STED Microscopy - Background

Background

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: D=λ/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. 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 the traditional confocal microscopy. Normal fluorescence occurs by exciting an electron from the ground state into an excited electronic state which, after relaxing back to the ground state, emits a photon. 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 below. 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.