Should the excitation wavelength exactly match the peak of the fluorochrome’s excitation spectrum in order to be excited?
No. However, the excitation efficiency is the highest when the excitation wavelength correlates closely with a fluorochrome’s excitation peak. The excitation spectra of most fluorochromes are rather broad. Some fluorochromes also have long “tails” in their excitation spectra or have additional excitation peaks. Therefore it is not mandatory that the excitation wavelength match exactly with a fluorochrome’s major excitation peaks in order to efficiently excite it. Note that a fluorochrome’s excitation peak may shift with the changes in the binding environment or solvent. A more detailed explanation can be found in Chapter 1 and Chapter 3 of this handbook.
