STED microscopy

Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimising the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin (Institute of Biophysics, USSR Academy of Sciences, Siberian Branch, Krasnoyarsk) had patented the STED idea. This patent was, perhaps, unknown to Hell and Wichmann in 1994. Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimising the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin (Institute of Biophysics, USSR Academy of Sciences, Siberian Branch, Krasnoyarsk) had patented the STED idea. This patent was, perhaps, unknown to Hell and Wichmann in 1994. STED microscopy is one of several types of super resolution microscopy techniques that have recently been developed to bypass the diffraction limit of light microscopy to increase resolution. 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. 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: 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. 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. 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. 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. 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. 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 a helical phase ramp. The lateral resolution of this DOE is typically between 30 and 80 nm. However, values down to 2.4 nm have been reported. Using different DOEs, axial resolution on the order of 100 nm has been demonstrated. A modified Abbe’s equation describes this sub diffraction resolution as: D = λ 2 n sin ⁡ α 1 + I I sat {displaystyle mathrm {D} ={frac {lambda }{2nsin {alpha }{sqrt {1+{frac {I}{I_{ ext{sat}}}}}}}}} Where n is the refractive index of the medium, I is the intracavity intensity and Isat is the saturation intensity.