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

Because there is a real population of "green" DsRed molecules that no sequential scanning can separate from FITC.  One way to go around this is to allow DsRed enough time to mature. We imaged 72 hours after transfection and 48 hours (I think) after adding an inhibitor of new protein synthesis.  

Anda

Anda Cornea Ph.D.
Head of the Imaging and Morphology Core
Oregon National Primate Research Center
Oregon Health & Science University
(503) 690-5293

>>> [log in to unmask] 05/12/06 9:23 AM >>>
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 

I'm not sure why the need to do spectral separation in the first place.  As 
someone mentioned, simply capturing each dye sequentially in different 
channels not only precludes the possibility of bleed-through, but uses more 
sensitive detectors.  Modern systems seem to have solved mis-registration 
problems associated with changing mirrors/filters, and the automated systems 
allow full scans of each channel before changing to another focal plane.

Just my two cents worth.
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX 520-621-3709
----- Original Message ----- 
From: "Stefan Terjung" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Friday, May 12, 2006 4:27 AM
Subject: Re: DSRed and green emission?


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

Switching to a more separated protein will help here, but mainly because
DsRed(2) has a green fluorescing maturation step. Using DsRed you will
always have a part of the DsRed molecules still in the green state.

You can read about the green maturation step in

Baird et al. 2000, PNAS 97, 11984-11989

DsRed2 still seems to have this green maturation step (see
http://www.clontech.com/clontech/archive/JUL03UPD/pdf/LC_RCFP_Vectors.pdf ).
There you can also read that "DsRed-Express" is "Preferred DsRed for FACS
due to diminished green emission, faster maturation".

As already recomended, I would also advise to use a different red
fluorescent protein (mRFP, mCherry...) or you could maybe exchange the FITC
against Cy5, to avoid the overlap in the green channel.

Regards,

Stefan



-------------------------
Dr. Stefan Terjung
EMBL, Meyerhofstr.1
D - 69117 Heidelberg
Cell Biology/Biophysics Program
Advanced Light Microscopy Facility
Phone ++49-6221-3878-467 FAX-242
www.embl.de/almf/ 
www.embl.de/eamnet/ 



> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[log in to unmask]] On Behalf Of Michael Weber
> Sent: Freitag, 12. Mai 2006 09:03
> To: [log in to unmask] 
> Subject: Re: DSRed and green emission?
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
>
> Switching to more separated proteins is the best way. Beside
> that, you could also try to unmix the spectral informations
> by using Leica's Lambda-Stack capability. For that it would
> be good to have controls with single stainings to record the
> spectra under the same conditions as your sample.
>
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
> >
> > see
> >
> http://www.aecom.yu.edu/aif/instructions/fluorescence/probes_gfp_dsred 
> > .htm
> >
> > dsRed has a large green component.
> >
> > switch to a monomeric red protein.
> >
> >> Search the CONFOCAL archive at
> >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
> >>
> >> Have you tried scanning sequentially rather than
> simultaneously? The
> >> Leica has a great protocol for doing this.
> >>
> >> Judy
> >>
> >>
> >> Judy Trogadis
> >> Bio-Imaging Coordinator
> >> St. Michael's Hospital, 7Queen
> >> 30 Bond St.
> >> Toronto, ON M5B 1W8, Canada
> >> ph:  416-864-6060  x6337
> >> pager: 416-685-9219
> >> fax: 416-864-6043
> >> [log in to unmask] 
> >>
> >>>>> Joel Sheffield <[log in to unmask]> 05/11/06 3:35 PM >>>
> >> Search the CONFOCAL archive at
> >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
> >>
> >> Greetings,
> >>
> >> I have been working with someone who is attempting to do
> double label
> >> studies using DSRed2-mito and an FITC-based antibody system.  We
> >> disscovered, using the spectral scan of the Leica SP system, that
> >> there is significant fluorescence of the DSRed in both the
> green and
> >> red wavelengths when we illuminate with a 488 argon line.  Indeed,
> >> the spectrum appears to have two peaks, of almost equal
> intensity, in
> >> response to this excitation.  As a result, we can not separate the
> >> DsRed Signal from the FITC when the FITC is weak.
> >>
> >> At the risk of sounding like a complete novice, has anyone
> worked out
> >> a way to deal with this problem?
> >>
> >> Joel
> >>
> >>
> >> Joel B. Sheffield, Ph.D.
> >> Biology Department, Temple University
> >> 1900 North 12th Street
> >> Philadelphia, PA 19122
> >> [log in to unmask] 
> >> (215) 204 8839, fax (215) 204 0486 http://astro.temple.edu/~jbs 
> >>
> >
> >
> > _________________________________________
> > Michael Cammer
> > Analytical Imaging Facility and
> > Dept. ASB Biophotonics Innovation Laboratory
> > Albert Einstein College of Medicine
> > 1300 Morris Park Avenue, Bronx, NY  10461
> > 718-430-2890  Fax 718-430-8996
> > work:  http://www.aecom.yu.edu/aif/ 
> > personal:  http://coxcammer.com/ 
> >
>
>
> _____________
>
> Michael Weber (B.Sc.)
>
> ..MTZ (before 12.30 pm)..
> Technische Universität
> Medical Theoretical Centre, Haus 91
> Fiedlerstr. 42
> D-01307 Dresden
> e-mail: [log in to unmask] 
> phone: +49 351 4586426
>
> ..MPI (after 12.30 pm)..
> Max Planck Institute of Molecular Cell Biology and Genetics
> Light Microscopy Facility Pfotenhauer Str. 108 D-01307 Dresden
> e-mail: [log in to unmask] 
> phone: +49 351 2102837
>