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Heather,

to get some help from the list members it would be helpful if you'd let 
us know in which corner of the world you are located. Your G-mail 
account doesn't give a clue and you don't have a signature. I get my 
non-fluorescent coverslips from http://www.hecht-assistent.de/ (Order 
no.:1014) but I am not sure if you would consider ordering in Germany.

Also, you might want to let people know which decon software you are using.

Steffen

On 16.07.2013 06:23, Heather Bowden wrote:
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> 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
>


-- 
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

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