STED microscopy

Ernst Abbe's formula for the diffraction limit, set in stone at a monument in Jena.

Jablonski diagram showing the redshift of the stimulated photon. This redshift allows the stimulated photon to be ignored.

Diagram of the design of a STED device. The double laser design allows for excitation and stimulated emission to be used together for STED.

In traditional microscopy, the resolution that can be obtained is limited by the diffraction of light. Ernst Abbe developed an equation to describe this limit. The equation is:

${\displaystyle \mathrm {D} ={\frac {\lambda }{2n\sin {\alpha }}}={\frac {\lambda }{\mathrm {2NA} }}}$

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. n describes the refractive index of the specimen, α measures the solid half‐angle from which light is gathered by an objective, λ is the wavelength of light used to excite the specimen, and NA is the numerical aperture. To obtain high resolution (i.e. small d values), short wavelengths and high NA values (NA = n sinα) are optimal.[6] This diffraction limit is the standard by which all super resolution methods are measured. Because STED selectively deactivates the fluorescence, it can achieve resolution better than traditional confocal microscopy. Normal fluorescence occurs by exciting an electron from the ground state into an excited electronic state of a different fundamental energy level (S0 goes to S1) which, after relaxing back to the ground state (of S1), emits a photon by dropping from S1 to a vibrational energy level on S0. STED interrupts this process before the photon is released. 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.[7] Because the electron is going to a higher vibrational state, the energy difference of the two states is lower than the normal fluorescence difference. This lowering of energy raises the wavelength, and causes the photon to be shifted farther into the red end of the spectrum. This shift differentiates the two types of photons, and allows the stimulated photon to be ignored.

To force this alternative emission to occur, an incident photon must strike the fluorophore. This need to be struck by an incident photon has two implications for STED. First, the number of incident photons directly impacts the efficiency of this emission, and, secondly, with sufficiently large numbers of photons fluorescence can be completely suppressed.[8] To achieve the large number of incident photons needed to suppress fluorescence, the laser used to generate the photons must be of a high intensity. Unfortunately, this high intensity laser can lead to the issue of photobleaching the fluorophore. Photobleaching is the name for the destruction of fluorophores by high intensity light.

Comparison of confocal microscopy and STED microscopy. This shows the improved resolution of STED microscopy over traditional techniques.

Excitation spot (2D, left), doughnut-shape de-excitation spot (center) and remaining area allowing fluorescence (right).

STED functions by depleting fluorescence in specific regions of the sample while leaving a center focal spot active to emit fluorescence. 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. The red zone is depleted, while the green spot is left active. This DOE is generated by a circular polarization of the depletion laser, combined with an optical vortex. The lateral resolution of this DOE is typically between 30 and 80 nm. However, values down to 2.4 nm have been reported.[12] Using different DOEs, axial resolution on the order of 100 nm has been demonstrated.[13] A modified Abbe's equation describes this sub diffraction resolution as:

${\displaystyle \mathrm {D} ={\frac {\lambda }{2n\sin {\alpha }{\sqrt {1+{\frac {I}{I_{\text{sat}}}}}}}}={\frac {\lambda }{\mathrm {2NA} {\sqrt {1+\sigma }}}}}$

Where ${\textstyle n}$ is the refractive index of the medium, ${\textstyle I}$ is the intracavity intensity and ${\textstyle I_{\text{sat}}}$ is the saturation intensity. Where ${\textstyle \sigma }$ is the saturation factor expressing the ratio of the applied (maximum) STED intensity to the saturation intensity, ${\textstyle \sigma =I_{\text{max}}/I_{\text{sat}}}$.[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. That imposes certain constraints on the optics that can be used.

Early on in the development of STED, the number of dyes that could be used in the process was very limited. Rhodamine B was named in the first theoretical description of STED.[2] As a result, the first dyes used were laser emitting in the red spectrum. To allow for STED analysis of biological systems, the dyes and laser sources must be tailored to the system. This desire for better analysis of these systems has led to living cell STED and multicolor STED, but it has also demanded more and more advanced dyes and excitation systems to accommodate the increased functionality.[7]

One such advancement was the development of immunolabeled cells. These cells are STED fluorescent dyes bound to antibodies through amide bonds. The first use of this technique coupled MR-121SE, a red dye, with a secondary anti-mouse antibody.[8] Since that first application, this technique has been applied to a much wider range of dyes including green emitting, Atto 532,[15][16][17] and yellow emitting, Atto 590,[18] as well as additional red emitting dyes. In addition, Atto 647N was first used with this method to produce two-color STED.[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.

Photobleaching can occur either from excitation into an even higher excited state, or from excitation in the triplet state. To prevent the excitation of an excited electron into another, higher excited state, the energy of the photon needed to trigger the alternative emission should not overlap the energy of the excitation from one excited state to another.[41] This will ensure that each laser photon that contacts the fluorophores will cause stimulated emission, and not cause the electron to be excited to another, higher energy state. Triplet states are much longer lived than singlet states, and to prevent triplet states from exciting, the time between laser pulses needs to be long enough to allow the electron to relax through another quenching method, or a chemical compound should be added to quench the triplet state.[20][42][43]

• Confocal microscopy
• Fluorescence
• Fluorescence microscope
• Laser scanning confocal microscopy
• Optical microscope
• Photoactivated localization microscopy
• Stochastic optical reconstruction microscopy
• Super-resolution microscopy
• Ground state depletion microscopy

Wikimedia Commons has media related to STED microscopy.

• Overview at the Department of NanoBiophotonics at the Max Planck Institute for Biophysical Chemistry.
• Brief summary of the RESOLFT equations developed by Stefan Hell.
• Stefan Hell lecture: Super-Resolution: Overview and Stimulated Emission Depletion (STED) Microscopy
• Light Microscopy: An ongoing contemporary revolution (Introductory Review)