Method for ascertaining the orientation of molecules in biological specimens

A method of ascertaining the orientation of molecules in a biological specimen by total internal reflection, the method comprising:

• focusing illuminating light through an objective in different positions in a plane of a pupil of the objective so as to generate a plurality of respective differently oriented evanescent fields in the specimen;
• and
• correlating, with respective different orientations of molecules in the specimen, respective different fluorescence intensities resulting from the differently oriented evanescent fields,
• wherein the focusing illuminating light is performed so as to illuminate the specimen in evanescent fashion so as to generate a sequence of the differently oriented evanescent fields and respective images resulting from the fluorescence, and further comprising determining a respective first orientation of a first molecule based on a respective changing fluorescence intensity within the sequence.