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

Doug,
If you decide to go back to inverted, use Gelrite (plantmedia.com) 
instead of agar as the hardener.  It is optically clear.  Also, to 
get a very thin layer, try dipping the slide or coverslip in the 
molten media, then let harden in a moist chamber.  scrape off the 
other side before imaging.  You may want to seal the mount with wax 
or Valap (vasoline, lanolin, parafin 1:1:1)  or the whole thing will 
dry out over 8 hours.  scrape away excess media to get a good seal. 
Of course, now you limit your gas exchange but maybe you have figured 
out this part already.

Good luck,
Carol

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>I could use some advice with a sample preparation question for 
>confocal that was recently posed to me by a plant scientist.  He 
>would like to look at the fertilization of egg containing ovules 
>using GFP tagged pollen tubes in arabidopsis.  The dissected 
>portions of the appropriate plant parts (I'm not a botanist) would 
>be placed on an agar surface; pollen tubes actively grow on the agar 
>surface and enter the ovule to fertilize the eggs .
>
>The question becomes how to best image this process on our upright 
>Zeiss LSM510NLO-Meta.  My colleague would like to avoid the use of a 
>culture liquid, since the liquid may diffuse whatever chemotactic 
>agent draws the sperm into the ovule.
>
>I'm thinking that a dry lens (somewhere between 20x & 40x), with 
>decent working distance might be in order.  A coverslip could be 
>placed over the plant parts and agar (to prevent them drying out 
>over the 8 hr time period).  What I can't figure out is what to do 
>with the probable air gap under the coverslip if we can't fill the 
>space with an aqueous solution.  Glycerine?
>
>Note, an inverted scope would mean imaging through the agar.  While 
>my colleague did this earlier with a wide-field microscope 
>time-lapse of the GFP labeled pollen tube, I'm wary of doing this 
>with a confocal.
>
>Any ideas?
>
>Thanks,
>Doug
>
>--
>^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>Douglas W. Cromey, M.S. - Assistant Scientific Investigator
>Dept. of Cell Biology & Anatomy, University of Arizona
>1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
>office:  AHSC 4212         email: [log in to unmask]
>voice:  520-626-2824       fax:  520-626-2097
>
>http://swehsc.pharmacy.arizona.edu/exppath/
>Home of: "Microscopy and Imaging Resources on the WWW"


-- 
<><><><><><><><><><><><><><><><><><><><><><>
Carol Bayles, Manager
Microscopy,  Imaging & Fluorimetry (MIF)
Biotechnology Resource Center
160a Biotech Bldg
607-254-4860
www.brc.cornell.edu

Confocal and Multiphoton Microscopy
Nanobiotechnology Center
www.nbtc.cornell.edu

Cornell University
Ithaca NY 14853