Fluorescence bleed-through

[harrison]

Hello,
Our team is usually using GFP at 450nm excite ~550 read, and then mScarlet at 595 excite and 670 read. Indeed the 620 read seems to bring a lot of GFP bleed-through, but this appears to be eliminated if you go even higher on the red edmission band.

[harrison]

AFter talking to our team they were surprised to hear you have GFP when using mScarlet only. What media and strain are you working with ? This might be down to autofluorescence of one or the other.

[msilva]

I’ve used LB and M9 medium with some trace elements for P. putida, the bleed-through also happens with the M9 medium. I believe this M9 medium should not be auto fluorescent. The strain is P. putida EM42, a derivative of P. putida KT2440.

• This reply was modified 1 year, 12 months ago by msilva.

[vuthuhien97]

Hi Msilva and Harrison, I am facing a similar struggle as well. In my experiment, I tested various plasmids, ranging from green to red fluorescent, based on this paper with different emission/excitation settings. Surprisingly, despite adjusting the emission/excitation (Em/Ex) parameters to be far apart, I obtained green fluorescent signals even when the plasmid in the culture is expected to be red.
Also, I tested a non-fluorescent strain but still detected some fluorescence.
For my experiments, I used M9 medium and LED intensities ranging from 0.2 to 0.5. The plasmid used is a high copy number plasmid.

[Author]

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