How does stimulated emission depletion microscopy work?

How does stimulated emission depletion microscopy work?

Stimulated emission depletion microscopy (STED) is a fluorescence microscopy technique that overcomes the diffraction limited resolution of confocal microscopes. Fluorescence from the remaining excited dye molecules in the center of the excitation focus is then detected and used to form the high resolution images.

What is depletion in microscopy?

Stimulated emission depletion microscopy is a technique that relies on the depletion of the excited state fluorophores surrounding the objective focal spot in order to significantly narrow the dimensions and increase resolution through point-spread function engineering.

What is the resolution of STED?

Using STED, a lateral resolution of 70 – 90 nm has been achieved while examining SNAP25, a human protein that regulates membrane fusion.

What is STED nanoscopy?

Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell.

What determines the spatial resolution of a STED microscope?

The spatial resolution of the STED microscopy is determined by the specially engineered beam profile of the depletion beam and its power.

What is point spread function in image processing?

The ideal point spread function (PSF) is the three-dimensional diffraction pattern of light emitted from an infinitely small point source in the specimen and transmitted to the image plane through a high numerical aperture (NA) objective.

Is STED microscopy confocal?

STED microscopy, but not confocal microscopy, allows discrimination of single features at high spatial densities. Specific patterns of movement within the confined space of the axon are revealed in STED microscopy, while confocal imaging is limited to reporting gross motion.

Who invented STED microscopy?

Stefan Hell
Here, Stefan Hell, who invented many of these techniques, gives an introduction to these super-resolution microscopy techniques, and a detailed discussion of two such techniques: STED (Stimulated Emission Depletion) and RESOLFT (REversible Saturable OpticaL Fluorescence Transitions).

How does expansion microscopy work?

Expansion microscopy is achieved by synthesizing a polymer system within a specimen. By then swelling this polymer network, the sample is expanded to be examined under conventional microscopic analysis tools without degrading the integrity of the sample.

Why the shape of STED beam is of Doughnut shape in STED microscope?

These donut-shaped beams are suitable for STED microscopy. The peak-to-peak distances of donut are 520 nm and 480 nm for right circular and azimuthal polarization light, respectively. Thus, the STED beam generated with azimuthally polarized light could achieve higher lateral resolution.