I am using the filter pair of 480/535 and trying to measure fluorescence. The instrument that I am using is Spectramax M2. The Cut-off filter for this pair is showing 530nm in this machine. So what would happen if we do not use the cut-off filter? The fluorescence values are not satisfactory. Can anyone help me?
The cutoff filter reduces the amount of excitation light that reaches the detector due to scattering and reflections. Without the cutoff filter, the background is higher.
The cut-off wavelength is used to filter out the excitation wavelength. In your case, the excitation might be around 480 nm. If you don't use it, the scattered/reflected excitation light will contribute to your measured fluorescence spectra, resulting a bump or very intense excitation peak in your spectrum.
If your goal is to record the full emission spectra of your dye, it is recommended to use a fluorimeter, which can tune your excitation/emission filter setting much more precisely (say you can set excitation of 485 nm, collect emission from 500-700 nm).
A quick fix in your fluorescence value (assuming you mean you get low intensity and noisy spectra) could be to increase the emission/excitation slit value to collect more photons or use higher concentration. If your emission spectra look like not starting from 0ish especially at the left shoulder, the filter sets may need to be changed.
As far as I understand, the SpectroMax M2 is a dual monochrometer spectroscopic plate reader, so there should be no need for filters. The first monochrometer selects your excitation, the second one your emission. I am not sure about this instrument, but usually either one can be fixed, while the other is scanned across the spectrum (excitation < emission wavelength, emission > excitation wavelength).
https://www.moleculardevices.com/sites/default/files/en/assets/data-sheets/br/spectramax-m2-m2e-microplate-readers.pdf
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