Improve fluorescence signal

[giacomo]

Hello,
I have started to play with chi.bio a couple of weeks ago and I am really happy of its turbidostat capacity.
This week I tried to use it to measure the signal of an oscillating circuit, the repressilator (https://www.nature.com/articles/nature19841). In this circuit 3 fluorescence proteins oscillates out of phase when we use the plate reader to measure a growing culture in exponential phase.

When I tried with chi.bio I saw that for each channel the measurement is flat. As culture media we use the same one described in the paper in previous link, that is called imaging medium that should have low autofluorescence).

I used the following setup for the FP measurements:
– sCFP3 (EX: 395/30, EM1: 470/36, EM2:510/40)
– mVenus (EX: 500/55, EM1: 550/42, EM2: 583/33)
– mKate2 (EX: 595/25, EM1: 620/53, EM2: 670/60)

with Baseband= Clear for all the channels.

Am I doing something wrong?
Or is there someway to improve the sensibility of the chi.bio, through code or changing some of the components?

[harrison]

Hello giacomo,
That setup sounds sensible.

How bright would you say it is as a function of “the brightest GFP cells you have ever seen”. If it is <0.5% of “very bright” it will be difficult to measure due to other autofluorescence and filter bleed-through. This consideration will depend on what the plasmid copy number/RBS/Promoter strengths all look like.
Also, what OD level are you regulating it at? Something like 0.5 is probably optimal, if you have it very low (e.g. 0.05) then the signal-to-noise may be more tricky as well.

[giacomo]

Hello Harrison, thanks for your quick reply.
Probably we are in the situation in which the concentration of fluorescence protein is too low to allow detection from the instrument as it is now.
Are you thinking in some implementation to improve the fluorescence sensibility? Could be possible to implement an optical fiber to substitute the actual led to have higher power from an external light source?

[harrison]

The LED is pretty high power. Likely significantly more powerful than most external light sources. The real problem is filter bleed-through i.e. the amount of excitaiton light that makes it through the emission filter. Thus if you want better sensitivity best approach is to use an off-the-shelf spectrometer (i.e. something much better than the AS7341 filtered photodiode array device in there) as this will give much better spectral resolution. Does this make sense?
If you do want to try that, there are some good ones from Ocean Optics for ~1k mark that are easy to use and good price vs performance

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