Dapi stain emits at 488,
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Hello
I know this is not strictly a confocal question but, we are staining with DAPI and alexa-488 and get a VERY strange bleed through. 1. We illuminate with 488 and observe cytoplasmic staining (nothing in the nucleus). 2. We then illuminate with UV light from the HBO and observe a nice DAPI stain. 3. If we then re-illuminate with the 488 laser, the nucleus is now lit! (these are PFA fixed cortical neuron primary cultures). We have diluted DAPI to the point of loosing the signal, and this still occurs. Any suggestions? Any explication on what is going on? Thank you all. |
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Try it without any DAPI - if it still occurs it must be
autofluorescence (probably from the pfa) which is being photoactivated by the UV light. If so, try methanol fixation instead and see if it goes away. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alejandro Roth Sent: Friday, 5 December 2008 6:24 AM To: [hidden email] Subject: Dapi stain emits at 488, Hello I know this is not strictly a confocal question but, we are staining with DAPI and alexa-488 and get a VERY strange bleed through. 1. We illuminate with 488 and observe cytoplasmic staining (nothing in the nucleus). 2. We then illuminate with UV light from the HBO and observe a nice DAPI stain. 3. If we then re-illuminate with the 488 laser, the nucleus is now lit! (these are PFA fixed cortical neuron primary cultures). We have diluted DAPI to the point of loosing the signal, and this still occurs. Any suggestions? Any explication on what is going on? Thank you all. |
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I cannot offer an explanation but I can corroborate we have also seen the same phenomenon in wide field epifluorescence. We never investigated the problem further, in our case it was enough to just get the DAPI image last and work around the problem.
Leoncio A. Vergara MD Assistant Professor Laboratory of Protein Misfolding Diseases (lab-PMD), George and Cynthia Mitchell Center for Neurodegenerative Diseases Research. Director of the Optical Imaging Lab. (OIL), Dept. of Neuroscience and Cell Biology University of Texas Medical Branch (UTMB) 301 University Blvd Galveston, Texas 77555-0641 OIL phone: 409-772-3970 Lab-PMD phone: 409-7470019 fax: 409-7470015 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox Sent: Thursday, December 04, 2008 7:00 PM To: [hidden email] Subject: Re: Dapi stain emits at 488, Try it without any DAPI - if it still occurs it must be autofluorescence (probably from the pfa) which is being photoactivated by the UV light. If so, try methanol fixation instead and see if it goes away. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alejandro Roth Sent: Friday, 5 December 2008 6:24 AM To: [hidden email] Subject: Dapi stain emits at 488, Hello I know this is not strictly a confocal question but, we are staining with DAPI and alexa-488 and get a VERY strange bleed through. 1. We illuminate with 488 and observe cytoplasmic staining (nothing in the nucleus). 2. We then illuminate with UV light from the HBO and observe a nice DAPI stain. 3. If we then re-illuminate with the 488 laser, the nucleus is now lit! (these are PFA fixed cortical neuron primary cultures). We have diluted DAPI to the point of loosing the signal, and this still occurs. Any suggestions? Any explication on what is going on? Thank you all. |
 
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McDonald, David L |
Re: Dapi stain emits at 488,
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In reply to this post by Alejandro Roth-2
I have seen the same phenomenon in our Molecular Probes FluoCells
Slide (#1) using our widefield scopes. Strangely, it seems to occur (or is at least much more obvious) only in certain areas of the slide. Dave Dave McDonald Scientific Imaging Lab Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, DE-512 Seattle, WA 98109 206-667-4205 http://www.fhcrc.org At 11:23 AM 12/4/2008, you wrote: >Hello >I know this is not strictly a confocal question but, we are staining with >DAPI and alexa-488 and get a VERY strange bleed through. >1. We illuminate with 488 and observe cytoplasmic staining (nothing in the >nucleus). >2. We then illuminate with UV light from the HBO and observe a nice >DAPI stain. >3. If we then re-illuminate with the 488 laser, the nucleus is now lit! >(these are PFA fixed cortical neuron primary cultures). >We have diluted DAPI to the point of loosing the signal, and this >still occurs. > >Any suggestions? Any explication on what is going on? > >Thank you all. |
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Brotchie, Daniel |
Re: Dapi stain emits at 488,
