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:[log in to unmask]] On Behalf Of Guy Cox
Sent: Thursday, December 04, 2008 7:00 PM
To: [log in to unmask]
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:[log in to unmask]]
On Behalf Of Alejandro Roth
Sent: Friday, 5 December 2008 6:24 AM
To: [log in to unmask]
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.