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) Hi Kevin,

Thanks for referencing my former colleagues at ASI. I also recommend 
Yuval and my 2006 review at
DOI: 10.1002/cyto.a.20311
and perusal of other articles (including Carl Boswell and I) in the rest 
of the 2006 spectral imaging issue
http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.v69a:8/issuetoc
(back issues of Cytometry are free online).

As for 5 colors (actually 6 with a separate filter cube for DAPI), the 
ASI SKY system has about 800 references:

http://www.ncbi.nlm.nih.gov/pubmed?term=spectral%20karyotyp*

using (essentially) Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594 
(or Texas Red), Cy5 (or Alexa fluor 647), Cy5.5

With the same instrument Tsurui et al 2007 (
PMID:
10769049) acquired seven colors (but with 3 filter cubes):
Alexa488 and Alexa532, for Alexa546, Alexa568, and Alexa594, and for Cy5 
and Cy5.5.

Not that I would buy a SKY instrument for one experiment (though my 
former colleagues at CHLA might be willing to sell theirs).

If I wanted to buy a new instrument, I would contact Jeremy Lerner at 
LightForm Inc, http://www.lightforminc.com/ to order a PARISS with solid 
state lighting (I would go for a Lumencor SPECTRA X) and excitation slit 
(ideally some light output from the X to closely match the slit). This 
would result in a pretty sweet linescan confocal (ditch the disc) - 
Jeremy routinely puts on a motorized stage for "pushbroom" acquisition.

If you want to go fast, Keith Lidke (UNM), updated from an earlier 
Sandia National Labs clone of Jeremy's PARISS, have developed a fast 
scanning module - see the "Hyperspectral Microscope Layout" page at
http://www.systemscenters.org/wordpress/wp-content/uploads/2011/01/nm-ncbs-aug-8-11-lidke.pdf
Not clear from that presentation, but Keith is tracking 8 colors quantum 
dots at 30 fps with his instrument (single excitation, 30 full field 
spectral images per sec).

***

A little chemistry can also go a long way ... if certain colors are NOT 
being colocalized, you could buy or make a FRET construct. Alan Waggoner 
had a presentation circa 1996 (a CSHL immunocytochemistry course) with 
nearly 100% FRET from Cy3 to Cy5. Likewise in the FP world, Miyawaki 
published CY11.5 with nearly 100% FRET, and CyPet-YPet was almost as 
high due to non-covalent heterodimerization.

Of course most core facility confocal microscopes (the Leica SP5's and 
Zeiss LSM710's I manage for examples) have 405, 458, 488, 561, 594, and 
633 nm lasers - can excite different dyes (or FPs) with each.

Getting better resolution out of existing (point scanning) confocals ... 
See  PMID 23361088 ("3B" ImageJ plugin, only just obtained the plugin, 
have not had time to get it running on my PC - some file location issue 
I have not figured out yet) and 22357945 (Munck PiMP ImageJ plugin ... 
but you'll need the plugin file locally, not the silly VIB Citrix 
server, to do PiMP usefully). I've done two color PiMP - works great 
(SP5 or LSM710, plan apo 63x/1.4NA, 30 nm pixel size, filter size = 1.6, 
16-bit output). Ther is a non-linear step (Rainer Heintzmann came up 
with that critical idea), so don't bother with intensity quantitation 
(thresholding and counting pixels should be fine).

George
p.s. best wishes with the controls. Unlikely I would be a reviewer (and 
don't suggest me!), but I would expect to see proof that both your 
"single marker' and your FMOs (fluorescence minus one) controls worked well.

On 2/8/2013 1:29 PM, Kevin Ryan wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> You might find Garini et al 1998 "Signal to Noise Analysis of Multiple Color Fluorescence Imaging Microscopy" (http://itmhrt.ca/students/files/April2008/pdfs_for_journal_clubs/pdf4_Signal_to_noise_analysis-Cytometry_1999-Y_Garini.pdf), a useful reference.
>
>
> Kevin Ryan
> Media Cybernetics, Inc.
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Aromis Storm
> Sent: Friday, February 08, 2013 9:13 AM
> To: [log in to unmask]
> Subject: 5 color imaging (6 color would be even better!)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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>
> Dear Everyone:
>
> I have been trying to visualize a macromolecular structure with 4 color imaging. I have been doing this with spinning disc microscopy as I need complete sections of cells also to know the distribution of the structure inside the cells in 3D. So far things are working properly BUT ...
>
> We now would like to upgrade to 5 color imaging as we want to visualize additional components of the structure. I checked the spinning disc setting myself and I asked several senior users of spinning disc and got the opinion that "5 color imaging with spinning disc without bleed-through is hardly possible, if possible at all". The main reason being that it is not very likely to set up emission filter combinations for 5 color imaging without bleed-through. Spectral mixing is technically possible but not very promising.
>
> I also learned that 5 color imaging is possible with laser scanning microscopy with which one can specifically define the emission band pass range. But with laser scanning microscope it will cost me much more time to collect enough data for statistical analysis.
>
> So my questions would be: can anyone of you share your experience/opinion/literatures on 5 color imaging? I also tried to look for literatures describing multi-color (>  4 color) imaging (I think there must be decent papers about it) but so far I have not found any. Maybe some of you have some nice papers about this?
>
> Thank you very much for your input.
>
> Nice weekend!
>
> Aromis
>
>