STED microscopy
It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system.
Okhonin[4] (Institute of Biophysics, USSR Academy of Sciences, Siberian Branch, Krasnoyarsk) had patented the STED idea.
STED is a deterministic functional technique that exploits the non-linear response of fluorophores commonly used to label biological samples in order to achieve an improvement in resolution, that is to say STED allows for images to be taken at resolutions below the diffraction limit.
This differs from the stochastic functional techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) as these methods use mathematical models to reconstruct a sub diffraction limit from many sets of diffraction limited images.
The equation is: where D is the diffraction limit, λ is the wavelength of the light, and NA is the numerical aperture, or the refractive index of the medium multiplied by the sine of the angle of incidence.
Because STED selectively deactivates the fluorescence, it can achieve resolution better than traditional confocal microscopy.
The excited electron is forced to relax into a higher vibration state than the fluorescence transition would enter, causing the photon to be released to be red-shifted as shown in the image to the right.
This lowering of energy raises the wavelength, and causes the photon to be shifted farther into the red end of the spectrum.
This focal area can be engineered by altering the properties of the pupil plane of the objective lens.
[9][10][11] The most common early example of these diffractive optical elements, or DOEs, is a torus shape used in two-dimensional lateral confinement shown below.
[6][14] To optimize the effectiveness of STED, the destructive interference in the center of the focal spot needs to be as close to perfect as possible.
[19] Over the last several years, STED has developed from a complex and highly specific technique to a general fluorescence method.
As a result, a number of methods have been developed to expand the utility of STED and to allow more information to be provided.
In addition, neurofilaments, actin, and tubulin are often used to compare the resolving power of STED and confocal microscopes.
[20][21][22] Using STED, a lateral resolution of 70 – 90 nm has been achieved while examining SNAP25, a human protein that regulates membrane fusion.
[23][24] Studies of complex organelles, like mitochondria, also benefit from STED microscopy for structural analysis.
Using custom-made STED microscopes with a lateral resolution of fewer than 50 nm, mitochondrial proteins Tom20, VDAC1, and COX2 were found to distribute as nanoscale clusters.
The resolution of both electron and atomic force microscopy is even better than STED resolution, but by combining atomic force with STED, Shima et al. were able to visualize the actin cytoskeleton of human ovarian cancer cells while observing changes in cell stiffness.
Using two fluorescent dyes and beam pairs, colocalized imaging of synaptic and mitochondrial protein clusters is possible with a resolution down to 5 nm [18].
[29] Combining STED with fluorescence correlation spectroscopy showed that cholesterol-mediated molecular complexes trap sphingolipids, but only transiently.
This method was shown to work at 50 nm lateral resolution within Citrine-tubulin expressing mammalian cells.
[34] Recently, multicolor live-cell STED was performed using a pulsed far-red laser and CLIPf-tag and SNAPf-tag expression.
[38] Super-resolution requires small pixels, which means more spaces to acquire from in a given sample, which leads to a longer acquisition time.
