GFP fluorescence in red channel?

Model Organism: Worms Methods & Reagents
1

Hi all,

I was trying to image a red-tagged fluorescent protein in the presence of a GFP marker. When looking though the TexasRed channel on my scope, I thought, “Wow, my transgene is really bright!”, when in fact I believe I was seeing the GFP in the red channel. What could be causing this and how can I avoid this happening in the future?

2

Hi,

What set up are you using? Knowing this might help narrow down the possible causes and help with a work around.

From your description, I guess bleed through/cross talk from the gfp fluorescence/channel into the red channel is the most likely reason. That said, lots of possibilities spring to mind;

  1. You could try using a narrower band pass filter of the gfp channel to reduce the bleed
    through.
  2. What filter are you using for the red channel? Careful choice here of a LP filter could
    separate the signals more effectively. You lose some sensitivity but also the background.
  3. Is the gfp signal very strong?
  4. What’s the excitation source? If you’re using a Ar/Kr/HeNe laser source then dropping the
    568nm and 633nm channels should give just the 488nm fluorescence and 2 black
    channels. If they’re not, then you can ‘tune’ the system until they are…
  5. You could use sequential imaging.

There are lots of possibilities…perhaps your set up would shed more light on the problem

Regards

Steve