Epi-fluorescence noise

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Epi-fluorescence noise

Postby genegun » Apr 07 2005 4:25 pm

Recently I seed the cell line on cover slip ( plastic) and then I transfect some EGFP plasmid into the cell. When I saw it under epi-fluroscence microscope, I found except the transfected cell glowing green, all other cell glowing yellow.
since I culture the cell in DMEM with phenol red, is that possible the phenol red goes into cell by transfection reagent treatment and keep glowing under microscope? or it is the cover slip that cause the yellow fluorescence ,which many people suggest the plastic will have inherent fluorecence?
But before this experiment, I did another time, I found no problem at all, just the green cell glowing. What 's wrong ?
Also long term exposure of energy will cause the yellow even stronger this time.
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Postby seankim » Apr 08 2005 7:56 pm

Recently I seed the cell line on cover slip ( plastic) and then I transfect some EGFP plasmid into the cell.


should be using glass cover slips. plastic diffracts UV a lot more than glass. unless you have other refernces that have used plastic cover slips.


I found except the transfected cell glowing green, all other cell glowing yellow.


this makes me suspect you are getting some autofluroescence. are the cells postmitotic? or do you get this in all the cells that are not tfx'd?

since I culture the cell in DMEM with phenol red, is that possible the phenol red goes into cell by transfection reagent treatment and keep glowing under microscope?


if so, why would it be yellow? I have never heard tissue culture dye causing problems in microscopy?

or it is the cover slip that cause the yellow fluorescence ,which many people suggest the plastic will have inherent fluorecence?
But before this experiment, I did another time, I found no problem at all, just the green cell glowing. What 's wrong ?


are you sure it was plastic cover slip? you can use plastic coverslips if youmean to do colorimetirc staining of the cells.

I havenever looked at plastic cover slip under a confocal or florescent scop for that matter. i don't know what color cells will glow in plastic.

in tissue culturte plates, they don't seem to mind too much. so you would think it will be ok... but to be sure always use glass cover slips

does the problem go away when using glass?
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Postby adi » May 04 2005 4:21 pm

phenol red used as an indicator in DMEM will give a whacking amount of autofloroscence. did you image your cells in other channels and see if the untransfected cells still light up?

best
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