GSD Microscopy - Principle

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), it cannot be excited from the ground state. Switching between these two states (bright and dark) by applying light fulfills all preconditions for the RESOLFT concept and subdiffraction imaging and images with very high resolution can be obtained. For successful realization, GSD microscopy requires either special fluorophores with high triplet yield, or removal of oxygen by use of various mounting medium like Moviol 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 brings 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.