interior of cells
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
The invention of Stimulated Emission Depletion Microscopy (STED) in 1994 highlighted the unexpected fact that the diffraction resolution barrier can be effectively overcome in a microscope that uses regular lenses and focused visible light [1,2]
Summary
Fluorescence microscopy is one of the most extensively used tools for the structural and functional investigation of the interior of cells. By selecting two optical glasses whose refractive indices match at the excitation wavelength but differ for the STED wavelength, they were able to design a phase plate that can be shared by both beams In their scheme, the detection beam path is coupled out between the objective lens and the phase plate using a dichroic mirror. We have built a simple, errorproof and easy-to-use beam-shaping device that, together with an appropriate laser source, can economically retrofit almost any standard scanning (confocal) fluorescence microscope and turn it into a full STED microscope (easySTED) providing subdiffraction resolution. With a minor change, the same beam shaping device can be tuned so that the image of a single fluorophore depends strongly on the fluorophore’s transition dipole orientation As a result, this Molecular Orientation Microscopy by STED (MOM-STED) allows one to assess the orientation of the transition dipole of the molecule in space. MOM-STED allows one to improve counting of molecules within subdiffraction sample volumes
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