I have performed scanning mode of fluorescence measurement at different excitation wavelengths from 310 nm to 410 nm and collected the emission wavelength of each excitation wavelength from 350 nm to 600 nm. The picture below is the obtained fluorescent spectra. However, I am only able to obtain typical scanning-type emission spectra at excitation wavelengths from 310 nm to 340 nm. The rest of the excitation wavelength gives strange spectra (like something missing at the middle region). Can anybody explain this phenomenon? Is it normal or can we fix it if this is abnormal?
The problem is that you allowed the excitation wavelength to cross the emission wavelength. The excitation wavelength must always be lower than the emission wavelength. When they are equal, scattered light from the excitation monochromator will pass directly into the emission monochromator and saturate the detector. It looks like your instrument tried to compensate for that automatically by lowering the sensitivity, causing the sudden drops.
Adam B Shapiro From the fluorescent spectra above, all of the values of excitation wavelength are lower than the peak of the emission wavelength (450 nm) for a spacing of 50 nm from the emission peak. In addition, I performed the measurement using a fluorescence microplate reader. Is it possible that this is one of the factor contribute to this kind of reading?
You also have to consider that the plate reader has an upper limit to the fluorescence intensity it is capable of measuring. Above that limit, it will either saturate (show the same intensity at every wavelength) or automatically reduce the gain to keep the reading in range. To avoid this problem, you can either lower the gain setting or use a less concentrated sample.
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