Hi everyone,
I would really appreciate it if someone can tell me when and why a raw fluorescence starts below zero. In the picture attached below the blue colour curve starts from a negative value. My delta Rn values are quite low and I belive the negative start of the blue curve (raw data) is effecting the normalisation.
Can anyone give some insight?
The only way I can think of to get a negative fluorescence reading is if the intensity of a reference is being subtracted, and the intensity of the reference is higher than the intensity of the sample.
Hi Poonam Singh you can do a base line correction prior to normalization This will not affect your data If you are using a software like Origin Lab, this would be very straight forward. I have experience this scenario in the past whenever it involves the protein or DNA. One o the possibility is light scattering associated with solubility issues. Especially this is possible to happen if you are using an instrument such as a plate reader where they have option to do a top or bottom reading It is ideal to record the fluorescence emission in a 90 degree angle to the excitation light. If you use quartz material for fluorescence reading, your spectra will be much smoother and in the base line. Good Luck!
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