Qualitative analysis of amyloid formation can be monitored by ThT fluorescence study(Ex:440nm and Em: 485nm). Usually, at a given time, I record the Emission spectra of the sample thrice and consider the average value. This was not a problem as the Em at 485nm didn't vary much. However, when I have changed the medium from phosphate buffer to cerebrospinal fluid, I am getting some strange peak intensities in all the three readings (some exceptionally high, while some normal/expected). I am attaching a screenshot of the result below.
Can anyone give me some valuable suggestions on it if it is an experimental error or an instrumental error?
Though I do not have experience with ThT or cerebrospinal I could make some general comments. How many times you have repeated this experiment? Do you always get similar variation in intensity? The first thing that comes to mind is that the system may not be at equilibrium. Is the solution homogeneous? Do you expect any type of aggregation formation? Aggregates could also give fluctuating signal. I also see a peak/shoulder around 520 nm. Is this expected? I hope you have made sure that the solvent do not have any fluorescence impurity.
@tuhin khan, I testing kinetics of aggregation so
Hi..
I am testing kinetics of aggregation so presence of aggregates is obvious. The peak at 520 is due to Raman Scattering I guess. I have repeted the experiment several times and I have noticed the same abnormality in all of them. Presence of fluorescent molecules in the medium can also be a considerable fact. However, I always substract the baseline given by the medium while ploting my graphs. So I dont think this should be affecting my result to such an extent.
Hope for further suggestions on it. Thanks!
Depending on the stability and size of the aggregates the fluorescence intensity may fluctuate. You may check the effect of aggregates by monitoring the Rayleigh scattering intensity. For a stable solution the Rayleigh scattering should not change with time. I do not think the 520 nm peak/shoulder is due to Raman scattering. Raman signal is sharp and will be too weak to detect here.
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