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). This diffraction limit is based on the wave nature of 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. This concept is different from for example electron microscopy where instead the used wavelength is much smaller.

RESOLFT microscopy is an optical microscopy with very high resolution that can image details in samples that cannot be imaged with conventional or confocal microscopy. Within RESOLFT the principles of STED microscopy and GSD microscopy are generalized. Structures that are normally too close to each other to be distinguished are read out sequentially. Within this framework all methods can be explained that operate on molecules that have at least two distinguishable states, where reversible switching between the two states is possible, and where at least one such transition can be optically induced. In most cases fluorescent markers are used, where one state (A) which is bright, that is, generates a fluorescence signal, and the other state (B) is dark, and gives no signal. One transition between them can be induced by light (e. g. A→B, bright to dark). The sample is illuminated inhomogeneously with the illumination intensity at one position being very small (zero under ideal conditions). Only at this place are the molecules never in the dark state B (if A is the pre existing state) and remain fully in the bright state A. 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 transition back from B to A can be either spontaneous or driven by light of another wavelength. The molecules have to be switchable several times in order to be present in state A or B at different times during scanning the sample. The method also works if the bright and the dark state are reversed, one then obtains a negative image.

Different processes are used when switching the molecular states. However, all have in common that at least two distinguishable states are used. Typically the fluorescence property used marks the distinction of the states, however this is not essential, as absorption or scattering properties could also be exploited.

Some fluorescent proteins can be switched on and off by light of an appropriate wavelength. They can be used in a RESOLFT type microscope. During illumination with light, these proteins change their conformation. In the process they gain or lose their ability to emit fluorescence. The fluorescing state corresponds to state A, the non fluorescing to state B and the RESOLFT concept applies again. 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.

Just as with proteins, also some organic dyes can change their structure upon illumination. The ability to fluoresce of such organic dyes can be turned on and off through visible light. Again the applied light intensities can be quite low (some 100 W/cm2).