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Dear Chris,

Does riboflavin increase autofluorescence of untreated cells or increase
background?

Thank you,

Heather


On Tue, Jul 16, 2013 at 2:23 AM, Christian Wilms <[log in to unmask]> wrote:

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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
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > 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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