The discussion revolves around the measurement of fluorescence intensity time traces, focusing on the selection of excitation and emitted wavelengths. Participants explore the criteria for choosing these wavelengths and their implications for accurate fluorescence measurement.
Participants express varying views on the importance of the excitation wavelength, with some suggesting it is less critical, while there is a consensus on the need to select an emitted wavelength based on the fluorophore's emission characteristics. However, the discussion does not reach a definitive conclusion on the best practices for wavelength selection.
There are assumptions regarding the fluorophore's properties and the specific applications being discussed, which may influence the choice of wavelengths. The discussion does not resolve the implications of these assumptions.
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.