The invention describes a method of determining the position of fluorescent markers in a sample (4), with a high spatial resolution. To this end, the sample (4) is illuminated with an exciting light beam (11), while the sample (4) is simultaneously scanned by a particle beam (3). During scanning, markers will be impinged upon by the particle beam (3) and will be damaged, in such a manner that the marker impinged upon will no longer emit fluorescence radiation. This leads to a reduction of the flux of fluorescence radiation. This reduction is detected. Seeing as the position of the particle beam (3) w.r.t. the sample is known at the moment that the marker is damaged, the position of the marker in the sample is, accordingly, also known.