Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Russ,

This is just a guess, but I suspect that you are photobleaching the
chlorophyl in the leaves with your setup.  The filter set you describe is
a DAPI/FITC/Tritc triple band pass filter, which passes exitation
wavelengths in chlorophyl's exitation range as well as UV.  You are also
using a 100 W mercury bulb which is quite a lot of energy for a living
biological system, especially with UV excitation.   My suggestions would
be to reduce the amount of light if possible if your HBO burner has an
adjustable power supply, or with neutral density filters.  Better yet, I
would suggest you use a filter set that only exites with blue light.
Hope this helps.

Other possibilities are that you are killing the leaf cells with UV
excitation, and possibly your GFP tagged fungi as well.  I would switch
to a different filter set as the first priority.

Wes

On Thursday, January 03, 2002 12:53 PM, Russell Spear wrote:
>
>Date: Thu, 3 Jan 2002 11:53:47 CST
>From: Russell Spear
>To: [log in to unmask]
>Subject: live imaging of gfp on leaves
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>We are doing live time lapse imaging of gfp tagged fungi growing on leaf
>surfaces.   On returning to areas previously imaged we find that the
>normally yellowish autofluorescent background of the leaf has turned a red
>color.  The set up is using a HBO 100 Hg arc, ex 488 nm / emission 515
>nm filter multi spectral filter block (Chroma 83000) setup.
>
>We'd like to find a method to prevent this color change unfortunately
>mounting in some media (antifades, antioxidants) is not an option.
>
>Has anyone got any ideas?
>
>Thanks.
>
>Russ
>Russell N. Spear
>Sr. Research Specialist
>Dept. of Plant Pathology
>Univ. of Wisconsin-Madison
>
>[log in to unmask]
>Phone 608 263-2093
>Fax   608 263-2626


Dr. Wesley M. Garrett, Ph.D.
Germplasm and Gamete Physiology Lab
USDA/ARS/ANRI
Building 200, Room 101 A
Beltsville, MD 20705

E-mail: [log in to unmask]
Phone:  301-504-7413
FAX: 301-504-5123