I have observed that in one of my sample, the fluorescence lifetime was 0.6ns when excited with 400nm laser but it change to 0.3ns at 532 nm excitation? Why?
Based on the theoretical foundations this happens because of the excitation energy. By exciting your sample at 400 nm (more energetic than 532 nm) the population (amount of excited species) is greater. In addition, the energy levels reached by the sample excited at 400 nm are higher, that is, the longer the time to decay (lifetime).
I think , as Vincent Lecoeuche answered, it may be due to different transitions. The different lifetime may be due to presence of multi fluorophore in the sample. At 400 nm once species may be giving fluorescence and have a decay lifetime and at 532 nm it may have another lifetime.
Hassan Ahmed can you please show the absorption spectra of your fluorophore ?
In my opinion, this is because of photoselection of different molecules by changing the excitation wavelength. Please check seminal work by A P Demchenko
Are the emission wavelengths in the same position for both the excitations?
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