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Dear heather ,
I hear people are solving similar problem using a certain LCTF  camera called Nuance .The main principle of that camera is based on the spectrum image capturing and unmixing .Since the autofluorescence is mainly caused by the cover slip or the poly-lysine in your case . I believe that is the right solution for you .And deconvolution  on the other hand is aiming to deconvolve the defocused signal ,so it will greatly increase the s/n ratio,but may not work  well on the autofluorescence  extraction .


Charlie 

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Heather Bowden
Sent: Tuesday, July 16, 2013 12:23 PM
To: [log in to unmask]
Subject: Decon wall

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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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