Hi, I have a problem concerning my LIF measurements, intensity Vs concentration fails to be linear, also some dilutions have spectra lower than blank. Im working with a bacterial suspension about 10^8 cfu/ml. diluted from 10^-1 to 10^-10. also I tried another dilution sequence 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, etc.... some dilutions have fluorescence intensity lower than blank which was normal saline. i need help, where am I going wrong.
My guess is that light scattering is the problem. Bacterial suspensions scatter light, which is why they are turbid. (Turbidity is actually used as a standard measurement of bacterial cell density. This is known as the McFarland measurement and there is a special instrument for measuring it.) The denser the bacterial suspension, the more light scattering interferes with detection of the laser-induced fluorescence. At a high enough density, there will even be multiple scattering as another effect. I would expect LIF to give a linear result only for very dilute bacterial suspensions.
Additional confounding effects may arise from differences in the growth phase of the bacteria, their shape, their degree of aggregation, their species, and whether they shed any outer membrane vesicles or other scattering substances into the medium.
I agree with Adam B Shapiro ,
Fluorescence is rarely as linear as absorption, as mathematical models predict. If your sample has a very high optical density, the Inner Filter effect may occur, where only a portion of the suspended particles will emit fluorescence. In addition, Rayleigh/Mie scattering can also occur, making it difficult to detect the signal, if the sample is not very homogeneous or if the bacterial cells are aggregated. To minimise these effects, I suggest checking the optical density; if the values are higher than 0.3, the linearity of the fluorescence may be affected. In addition, measuring fluorescence at 90° can reduce interference from scattering.
I hope I've been of some help,
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