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Well, this was hard to read...
As to FRET, "FRAP approach" (Acceptor photobleaching) in FRET sounds to be
very destructive, and who knows how to correct for the damage the cell has
encountered (increased autofluorescence, intensity pixel shift due to the
laser, of high power, induced re-localization of molecules, etc.).
And confocal FRET is very disappointing, as it is very VERY!!! noisy, due
to?:
1. a shameful noise of PMTs (I do not mention the junky META detectors of
Zeiss - FRET incompatible!).
2. High autofluorescence of the cell, which overlaps with the Acceptor
spectra or donor spectra when Acceptor is photobleached, CFP and YFP pair is
one of the best examples (to avoid?). And no correction has been offered for
these artefacts either.
3. 12 bit dynamic range of Zeiss confocals seems to be another "serious"
joke that has not to be ignored.
4. Lipofectamine or FuGene6 transfection gives rise to non-specific
autofluorescence across the spectra, another problem for those who prefer
live samples for FRET analysis.
5. This list is obviously incomplete, I would welcome all people who have
extensive experience in physics and little in biology, and vise versa to
contribute to this very important issue, such as noiseless, quantitative
FRET.
Biotech people are welcome as well, especially those who are able to make
money out of the true basis, not out of the speculation.
Therefore,
1. wide-field microscopy may provide advantage over confocal one, as there
are a large number of good CCDs on the market (one may need to avoid noisier
EM-CCDs).
2. Using Acceptor with the emission spectra of ca. 580-700 nm is expected to
reduce noise due to very low cell autofluorescence in the red spectra.
And I do hope that FLIM and FRET would become much more useful as well.
It remains to see if Zeiss (or Germany headed by Mrs. M.) would bring
talented people and/or the production of their scopes back to Germany,
functional young brains inclusive.
Best,
Vitaly Boyko
NCI-Frederick,
301-846-6575
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Robert Zucker
Sent: Thursday, November 17, 2005 11:43 PM
To: [log in to unmask]
Subject: Re: FRET in Confocals
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
<FONT face=3D"Default Sans Serif,Verdana,Arial,Helvetica,sans-serif" size=
=3D2><DIV>Hi Julio </DIV><DIV>the test you describe is for Gross FRET. </DI=
V><DIV>Am I correct that when one does sensitive FRET, you =
look for the overlapping of pixels or pixels that are adjacent to one =
another. This is the test that molecules are indeed interacting on a molec=
ular level. </DIV><DIV> </DIV><DIV>Is it not essential to test for col=
ocalization of your confocal system using small submicron beads? =
This insures that the machines are registering spectra correctly.=
Without doing this test, FRET data may be misinterpreted. </DI=
V><DIV>It has been reported that many systems have problems colocalizing&nb=
sp;UV laser lines with the visible laser lines.</DIV><DIV>Bob</DIV><DI=
V> <BR></DIV><DIV><BR>Robert M. Zucker, PhD<BR>U.S. Environmental Prot=
ection Agency <BR>Office of Research and Development<BR>National Health and=
Environmental Effects Research Laboratory<BR>Reproductive Toxicology Divis=
ion, MD 72<BR>Research Triangle Park, North Carolina, 27711<BR>Tel: 919-541=
-1585; fax 919-541-4017<BR>e-mail: <A href=3D"mailto:[log in to unmask]"=
target=3Dblank >[log in to unmask]</A><BR><DIV><BR></DIV><FONT color=3D=
#990099>-----Confocal Microscopy List <[log in to unmask]>=
wrote: -----<BR><BR></FONT><blockquote style=3D"PADDING-RIGHT: 0px; PADDIN=
G-LEFT: 5px; MARGIN-LEFT: 5px; BORDER-LEFT: #000000 2px solid; MARGIN-RIGHT=
: 0px">To: [log in to unmask]<BR>From: Julio Vazquez <jvazque=
[log in to unmask]><BR>Sent by: Confocal Microscopy List <[log in to unmask]
BUFFALO.EDU><BR>Date: 11/17/2005 03:06PM<BR>Subject: Re: FRET in Zeiss<B=
R><BR>Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-b=
in/wa?S1=3Dconfocal <DIV>Alejandro, </DIV><DIV><BR class=3Dkhtml-bloc=
k-placeholder></DIV><DIV>There are different methods for doing FRET. In the=
most basic method, you image the donor, acceptor and FRET channels, in you=
r sample, as well as donor only and acceptor only samples. Then, you need t=
o perform some calculations to remove the bleed-through components and obta=
in the true FRET channel. Another approach uses acceptor photobleachi=
ng: you image your sample in the donor channel, bleach the acceptor, and im=
age the donor channel again. If FRET was occurring, bleaching the acceptor =
