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Hi Heather,

in our hands poly-lysine seemed to increase autofluorescence by binding riboflavin. So when using culture media with high riboflavin levels the autofluorescence levels were much higher than when using low riboflavin solutions. Importantly this binding seems to be quite stable: the difference persisted even after multiple wash steps with riboflavin free imaging solutions. 

Depending on the culture medium you are using, you might want to switch to a low riboflavin medium at least a day or two prior to imaging.

Hope to help,

Chris

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> 
> Dear Confocal Microscopy List,
> 
> I have hit another wall with a very old deconvolution microscope (late
> 90's).
> 
> I have images from fixed slides that have DAPI and a green dye.  I grow the
> cells on round coverslips that are pre-coated with Poly-D-Lysine or
> Poly-L-Lysine from the supplier (BD).
> 
> 1) I'm having issues with green autofluorescence. As a result, my
> deconvolved images don't look very good. They are very grainy and almost
> look homogeneous. I'm confident it's the coverslip that's causing it but I
> can't buy them without the poly-lysine and I can't grow the cells without
> poly-lysine either. I tried a round Fisher coverslip with no coating, but
> that had autofluorescence too. I have been using extra long rectangular
> coverslips for mounting because those don't have autofluorescence. Any
> suggestions would be incredibly appreciated.
> 
> 2) When I deconvolve a slide I made, it cuts off the right side of the
> image and places it on the left. There's usually a big line where it made
> the cut. Why would this occur? When I use a prepared fixed slide from
> Invitrogen (Fluocell #2), it cuts off the left side and puts it on the
> right! This only happens when I deconvolve wavelengths separately. I do not
> touch the "pass wave" function (what does that do?).
> 
> 3) What glass slides do you use? Cat #'s would be greatly appreciated as
> the ones Invitrogen recommended aren't available anymore and Fisher does
> not know (and their slides autofluoresce).
> 
> Thank you so much for any help!
> 
> Heather
> 
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