In my research I'm enumeraring the florescence intensity (FI) Vs E-coli concentration in river water. I measure FI by Tecan I control 200 pro and E-coli by Membrane filtration.
Its likely because your fluorescent intensity readings are lower than background (or standard curve lowest FI numbers). You need to either make a new standard curve with more diluted samples, either increase amount of measured material (by centrifugation, etc) so readings from it will fall within the standard curve readings.
There is similar discussion here:
https://www.reasearchgate.net/post/Calibration_curve_of_linear-regression_Negative_slope_Should_the_I_ignore_the_negative_sign
This happens when the reader is not sensitive enough to detect the fluorescence of the samples. You can try increasing the concentration of the measured substance
One of the possibility is you may have problems with the solubility of your probe. This can lead to significant deviations in the recorded optical spectra (absorbance, fluorescence). Did you check your material is well soluble ?
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