Method of fluorescence-microscopically imaging a structure in a sample with high three-dimensional spatial resolution

A method of imaging a structure in a sample with three-dimensional spatial resolution, the method comprising the steps of:

• selecting a fluorophore from a group of photochromic fluorophores
• which are repeatedly temporarily transferred out of a first photochromic state into a second photochromic state by means of an optical transfer signal by a process selected from a group of processes including:
• cis-/trans-isomerisation,
• proton transfer,
• atom group transfer,
• electron transfer,
• ring-opening/closing, and
• dimerisation,
• which are at least fluorescent in their second photochromic state and have fluorescence properties specific to the second photochromic state, and
• which display a return rate out of the second photochromic state into the first photochromic state, the return rate being inducible by means of a physical return signal;
• labelling the structure with the fluorophore;
• via a common objective, subjecting the sample in a spatially limited transfer-volume to the optical transfer signal transferring a portion of the fluorophore located in the transfer-volume into its second photochromic state;
• via the common objective, subjecting the sample in a spatially limited excitation-volume to an optical excitation signal exciting a portion of the fluorophore located in the excitation-volume and being in its second photochromic state for fluorescence, the transfer-volume and the excitation-volume having a common centre of maximum intensity of the transfer signal and of the excitation signal;
• via the common objective, subjecting the sample to the return signal, the intensity of the return signal having no minimum at the common centre of maximum intensity of the transfer signal and the excitation signal;
• wherein, at least in the direction of the optical axis of the common objective, a decrease of intensity of the transfer signal with the distance to the common centre of maximum intensity is substantially stronger than any decrease of the effective return rate of the fluorophore;
• detecting fluorescence light emitted by the excited fluorophore in its second photochromic state located in and about the common centre of maximum intensity of the transfer signal and the excitation signal;
• shifting the common centre of maximum intensity of the transfer signal and the excitation signal with regard to the sample;
• and
• repeating the preceding steps of subjecting and detecting for the shifted common centre of maximum intensity of the transfer signal and the excitation signal for three-dimensionally scanning the sample with the common centre of maximum intensity of the transfer signal and the excitation signal.