Method and apparatus for detecting fluorescence signals in a three-dimensional region of a sample

A method of detecting an antigen on a biological surface which is incubated with at least one antibody that is compatible with the antigen and which is provided with photoswitchable marker molecules suitable for induced emission of signals as fluorescence signals and/or induced light signals, the method comprising:

• exciting different subsets of the photoswitchable marker molecules associated with the at least one antibody marked by the photoswitchable marker molecules and bound to the antigen in each case to emit signals;
• guiding detection radiation corresponding to the signals emitted from the excited marked at least one antibody along a detection beam path,
• imaging the detection radiation with an objective having an original depth of field in a direction of a detection axis in the detection beam path onto a detector, wherein the detector is configured for two-dimensionally resolved capture of the detection radiation and wherein the detection radiation corresponding to the signals from the different subsets are imaged in a time series;
• extending the original depth of field with an optical element located in a pupil of the detection beam path;
• evaluating image data read from the detector, the image data corresponding to two-dimensionally super-resolved detection radiation captured with the extended depth of field of the objective, wherein the super-resolved detection radiation includes fluorescence signals from individual photoswitchable marker molecules that are located relative to one another by distances below a resolution limit of the objective, to ascertain a number of the captured signals, wherein evaluating the image data read from the detector to ascertain the number of the captured signals emitted from the excited marked antibodies includes ascertaining a number of the captured signals emitted from the excited marked antibodies captured from each subset of the time series, and determining the number based on the numbers of captured signals captured from each subset of the time series;
• and
• storing and providing the number of the captured signals.

An apparatus for detecting fluorescence signals and/or induced light signals emitted from a three-dimensional region of a sample, the apparatus comprising:

• a detection beam path configured for guiding detection radiation corresponding to the signals emitted from different subsets of photoswitchable marker molecules in the three- dimensional region of the sample;
• a detection objective having an original depth of field in a direction of a detection axis in the detection beam path and configured for imaging the detection radiation;
• an optical element located in a pupil of the detection beam path, the optical element being configured to extend the depth of field over the original depth of field;
• a detector configured to capture from the detection radiation guided along the detection beam path, and to resolve in two-dimensions, the signals as image data, wherein the detection objective is configured to image the detection radiation onto the detector;
• and
• a computer configured to:
• cause the signals emitted from the different subsets of photoswitchable marker molecules to be captured by the detector in a time series;
• process the image data to generate two-dimensionally super-resolved image data, wherein the super-resolved image data includes fluorescence signals from individual photoswitchable marker molecules of the subsets of photoswitchable marker molecules, which are located relative to one another by distances below a resolution limit of the optical apparatus, and
• evaluate the image data read from the detector, based on a point spread function (PSF) of the detection objective, which is modified along the detection beam path by the depth of field that is extended over the original depth of field, to ascertain a number of the captured signals, to assign the number of signals to an image region, and to store the number of signals assigned to the image region, wherein evaluating the image data read from the optical apparatus to ascertain the number of the captured signals includes ascertaining a number of the captured signals emitted from individual photoswitchable marker molecules from each subset of the time series, and
• determine the number based on the numbers of captured signals captured from each subset of the time series.