Details can be found at this paper:
Fluorescence Correlation Spectroscopy: An Introduction to its Concepts and Applications
Fluorescence Correlation Spectroscopy: An Introduction to its Concepts and Applications
Now, two emission filters are required, whose transmission spectra ideally should not overlap to minimize crosstalk. As a rule of thumb, the transmission maxima of the bandpass filters should be at least 100 nm apart, if possible.
Checking and optimizing the alignment either directly by measuring the illumination profiles with a specifically designed focus scanner or indirectly by FCS calibration measurements can be quite tedious. One of the solutions to this problem is the use of a multi-linelaser as suggested by Winkler et al.[Winkler 1999]. Among the severe drawbacks of this technique, however, is the need for perfectly corrected microscope objectives. Moreover, to achieve focal spots of the same size, selective filters that reduce the diameter of the green beam have to be employed. Only recently, another elegant solution has been established. Using two-photon excitation, it is possible to excite two carefully selected spectrally different dyes with only one IR laser line. This technique will be discussed below in more detail.
Checking and optimizing the alignment either directly by measuring the illumination profiles with a specifically designed focus scanner or indirectly by FCS calibration measurements can be quite tedious. One of the solutions to this problem is the use of a multi-linelaser as suggested by Winkler et al.[Winkler 1999]. Among the severe drawbacks of this technique, however, is the need for perfectly corrected microscope objectives. Moreover, to achieve focal spots of the same size, selective filters that reduce the diameter of the green beam have to be employed. Only recently, another elegant solution has been established. Using two-photon excitation, it is possible to excite two carefully selected spectrally different dyes with only one IR laser line. This technique will be discussed below in more detail.