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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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