I have performed 3D scans for fluorescence studies, which consist of contours at the emission wavelength. how to draw useful information from these 3D maps and to understand the dynamics of the reaction? Kindly give the possible explanation for the image file attached.
My interpretation of this figure is that you used excitation wavelengths separated by 10 nm, including 450, 460 and 470 nm, and measured fluorescence emission at wavelengths offset from the excitation wavelength by +/- 5 steps of 1.25 nm, and vice versa. If that is right, then all the figure shows is the detection of the excitation light when the wavelength settings of the excitation and emission monochromators overlap (which is something that should not be done because it is bad for the detector). If there was any fluorophore present, there is no signal from it.
Fluorescence emission of fluorophores always occurs at wavelengths that are longer than the excitation wavelengths (an exception would be for dual-photon laser excitation), so the emission monochromator should always be set for a longer wavelength than the excitation monochromator.
Yes! you're right. this is the case of complete quenching or turn off situation. According to your explanation, I guess 3D scan just shows the emission of fluorophore at its specific wavelength. Means no other qualitative information can be drawn from it.
Hi Shubhangi,
If there is only one flourophore in your sample or the fluorescence is quenched completely, 3D Scans do not provide any additional information.
It can, however, become a useful tool if you have several different fluorophores in your solution. In that case, you can use the 3D scan to determine the fluorescence spectra of each single fluorophore in the sample. It is also often used to detect excitation energy transfer between fluorophores.
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