Fluorescence and OD

[marco.fondi]

Hi. Sorry if this question has been answered elsewhere but I could not find a clear response.

The fluorescence that chi.bio measures is somehow normalized by OD? In other words, the output is a fluorescence/cell value or the overall fluorescent signal of the culture? Specifically, we are measuring the fluorescence of an mCherry constitutively expressed in E. coli (starting OD = 0.1) and we are letting the culture grow from OD=0.1 to OD=1. Should we expect the signal to increase together with OD (as in the case of a measured cumulative fluorescence) or should we expect the signal to remain constant (as in the case of measured fluorescence/cell)? Also, the emit FP2 values are in the range of 1.6 while in most of the documentation of chi.bio typical value are in the order of 0.01 or even lower. Is this indicative of something strange?

Thanks in advance for your assistance.
best
m.

[harrison]

Hello Marco,

Yes, if you see the supplementary of the PLOS Biology paper it details how the “normalisation” works. Basically it is looking at the amount of scattered (excitation) light and dividing the emission light by this to try to mitigate the OD effect. This reduces (by say ~90%) the influence of OD but it is not perfect, so the fluorescence vs cell at OD 0.5 vs 0.1 may be somewhat different.
In terms of the large FP2/X values it is entirely dependent on what the excitation and emission wavelength bands are. It sounds like your emission wavelength band is not far enough away (e.g. not high enough) compared to the excitation wavelength, hence loads of the excitation light is getting through and this will mean a very poor signal-to-noise ratio in the measurement.

[Fabian.MA]

Hello,

According to Harrison’s answer, as Marco said, if it is normalized by the OD, constant GFP production will return flat curves instead of exponential profiles, right?. In my case, this is not very convenient, can i get the direct F.A.U units in some way?. I work with constitutive promoters and I need to test them as a proxy of the fluorescent population.

Best.

Fabian

[harrison]

Hello,
Yes, indeed it will return flat curves. But, the LEVEL of that flat curve should reflect the constant production rate. I.e. if you have twice the production rate you should get twice the measured fluorescene signal.
You can get the raw info from the .csv file by multiplying the FP reading by the baseband signal which gives you absolute amount of light/fluorecence that has been delivered.
However, there may be greater inter-reactor variability due to any change in LED intensity now not being normalised away…

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