*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Michal,

You have a META spectral detector - use the spectral unmixing 
capability. If necessary, you can acquire sequentially with different 
lasers and combine the images later.

PMT's have poor quantum efficiency in the far red, so even if Cy5.5, 
AF680 or AF700 dye, or QD705 quantum dot conjugates work well at the 
specimen level the signal might be weak. QD705 might have an advantage 
in being excitable with the same laser as DAPI.

Tsurui et al published 7 color immunofluorescence eleven years ago:

    Tsurui H, Nishimura H, Hattori S, Hirose S, Okumura K, Shirai T.
    Seven-color fluorescence imaging of tissue samples based on Fourier
    spectroscopy and singular value decomposition. </pubmed/10769049> J
    Histochem Cytochem. 2000 May;48(5):653-62. PMID: 10769049

You might take a dye from them and add AF532 to your mix (AF546 is not 
as useful a dye as AF555 or AF568). When you have an opportunity, 
replacing Cy3 with any of Cy3B (B=Brighter), AF555 or AF568 should 
increase brightness.

If any of your reagents are dim, you can use tyramide signal 
amplification to boost the signal between 10x and 100x. PerkinElmer has 
a limited TSA line-up, Invitrogen/Molecular probes has many fluorescent 
TSA kits.

Anothre possibility (instead of NIRF or AF532) would be Lucifer Yellow 
... AF430, excited with the same laser as DAPI.

If you need to see spectra of any of these, see 
http://www.mcb.arizona.edu/ipc/fret/index.html
If you want the spectra data, see the XLSX file inside the PubSpectra 
zip file at the top of 
http://sylvester.org/research/shared-resources/laboratory-resources/analytical-imaging-core-facility/analytical-imaging-core-facility-links-forms/pubspectra-data 


Best wishes,

George
p.s. there are over 700 spectral karyotyping papers that use five 
FISH/immuno reagents acquired simultaneously plus DAPI (acquired 
separately with a DAPI filter set). The dye-detection reagents used 
include DNA probes conjugated with Rhodamine 110 (green fluorophore), 
spectrum orange, spectrum Red (Texas Red), biotin and digoxigenin, with 
detection reagents Cy5-streptavidin and Cy5.5-anti-mouse/mouse anti-dig. 
I will guess here that over 10,000 specimens have been acquired and 
analyzed for clinical + research use. The META detector ought to be able 
to handle five color unmxing as well as the SKY camera.


On 2/20/2011 9:01 AM, Michal Gdula wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Confocalists,
>
> I am in desperate need for a new fuorophores for my multicolor FISH-
> immunofluorescence experiments. I am  using at the moment DAPI, Alxea488, Cy3,
> Texas Red and Cy5. I use for imaging Zeiss microscope LSM510meta.
> I am thinking about purchasing antibodies conjugated with Alexa 680 or Alexa
> 700. According to olipmpus fluorophore tutorial web page my 633 nm laser should
> be able to excite both although its quite far away from excitation maximum.
> Has anybody used successfully any of these fluorophores with LSM510 meta?
> Is it easy to separate them from Cy5 using meta detector?
> Molecular mass of of these fluorophores is relatively high (1.2-1.4 kDa) compared
> with the fluorophores which I am using right now, will it have effect on the
> antibody penetration ?
>
> Kind regards,
>
> Michal Gdula
> PhD student
> Bradford University
> Uk
>
>    


-- 


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami