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 diffraction limit down to molecular scales can be obtained.

With conventional microscopy techniques, it is not possible to distinguish features that are located at distances less than about half the wavelength used (i.e. about 200 nm for visible light).

In conventional microscopes the limit is determined by the used wavelength and the numerical aperture of the optical system.

The RESOLFT concept surmounts this limit by temporarily switching the molecules to a state in which they cannot send a (fluorescence-) signal upon illumination.

The area where molecules are mostly in the bright state can be made very small (smaller than the conventional diffraction limit) by increasing the transition light intensity (see below).

Any signal detected is thus known to come only from molecules in the small area around the illumination intensity minimum.

A high resolution image can be constructed by scanning the sample, i.e., shifting the illumination profile across the surface.

The method also works if the bright and the dark state are reversed, one then obtains a negative image.

Increasing the illumination brightness already results in a smaller area where the intensity is below the amount for efficient switching to the dark state.

The (fluorescence) signal during a following readout originates from a very small spot and one can obtain very sharp images.

The diffraction-unlimited nature of the RESOLFT family of concepts is reflected by the fact that the minimal resolvable distance

This is the standard operation mode in fluorescence microscopy and depicts state A.

SPEM (Saturated Pattern Excitation Microscopy)[5] and SSIM (Saturated Structured Illumination Microscopy)[6] are exploiting the RESOLFT concept using saturated excitation to produce "negative" images, i.e. fluorescence occurs from everywhere except at a very small region around the geometrical focus of the microscope.

The reversible transition (e.g. from B back to A) takes place either spontaneously or again driven by light.

Inducing conformational changes in proteins can be achieved already at much lower switching light intensities as compared to stimulated emission or ground state depletion (some W/cm2).

In combination with 4Pi microscopy images with isotropic resolution below 40 nm have been taken of living cells at low light levels.

[9][10] The ability to fluoresce of such organic dyes can be turned on and off through visible light.

Switching fluorescence on and off in specific areas results in enabling fluorescence in areas smaller than the diffraction limit. Rasterisation of the whole sample results in a pixel image with extremely high resolution.
RESOLFT principle: The sample is inhomogeneously illuminated (red line). The intensity is reduced in an area comparable to the classical resolution limit, but is (ideally) zero at only one position. The threshold intensity (blue line) required to switch the molecules to the dark state is only a fraction of the maximum intensity applied, so only molecules very close to the position of minimum intensity can be in the bright state. The green area in the right frame illustrates the size of the area where molecules are able to generate a signal.