This discussion focuses on measuring fluorescence intensity time traces, emphasizing the importance of selecting appropriate excitation and emitted wavelengths. The excitation wavelength should ideally be chosen based on the fluorophore's absorbance spectrum, specifically at the wavelength with the highest absorbance, provided there is sufficient separation from the emission wavelength. The emitted wavelength is critical and should be selected based on the highest emission from the fluorophore, although alternative wavelengths may be used in specific applications like FRET to reduce crosstalk.
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Ygggdrasil said:The excitation wavelength does not matter so much as long as the fluorophore's extinction coefficient in that region is reasonable. You could take an absorbance spectrum of your fluorophore, and excite at the wavelength with the highest absorbance as long as there is reasonable separation between that wavelength and the wavelength at which you're monitoring emission.