STED - Stimulated Emission Depletion
Short description of the method
STED microscopy is a process that provides super resolution imaging by selectively
deactivating fluorophores, so as to enhance the resolution in an area of a sample. It
was developed by Stefan W. Hell and Jan Wichmann in 1994, Hell was awarded the
Nobel Prize in Chemistry in 2014 for its development.
A STED microscope is built on the basis of a confocal laser scanning microscope
(CLSM). The light intensity of the focal spot distributes according to the point spread
function (PSF). For a circular aperture in ideal situation, the PSF displays a pattern
called “Airy disk”, with a size proportional to λ/NA (λ is the wavelength of light, and
NA is numerical aperture). The resolution of CLSM is determined by the size of the
PSF: The smaller the focal spot is, each pixel in the acquired image will be corresponding to a smaller region in the sample, and thus the image will be less blurred.
The physicist Ernst Abbe pointed out that the PSF size has a lower limit proportional
to λ/NA (circular aperture) due to diffraction. He developed an equation to describe
this limit:
D =
λ
2 NA
=
λ
2n sin α
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.
The basic idea of STED microscopy is to utilize nonlinear optics to engineer a smaller
PSF below such limit. The STED microscope operates by using two laser beams to
illuminate the specimen. An excitation laser is closely followed by a doughnut-shaped
red-shifted laser beam that is termed the STED beam (depletion laser). The excitation beam switches the marker molecules into a bright state and these excited molecules can emit fluorescence light (spontaneous emission). The STED beam essentially brings the molecules back into its ground state (stimulated Emission) and thus
prevents fluorescence to occur. This mechanism is an effective implementation of a
controllable switch from "on" to "off" mode of the fluorescent molecule.
The STED beam has an annular shape ("donut"-shape) as depicted by the red ring in
the next Figure. When the two laser pulses are superimposed, only molecules that
reside in the center of the STED beam are able to emit fluorescence, thus signifycantly restricting emission. This action effectively narrows the point spread function
and ultimately increases resolution beyond the diffraction limit.
A modified Abbe’s equation describes this sub diffraction resolution as:
λ
D =
2 NA 1 +
I
I SAT
=
λ
2n sin α 1 +
I
I SAT
where n is the refractive index of the medium, I is the intracavity intensity and ISAT is
the saturation intensity.
STED microscopy is capable of 20 nanometer (or better) lateral resolution and 40 to
50 nanometer axial resolution.
Confocal (left) and the corresponding STED (right) microscopy image (tubulin, label ATTO647N)
Confocal (left) and the corresponding STED (right) microscopy image (tubulin, label ATTO647N)
Line profile at the confocal (left) and the corresponding STED (right) microscopy image