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Hi,
By chance I came across the same question today when searching the 2004 Archive. Lots of useful comments. One subscriber - Xuejun - suggested it was photoactivation of DAPI causing the nuclei to emit green light when previously non had been detected. Xuejun solved the problem by reducing DAPI concentration and using NDFs: http://lists.umn.edu/cgi-bin/wa?A2=ind0406&L=confocalmicroscopy&D=0&P=6493 Daniel ------------------------------------------------ Daniel Brotchie Ophthalmology School of Clinical Sciences University Clinical Departments The Duncan Building Daulby Street LIVERPOOL L69 3GA UK Tel: +44 (0) 151 706 4017 Fax: +44 (0) 151 706 5934 E-mail [hidden email] -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dave McDonald Sent: 05 December 2008 15:34 To: [hidden email] Subject: Re: Dapi stain emits at 488, I have seen the same phenomenon in our Molecular Probes FluoCells Slide (#1) using our widefield scopes. Strangely, it seems to occur (or is at least much more obvious) only in certain areas of the slide. Dave Dave McDonald Scientific Imaging Lab Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, DE-512 Seattle, WA 98109 206-667-4205 http://www.fhcrc.org At 11:23 AM 12/4/2008, you wrote: >Hello >I know this is not strictly a confocal question but, we are staining with >DAPI and alexa-488 and get a VERY strange bleed through. >1. We illuminate with 488 and observe cytoplasmic staining (nothing in the >nucleus). >2. We then illuminate with UV light from the HBO and observe a nice >DAPI stain. >3. If we then re-illuminate with the 488 laser, the nucleus is now lit! >(these are PFA fixed cortical neuron primary cultures). >We have diluted DAPI to the point of loosing the signal, and this >still occurs. > >Any suggestions? Any explication on what is going on? > >Thank you all. |
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We have seen this too. I favour the photoconversion theory. Just always image DAPI last and you will not have a problem.
Cheers
Stephen Cody
2008/12/6 Brotchie, Daniel <[hidden email]>
Hi, -- Stephen Cody |
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In reply to this post by leoncio vergara
Hi,
I also had the same problem in widefield epifluorescence. With a long-pass filter you could clearly see the DAPI changing color from bright blue to greenish even after short exposures. In my case the user was mounting her slides in PBS/glycerol without any antifade agent. We solved the issue using very diluted DAPI and mounting in ProLong (I assume other antifade mediums will work as well). Cheers, Tommaso -- Tommaso Mello, PhD. Gastroenterology Unit Dept. of Clinical Pathophysiology University of Florence, Italy Phone: +39.055.4271-294 FAX: +39.055.4271-297Il giorno gio, 04/12/2008 alle 19.05 -0600, Vergara, Leoncio A. ha scritto: I cannot offer an explanation but I can corroborate we have also seen the same phenomenon in wide field epifluorescence. We never investigated the problem further, in our case it was enough to just get the DAPI image last and work around the problem. Leoncio A. Vergara MD Assistant Professor Laboratory of Protein Misfolding Diseases (lab-PMD), George and Cynthia Mitchell Center for Neurodegenerative Diseases Research. Director of the Optical Imaging Lab. (OIL), Dept. of Neuroscience and Cell Biology University of Texas Medical Branch (UTMB) 301 University Blvd Galveston, Texas 77555-0641 OIL phone: 409-772-3970 Lab-PMD phone: 409-7470019 fax: 409-7470015 -----Original Message----- From: Confocal Microscopy List [[hidden email]] On Behalf Of Guy Cox Sent: Thursday, December 04, 2008 7:00 PM To: [hidden email] Subject: Re: Dapi stain emits at 488, Try it without any DAPI - if it still occurs it must be autofluorescence (probably from the pfa) which is being photoactivated by the UV light. If so, try methanol fixation instead and see if it goes away. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ -----Original Message----- From: Confocal Microscopy List [[hidden email]] On Behalf Of Alejandro Roth Sent: Friday, 5 December 2008 6:24 AM To: [hidden email] Subject: Dapi stain emits at 488, Hello I know this is not strictly a confocal question but, we are staining with DAPI and alexa-488 and get a VERY strange bleed through. 1. We illuminate with 488 and observe cytoplasmic staining (nothing in the nucleus). 2. We then illuminate with UV light from the HBO and observe a nice DAPI stain. 3. If we then re-illuminate with the 488 laser, the nucleus is now lit! (these are PFA fixed cortical neuron primary cultures). We have diluted DAPI to the point of loosing the signal, and this still occurs. Any suggestions? Any explication on what is going on? Thank you all.
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