Abberior Instruments

RESOLFT

RESOLFT implements a general switching principle: the switching mechanism does no longer need to be purely electronically, but includes as switching mechanism for example the possibility of conformational changes of a molecule. Strictly speaking, the STED method is one implementation variant of the RESOLFT concept.

The implementation approach is similar as shown for STED - a beam scanning setup with overlapping beams of the excitation and the switching beam.

Molecule off (non-fluorescent conformation)
Molecule on (fluorescent conformation)
Diffraction limited excitation area
Area forcing molecules off via switching light

Depending on the implementation of the switch, typical saturation intensities and typical switching times are characteristically - e.g. conformational changes feature much lower switching intensities, while the switching time is much longer compared to electronical states (as for STED).

Below is shown exemplarily the implementation method of RESOLFT via light induced chemical reaction which generates the highly fluorescent dye from the colorless and non-fluorescent precursor.


 

Depending on the implementation of the switch, typical saturation intensities and typical switching times are characteristically - e.g. conformational changes feature much lower switching intensities, while the switching time is much longer compared to electronical states (as for STED).

Below is shown exemplarily the implementation method of RESOLFT via light induced chemical reaction which generates the highly fluorescent dye from the colorless and non-fluorescent precursor.

1: Phaseplate is responsible to redistribute the switching light in the focal plane, e.g. create "donut" shaped light

The implementation approach is similar as shown for STED - a beam scanning setup with overlapping beams of excitation and switching beam. In a STED setup the switching beam would be denoted as STED beam.

Images of living PtK2 cells using Dreiklang. Cells expressing Keratin19-Dreiklang in living cells and imaged both confocally (left) and by RESOLFT imaging (right). Scale bar: 1 μm.
Images of living PtK2 cells using Dreiklang. Cells expressing Keratin19-Dreiklang in living cells and imaged both confocally (left) and by RESOLFT imaging (right). Scale bar: 1 μm.

RESOLFT features theoretically unlimited resolution (see equation from the STED section) since only the molecule in the center of the focus is left in the on-state.

The detector only needs to see at minimum one photon to detect individual molecule (clusters) on the nanoscale resolution. 

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