Automatic G-factor calibration

A method of determining a G-factor correction to the measured fluorescence polarization of a first sample which contains at least a first probe comprising the following steps:

• illuminating the sample to effect fluorescence emission from the first probe with a beam of excitation light that is linearly polarized along a first axis;
• measuring the intensities of a first component of the fluorescence emission that is polarized along the first axis and a second component of the fluorescence emission that is polarized orthogonal to the first axis while the sample is illuminated with the beam of excitation light to obtain a measured fluorescence polarization of the first probe;
• switching the state of polarization of the beam of excitation light to a polarization state wherein said beam is linearly polarized along a second axis substantially orthogonal to the first axis;
• measuring the intensities of a third component of fluorescence emission that is polarized along the first axis and a fourth component that is polarized orthogonal to the first axis while the sample is illuminated with the beam of excitation light that is linearly polarized along the second axis;
• and
• calculating the absolute fluorescence polarization of the first probe based on the measurements of the intensities of the first, second, third and fourth components;
• and
• calculating the G-factor correction for the first probe from the absolute fluorescence polarization and the measured fluorescence polarization.