I am doing an experiment about measuring quantum yield of fluorophores and I have some questions about it:
May I use fluorescence intensity instead of integrated fluorescence intensity to find quantum yield of fluorophores?
Also, should the absorbance measured be at the same wavelength as the excitation wavelength of fluorescence for both reference standard and sample?
Grateful if anyone can provide some suggestions to me!
Here is a useful reference on the technique of measuring fluorescence quantum yield.
http://www.fluortools.com/software/ae/documentation/qy
But how to get the integrated fluorescence intensity? The instrument I used doesnt give me the area and I tried to get the fluorescence emission spectrum of both sample and reference standard, but their spectrum aren't typical nice peaks.
If you have your spectrum as a text file, then you can integrate the area under the curve with a computer program. Here is a way to use Excel to do it my the old trapezoid method, which is basically the same as counting the squares on graph paper.
http://www.statisticshowto.com/how-to-find-the-area-under-a-curve-in-microsoft-excel/
Collect the spectrum at as many nm steps as possible to maximize the accuracy.
I've also used the cut-and-weigh method, although not since 1985.
A bigger problem is why the emission spectra aren't nice peaks. Is there a problem with the sensitivity of the instrument? Can you show a picture of the spectra?
The picture I attached is one of the examples of my fluorescence spectrum using different concentrations. I am not sure whether I used a wrong excitation wavelength or not? In this picture, should I use a lower excitation wavelength to get the full spectrum or others? And also, can I just use this graph to measure the area? As the measurement of quantum yield is just using the factor of gradient of both reference and sample.
I would suggest using a lower excitation wavelength in order to get the whole emission spectrum. Of course, the emission intensity will decrease if the excitation wavelength is not at the peak of the excitation spectrum.
I think selection of wavelength range for recording of the fl. spectra has been improper, it should be selected in a manner so as to cover the full range of fluorescence with no significant contribution from scattered light and other artifacts. After this, you can find the area under the curve simply on any std. software program, like excel/origin/matlab or any graphical program (some of which may also be free downloadable). However, use of fl. intensity in the place of area under the curve will incorporate errors in your results (the same is true for area calculation with significant non-fluorescence contribution in your spectra as well).
The fluorescence intensity values must be corrected via using the correction values of the fluorometer you used for the measurements. Also, the absorbance of your sample at the excitation wavelength must be 0.1 or lower (if you use 1cm path length fluorescence cell) to avoid the inner filter effect. you can use excel to calculate the area under the curve (refer to some tutorials on youtube). If I were you, I would calculate the QY by hand first, and then use the computer (e.g. excel) to calculate it. Good luck
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