FCS method

A fluorescence correlation spectroscopy (FCS) system, comprising:

• a light source configured to emit excitation radiation;
• an excitation beam path optically coupled to the light source and configured to direct the excitation radiation toward a sample that contains fluorescent markers, the excitation beam path comprising an optical system configured to selectively provide at least two different numerical apertures (NAs), including:
• a first numerical aperture configured to define a first measurement volume during a bleaching phase;
• and
• a second numerical aperture, different than the first, configured to define a second measurement volume during a subsequent measurement phase;
• a detector configured to detect fluorescence radiation emitted from the sample in response to the excitation radiation;
• a control unit operatively coupled to the detector, the light source, and the optical system, the control unit configured to:
• control the optical system to apply the first numerical aperture during the bleaching phase;
• initiate the bleaching phase by controlling the light source to illuminate the sample with excitation radiation to bleach selected fluorescent markers;
• continuously or repeatedly acquire intensity values of the fluorescence radiation during the bleaching phase via the detector;
• compare the acquired intensity values to a predefined threshold value;
• and
• upon determining that the threshold value has been reached, control the optical system to apply the second numerical aperture, and initiate acquisition of FCS measurement data from the sample by continuing illumination with the excitation radiation and detecting resulting detection radiation via the detector.