will result in an enhanced fluorescence of the donor. </DIV><DIV><BR =
class=3Dkhtml-block-placeholder></DIV><DIV>Both techniques can be performed=
with a Zeiss 510 using the standard channels, or with the META detector an=
d spectral unmixing, (assuming your signal is strong enough...). </DIV><DIV=
><BR class=3Dkhtml-block-placeholder></DIV><DIV>The FRET macros that Zeiss =
provides are designed to streamline the image acquisition and calculations =
necessary to perform your FRET experiment, but in principle you could do ev=
erything manually. As a matter of fact, if you (or the other user) have lit=
tle experience with FRET, I would probably recommend doing everything manua=
lly, since this will force your user to do all the controls, take the time =
to look at the individual images, and understand what is going on. &nb=
sp;The basic Zeiss software should have all the tools you need to perform t=
he image calculations (subtractions and ratios), as would something such as=
ImageJ. I think there are some relatively inexpensive packages available t=
hat will facilitate those calculations- you just specify all the images inv=
olved and the software will calculate the final result, instead of doing ev=
erything manually. </DIV><DIV><BR class=3Dkhtml-block-placeholder></D=
IV><DIV>Once you are comfortable with the entire FRET procedure, it may be =
worth getting a demo of the FRET macro and seeing for yourself how well it =
performs, and how much (or whether) it simplifies your life. </DIV><D=
IV><BR class=3Dkhtml-block-placeholder></DIV><DIV><BR class=3Dkhtml-block-p=
laceholder></DIV><DIV>Julio. </DIV><DIV><BR class=3Dkhtml-block-placeholder=
></DIV><BR><DIV><SPAN class=3D""><SPAN class=3D""><P style=3D"MARGIN: 0px">=
<FONT style=3D"FONT: 12px Helvetica" face=3DHelvetica size=3D3>-- </FONT></=
P><P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" face=3DHelv=
etica size=3D3>Julio Vazquez, PhD </FONT></P><P style=3D"MARGIN: 0px"><FONT=
style=3D"FONT: 12px Helvetica" face=3DHelvetica size=3D3>Fred Hutchinson C=
ancer Research Center </FONT></P><P style=3D"MARGIN: 0px"><FONT style=3D"FO=
NT: 12px Helvetica" face=3DHelvetica size=3D3>1100 Fairview Ave. N., <SPAN =
class=3DApple-converted-space> <SPAN class=3DApple-converted-space>&n=
bsp; </SPAN></SPAN>DE-512 </FONT></P><P style=3D"MARGIN: 0px"><FONT style=
=3D"FONT: 12px Helvetica" face=3DHelvetica size=3D3>Seattle, WA 98109-1024 =
</FONT></P><P style=3D"MIN-HEIGHT: 14px; MARGIN: 0px; FONT: 12px Helvetica"=
><BR></P><P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" face=
=3DHelvetica size=3D3>Tel: Office: 206-667-1215/ Lab: 206-667-4205 </FONT><=
/P><P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" face=3DHel=
vetica size=3D3>FAX: 206-667-4029 </FONT></P><P style=3D"MARGIN: 0px"><BR c=
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class=3DApple-style-span size=3D4><SPAN class=3DApple-style-span style=3D"F=
ONT-SIZE: 13px"><SPAN class=3D""><SPAN class=3D"">-------------------------=
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e-style-span face=3DArial size=3D4><SPAN class=3D""><SPAN class=3D""><SPAN =
class=3D""> </SPAN></SPAN><BR></SPAN></FONT><BR class=3Dkhtml-block-p=
laceholder></DIV><BLOCKQUOTE type=3D"cite"><FONT class=3DApple-style-span f=
ace=3D"Tempus Sans ITC" size=3D4><SPAN class=3D""></SPAN></FONT></BLOCKQUOT=
E><BR class=3DApple-interchange-newline></SPAN></SPAN></DIV><BR><DIV><DIV>O=
n Nov 16, 2005, at 10:53 PM, Alejandro Roth wrote: </DIV><BR class=3DApple-=
interchange-newline><BLOCKQUOTE type=3D"cite">Search the CONFOCAL archive a=
t <A href=3D"http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal" =
target=3Dblank >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfoc=
al </A><DIV id=3DRTEContent>Dear List <BR>A co-user of our Zeiss 510-Meta w=
ould like to do FRET with our software version 3.0. <BR>The people from Zei=
ss said that we needed to upgrade the software to version 3.2 to be able to=
download the FRET macro they have available (-6.000 U$) <BR>Is there a way=
to get around and do FRET with the software version we have. <BR><BR>Thank=
you <BR><BR></DIV><BR><BR><DIV><DIV><DIV><DIV>Alejandro D. Roth  =
; PhD. </DIV><DIV>Post-Doctoral Fellow </DIV><DIV><DIV>Brain Tumor Research=
Group </DIV></DIV><DIV>Montreal Neurological Institute. Room BT112 <=
/DIV><DIV>McGill University. </DIV><DIV>3801 University St. Montreal, QC, H=
3A 2B4 </DIV><DIV>Phone: (514) 398-5346 </DIV><DIV> <BR class=3Dkhtml-block=
-placeholder></DIV></DIV></DIV></DIV></BLOCKQUOTE></DIV><BR></blockquote><b=
r></DIV></FONT>=
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