Method for Spatially High-Resolution Investigation of a Structure, Marked With a Fluorescing Substance, of a Specimen

A method for spatially high-resolution investigation of a structure, marked with a fluorescing substance, of a specimen, the substance being capable of being converted from a first state into a second fluorescing state, and a spatially highly resolved image of the structure being prepared, the method comprising the acts of:

• irradiating the specimen with a predefinable small quantity of excitation light so that a small percentage of the fluorescent molecules transitions into the second state;
• detecting emitted light resulting from spontaneous decay of the individual excited fluorescent molecules in the second state via a detection device;
• calculating a center point of the emitted light for each of said fluorescent molecules with the aid of suitable statistical methods;
• at least one of returning the fluorescent molecules in the second state to the first state, and bleaching the fluorescent molecules;
• repeating the acts of irradiating, detecting, center-point calculating, and returning to the first state and/or bleaching a large number of times, for a different subset of fluorescent molecules in each case;
• and
• preparing an overall image from the individual images thus prepared, wherein a target protein in living cells is used as the structure marked with the fluorescing substance, in that a ligand complex encompassing the fluorescing substance is bound to an enzyme via an enzymatic reaction in the cell, the enzyme being expressed as a fusion protein together with the target protein to be investigated.

A method for spatially high-resolution investigation of a target protein in living cells marked with a fluorescing substance, the substance being capable of being repeatedly converted from a first state into a second state, the first and second states differing from one another in terms of at least one optical property, the method comprising the acts of:

• binding a ligand complex encompassing the fluorescing substance to an enzyme via an enzymatic reaction in the living cell, wherein the enzyme is expressed a fusion protein together with the target protein to be investigated;
• bringing the fluorescing substance, in a region of the target protein to be sensed, into a first state;
• and
• inducing the second state by way of an optical signal, spatially delimited subregions within the region of the target protein to be sensed being blanked out in controlled fashion.