Ground state depletion microscopy (GSD Microscopy) is an implementation of the

RESOLFT RESOLFT, an acronym for REversible Saturable OpticaL Fluorescence Transitions, denotes a group of optical fluorescence microscopy techniques with very high resolution. Using standard far field visible light optics a resolution far below the diffra ...

concept. The method was proposed in 1995 and experimentally demonstrated in 2007. It is the second concept to overcome the diffraction barrier in far-field optical microscopy published by

Stefan Hell Stefan Walter Hell HonFRMS (: born 23 December 1962) is a Romanian-German physicist and one of the directors of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany. He received the Nobel Prize in Chemistry in 2014 "for the ...

. Using nitrogen-vacancy centers in diamonds a resolution of up to 7.8 nm was achieved in 2009. This is far below the

diffraction limit The resolution of an optical imaging system a microscope, telescope, or camera can be limited by factors such as imperfections in the lenses or misalignment. However, there is a principal limit to the resolution of any optical system, due to t ...

(~200 nm).

Principle

In GSD microscopy, fluorescent markers are used. In one condition, the marker can freely be excited from ground state and returns spontaneously via emission of a fluorescence photon. However, if light of appropriate wavelength is additionally applied the dye can be excited to a long-lived dark state, i.e. a state where no fluorescence occurs. As long as the molecule is in the long-lived dark state (e.g. a

triplet state In quantum mechanics, a triplet is a quantum state of a system with a spin of quantum number =1, such that there are three allowed values of the spin component, = −1, 0, and +1. Spin, in the context of quantum mechanics, is not a mechanical ...

), it cannot be excited from the ground state. Switching between these two states (bright and dark) by applying light fulfills all preconditions for the

concept and subwavelength scale imaging, and therefore images with very high resolution can be obtained. For successful implementation, GSD microscopy requires either special fluorophores with high triplet yield, or removal of oxygen by use of various mounting media such as Mowiol or Vectashield. The implementation in a microscope is very similar to stimulated emission depletion microscopy, however it can operate with only one wavelength for excitation and depletion. Using an appropriate ring-like focal spot for the light that switches the molecules into the dark state, the fluorescence can be quenched at the outer part of the focal spot. Therefore, fluorescence only still takes place at the center of the microscope's focal spot and the spatial resolution is increased.

References

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