PSF coloc2 colocalization

Dear all,

I’m trying to measure colocalization with coloc2, and we are using 2D pictures. Can someone explain to me how I can calculate the Psf? I know the approximation: PSF = lamda / (2 x NA x pixel spacing in nm ). But what is the correct lamda value when we use two different laser excitation wavelengths?

Thank you very much!