FCS method
The invention relates to an FCS method in which a sample that is to be measured and has fluorescent markers is illuminated with excitation radiation over a bleaching time in order to bleach selected fluorescent markers; and after bleaching has been carried out over at least one measurement period, FCS measurement data of the sample are acquired by illuminating the sample with excitation radiation and by detecting detection radiation brought about by the excitation radiation. The invention is characterized in that during the bleaching time, intensity values of fluorescence radiation that has been brought about by the excitation radiation which is directed at the sample for bleaching purposes are continuously or repeatedly acquired and compared with a threshold value, and the acquisition of the FCS measurement data is started when the threshold value has been reached.
1 . A fluorescence correlation spectroscopy (FCS) method, the method comprising:
using a light source to emit excitation radiation;
directing the excitation radiation toward a sample that contains fluorescent markers via an 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, smaller than the first, configured to define a second, larger measurement volume during a subsequent measurement phase;
detecting fluorescence radiation emitted from the sample in response to the excitation radiation using a detector;
operating a control unit operatively coupled to the light source, the optical system, and the detector 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.
2 . The method according to claim 1 , wherein the control unit is further configured to set the threshold value on the basis of a maximum permissible rate of change in the intensity values of the detection radiation per unit time.
3 . The method according to claim 1 , wherein the control unit is further configured to adapt a decay function to the acquired intensity values of the fluorescence radiation during the bleaching phase in order to predict when the threshold value will be reached.
4 . The method according to claim 3 , wherein the threshold value is set by the control unit by ascertaining a limit value of the decay function offset and adding to the offset a value of 0.5%, 1%, 2% or up to 5% of the offset.
5 . The method according to claim 1 , wherein the same detector is used to acquire both the intensity values during the bleaching phase and the FCS measurement phase.
6 . The method according to claim 5 , wherein the detector comprises a detector array with a plurality of detector elements.
7 . The method according to claim 6 , wherein the plurality of detector elements of the detector are configured to be:
connected and additively read during the bleaching phase; and
read individually or in predefined groups and evaluated during the acquisition of the FCS measurement data during the measurement phase.
8 . The method according to claim 3 , wherein the control unit is further configured to determine filter parameters for downstream processing of the acquired measurement data on the basis of the decay function.
9 . 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, smaller than the first, configured to define a second, larger 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.
10 . The system according to claim 9 , wherein the control unit is further configured to set the threshold value based on a maximum permissible rate of change in the intensity values of the detection radiation per unit time.
11 . The system according to claim 9 , wherein the control unit is further configured to adapt a decay function to the acquired intensity values of the fluorescence radiation during the bleaching phase in order to predict when the threshold value will be reached.
12 . The system according to claim 11 , wherein the threshold value is set by the control unit by ascertaining a limit value of the decay function (offset) and adding to the offset a value selected from 0.5%, 1%, 2%, or up to 5% of the offset.
13 . The system according to claim 9 , wherein the same detector is configured to acquire both the intensity values during the bleaching phase and the FCS measurement data during the measurement phase.
14 . The system according to claim 13 , wherein the detector comprises a detector array with a plurality of detector elements.
15 . The system according to claim 14 , wherein the plurality of detector elements of the detector are configured to be:
connected and additively read during the bleaching phase; and
read individually or in predefined groups and evaluated during the acquisition of the FCS measurement data during the measurement phase.
16 . The system according to claim 11 , wherein the control unit is further configured to determine filter parameters for downstream processing.