Decon wall
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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 -- ---------------------------------------------------------- |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** 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: > ***** > 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 Mail room: Marchioninistr. 15, D-81377 München Building location: Marchioninistr. 27, München-Großhadern |
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In reply to this post by Heather Bowden
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** 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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In reply to this post by Steffen Dietzel
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Steffen I am located in California. As for the software, it's SoftWorx. But I have been playing around with ImageJ to troubleshoot because it's faster. The cutting off problem only occurs when I deconvolve with apply correction in SoftWorx. I don't see it cut off at any other point. Chris: Wow what an interesting suggestion! I will check it out. Tom: Thanks! I'll check those out immediately. You're right that the signal isn't very good but I was hoping that it wouldn't make such an enormous difference. Thank you, Heather On Tue, Jul 16, 2013 at 1:06 AM, Steffen Dietzel <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > ***** > > 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: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<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 > > Mail room: > Marchioninistr. 15, D-81377 München > > Building location: > Marchioninistr. 27, München-Großhadern > -- ---------------------------------------------------------- E: [hidden email] GoogleVoice: 408-840-3643 http://www.arcbio.com |
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In reply to this post by Heather Bowden
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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 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:[hidden email]] On Behalf Of Heather Bowden Sent: Tuesday, July 16, 2013 12:23 PM To: [hidden email] Subject: Decon wall ***** 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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In reply to this post by ChrisWilms
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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 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 <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > 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 > > > > -- > > ---------------------------------------------------------- > > > -- ---------------------------------------------------------- E: [hidden email] GoogleVoice: 408-840-3643 http://www.arcbio.com |
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