bacteria and plant roots
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I have a user that I’ve been training on our Zeiss LSM
510-Meta. Her samples are plant root tips (a native desert plant,
“quail bush”) and she is looking for a specific type of bacteria
that may be located on the surface of the roots. To differentiate the one
type of bacteria from all the others she has done FISH using CY5. Her
expectation is that these specific bacteria will only constitute a subset of
the entire bacterial population, consequently we may have to look around to
find them. Wow, plant tissue certainly has a lot of non-specific
background fluorescence throughout the visible range (I usually work with
mammalian tissues)! Is there a way to reduce the plant autofluorescence? Is it even reasonable to think that we could image a small
spot of FISH fluorescence with enough signal to overcome the background autofluorescence? Is there a better/different way to do this? 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: [hidden email] voice: 520-626-2824 fax: 520-626-2097 http://swehsc.pharmacy.arizona.edu/exppath/ Home of: "Microscopy and Imaging Resources on the
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Mayandi Sivaguru |
Re: bacteria and plant roots
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Doug, You can do many things to overcome these problems. 1) Usually, I have not seen autofluorescence in plant roots at cy5 channel Ex 640 nm and beyond, and emission Beyond 650 (but band pass) . However, if you do see in your roots you can immerse them with 0.1% Toluidine blue (15 min) before mounting followed by a 5 min wash in PBS. 2) you should be able to see the cy5 spots in the bacteria, if the bacteria is present on the surface and not washed away during the procedures. You can perform this in the type of bacteria only sample to see whether the hybridization works before performing on a plant sample. 3) You can hybridize them also with an antibody dual conjugated with Alexa 647 and nanogold(5-10 nm), and if the fluorescence fails (or to double confirm the same fluorescent spot to gold particle) you can still use the reflectance mode of the confocal to image the gold particles (in this way you can also take the same sample to TEM if needed to image the gold particles). 4) immobilization of the bacteria on the surface and holding all of them during the process is the key. Good luck Shiv At 10:11 AM 7/10/2008, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Microscopy Facility Manager 8, Institute for Genomic Biology University of Illinois at Urbana-Champaign 1206 West Gregory Dr. Urbana, IL 61801 USA Office: 217.333.1214 Fax: 217.244.2496 [hidden email] http://core.igb.uiuc.edu |
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In reply to this post by cromey
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Hi, Doug – You should have your user record the
autofluorescent spectra from the sample on the If you want more details on how to do
that, let me know. Best, Holly From: I
have a user that I’ve been training on our Zeiss LSM 510-Meta. Her
samples are plant root tips (a native desert plant, “quail bush”)
and she is looking for a specific type of bacteria that may be located on the
surface of the roots. To differentiate the one type of bacteria from all
the others she has done FISH using CY5. Her expectation is that these
specific bacteria will only constitute a subset of the entire bacterial
population, consequently we may have to look around to find them. Wow,
plant tissue certainly has a lot of non-specific background fluorescence
throughout the visible range (I usually work with mammalian tissues)! Is
there a way to reduce the plant autofluorescence? Is
it even reasonable to think that we could image a small spot of FISH
fluorescence with enough signal to overcome the background autofluorescence? Is
there a better/different way to do this? Doug ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Douglas
W. Cromey, M.S. - Assistant Scientific Investigator Dept.
of Cell Biology & Anatomy, office:
AHSC 4212 email:
[hidden email] voice:
520-626-2824 fax: 520-626-2097 http://swehsc.pharmacy.arizona.edu/exppath/ Home
of: "Microscopy and Imaging Resources on the WWW" |
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