How to measure fluorescence intensity time trace?

[Yinxiao Li]

I understand that fluorescence intensity time trace is constantly monitor the fluorescence intensity and plot it over time. But the question is at which excitation wavelength? Also, what is the emitted wavelength that is being measured? I suppose it should be two particular wavelengths, but how is the wavelength selected?

 

[Ygggdrasil]

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.

 

[Yinxiao Li]

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.

Your answer is very helpful..But--Sorry I missed the part of emitted wavelength...I just changed the question. The extra question is what is the emitted wavelength being measured? The fluorescence intensity should be measured under one particular emitted wavelength, right?

Sorry that this is a simple question...

 

[Ygggdrasil]

Usually you would measure the emission spectrum of the fluorophore you're using, then choose the wavelength that shows the highest emission. However, for certain applications (e.g. measuring FRET), you might choose a different wavelength to minimize crosstalk from other fluorophores in your sample.