Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit

A STED MICROSCOPE DESIGNED FOR ROUTINE BIOMEDICAL APPLICATIONS (Invited Paper)

“…Examples comprise transferring the molecules to a transient dark state [5], optical transfers between two distinguishable fluorophore isomers (e.g., 'photoswitching' fluorophores), and others [6]. Separating features by states rather than by waves, and more specifically, preventing the molecules of neighboring features to occupy their fluorescent state at the same time, is also the basis of the stochastic nanoscopy approaches that detect and localize individual molecules (PALM/STORM) [7][8][9].…”

Fluorescence nanoscopy. Methods and applications

“…When this spot of subdiffraction dimensions is scanned across the specimen, a super-resolution image is obtained. Subdiffraction imaging techniques that share this working principle are termed reversible saturable optical transition (RESOLFT) [2]; depending on the particular molecular transition exploited to switch fluorophores to a dark state, the modalities are termed stimulated emission depletion (STED) [3][4][5], ground stated depletion (GSD) [6,7] or, simply, RESOLFT [8].…”

“…Optical shelving or GSD imaging [6,7] is another superresolution imaging technique based on the RESOLFT concept: Molecules in the periphery of the focal spot are driven to the lowest triplet state (optical shelving) depleting the fluorophores ground state and transiently confining the fluorescence emission to the unaffected central spot of subdiffraction size. In practice, recovery of the fluorescence is essential as the technique relies on scanning.…”

“…In practice, recovery of the fluorescence is essential as the technique relies on scanning. The challenge is to avoid irreversible photobleaching due to photoionisation and reaction with molecular oxygen while the molecule is in a dark state [25], which accounts for the fact that GSD had been suggested [6] more than a decade before it was demonstrated [7], when the conditions for optical shelving compatible with scanning microscopy (microsecond-long transient shelving) were eventually established.…”

3‐D stimulated emission depletion microscopy with programmable aberration correction

“…In the past two decades a number of super-resolved microscopy (SRM) techniques have emerged for which the resolution is not limited by diffraction. Following the invention of stimulated emission depletion (STED) microscopy [4,5] and ground state depletion (GSD) microscopy [6] these laser scanning approaches were generalised as "RESOLFT" [7] techniques and were later complemented by stochastically switched single molecule localisation techniques such as PALM [8,9] and STORM [10]. While all these SRM techniques can provide excellent laterally super-resolved images of cells near the microscope coverslip, it is more challenging to realise SRM in the axial direction, particularly inside biological samples that present optical aberrations and scattering.…”