A simplistic answer: do not use UV excitation with GFP since it is designed
to be excited with blue laser and emit green. What you see with UV
excitation is most probably auto-fluorescence, that you should see as faint
green in blue laser configuration.
About the emission filter you should use a band pass filter like BP515-540
instead of LP515 (> 515) to make sure only GFP fluorescence spectrum is
"seen" by the detector.


***********************
Hugo Dilhuydy, ing.
Laboratoire de microscopie
Centre de recherche
Institut universitaire de gériatrie de Montréal
4565 chemin Queen-Mary
Montréal (Québec)
H3W 1W5 CANADA
Tél: (514)-340-3540 poste 3222
Fax: (514)-340-2801
http://www.criugm.qc.ca

----- Original Message -----
From: Shea Miller <[log in to unmask]>
Newsgroups: bit.listserv.confocal
To: <[log in to unmask]>
Sent: Wednesday, August 02, 2000 11:20 AM
Subject: GFP


> Hello everyone;
>    I have just started looking at a GFP-transformed fungus growing on
plant surfaces, using regular fluorescence microscopy.  I can clearly see
the fungus using two filter sets: a UV set (ex/em 365/>420) and a blue
filter set (ex/em 470/>515).  While the fluorescence in blue light is much
brighter, stuff that is blue/blue-white with UV illumination is green in the
blue light.  is the "extra" green in the blue light likely to be where the
GFP is not as concentrated, and hence doesn't show up in the UV, or is it
more likely to be some other compound that has similar excitation/emission
characteristics?  I have done a fair bit of reading on the subject, but am
still feeling a bit vague, and would really appreciate hearing from those of
you who have some first hand experience with GFP.
>
> thanks in advance
> shea
>
>
>
> Dr. S.Shea Miller
> Agriculture & AgriFood Canada
> Eastern Cereal and Oilseed Research Centre
> Rm. 2068 Neatby Building
> Central Experimental Farm
> Ottawa, Ontario
> Canada   K1A 0C6
> Phone:  (613) 759-1760
> Fax:  (613) 759-1701
> E-mail:  [log in to unmask]