Fluorescence bleed-through

[msilva]

Hello,

When culturing bacteria with GFP and mScarlet fluorescence there is a high level of bleed-through. I get readings at Ex: 457 / Em: 510 (that I am using to detect GFP) when growing cultures with mScarlet only, and vice-versa. The problem is much pronounced when trying to read mScarlet fluorescence, that is because cultures with GFP only read as much fluorescence as cultures with mScarlet only. This happens when trying to read mScarlet fluorescence at Ex: 523 nm / Em: 583 nm or Ex: 595 nm / Em: 620nm. As I want to do co-cultivation experiments with both strains simultaneously this makes the quantification of each strain very challenging. Is there any way to solve this issue?

[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 2 years, 10 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.

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