Advice sought for imaging Metasystems 24XCyte DNA Probe Kit

[Andrew Vaughan-2]

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Hello All,

Does anyone have any experience imaging Metasystems 24XCyte DNA Probe Kit (or any other Metasystems kit) using microscopes other than the company's own Isis imaging system? The kit allows identification of the 24 human chromosomes by painting them with combinations of 5 different fluorophores. There is significant spectral overlap so the recommended filter sets have narrow bands. I would like to know whether anyone has tried imaging these (or other similar chromosome painting products and protocols) using other filter sets or confocal microscopes and perhaps using spectral imaging and un-mixing to separate the fluorophores.

Here's the link to Metasystems page:

http://www.metasystems-international.com/index.php?option=com_joodb&view=article&joobase=5&id=1%3Ad-0125-060-di&Itemid=242

Any advice would be much appreciated.

Thanks,

Andrew Vaughan.

[George McNamara]

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Hi Andrew,

Yes, in 2016 any spectral confocal microscope had better work.

The Applied Spectral Imaging Ltd/Inc spectral imager (mostly SD-300
model) has been used for ~900 SKY spectral karyotyping publications (I
estimate 10% are reviews, PubMed search: spectral karyotyp* ). There may
be one or more SKY systems in your town, see PubMed search  spectral
karyotyp* london.

See the image at the top of

https://www.linkedin.com/pulse/tattletales-t-bow-math-100-x-3-33-10000-brighter-than-george-mcnamara

For my favorite SKY image: 400 chromosome spread from one human breast
cancer cell, slide made by Carrie Viars and Steve Goodison, acquired
when I worked for ASI. For those curious what the chromosome
translocations are, see Goodison S, Viars C, Urquidi V. Molecular
cytogenetic analysis of a human breast metastasis model: identification
of phenotype-specific chromosomal rearrangements. Cancer Genet
Cytogenet. 2005 Jan 1;156(1):37-48. PubMed PMID: 15588854.
http://www.ncbi.nlm.nih.gov/pubmed/15588854. They left out the detail
that both M4A4 and NM2C5 cell lines (single cell cloned from MDA-MB-435)
have some large interphase nuclei and metaphase spreads with ~200 and
~400 chromosomes.

The ASI SKY reagents had (and still have) two of the SKY fluorophores
(orange, red) were placed deliberately close together to make it
difficult for early competing filter based systems (the other
fluorophores are green, Cy5, Cy5.5 all acquired in "one click", DAPI
acquired separately).

See the first SKY publication, Schröck E, du Manoir S, Veldman T,
Schoell B, Wienberg J, Ferguson-Smith MA,Ning Y, Ledbetter DH, Bar-Am I,
Soenksen D, Garini Y, Ried T. Multicolor spectral karyotyping of human
chromosomes. Science. 1996 Jul 26;273(5274):494-7. PubMed PMID: 8662537.
http://www.ncbi.nlm.nih.gov/pubmed/8662537

Now, in 2016, any of the commercial spectral confocal microscopes should
be able to spectrally unmix the MetaSystems or ASI 5 color combinations.
If it can't, either arrange more training or get your money back. The
MetaSystems 24XCyte page you cite states the fluorophores are compatible
with: FITC, Spectrum Orange™, TexasRed®, DEAC (Diethylamino-coumarin),
and Cy5®, so you will likely get better results with sequential
acquisition. You can model the spectra and laser lines on UA Spectra
Database http://spectra.arizona.edu/ or Semrock Searchlight
http://searchlight.semrock.com/ with coumarin(s) (or Spectrum Aqua or
Blue), FITC, Spectrum Orange, Texas Red, Cy5.

Even simpler: the chromosome chart shows chr 1-5 are the single color
paints, and are large chromosomes, so should be easy to find well
separated chromosomes to obtain "pure spectra" from, and use that
directly on the spectral confocal.  Filter based systems should also
work - I suggest on these matching signal intensity of the single
labeled chromosomes (after shading correction and background
subtraction). Don't be surprised if the other chromosomes have different
intensities per channel than the single dye chromosomes.



George
p.s. non-financial disclosure: I am coinventor on three spectral
unmixing patents. All assigned to ASI. No bonuses or royalties for them.
I also organized the data -- downloadable from
https://works.bepress.com/gmcnamara/9/   --  that Carl Boswell organized
the UA Spectral Database http://spectra.arizona.edu/

On 6/3/2016 12:12 PM, Vaughan, Andrew wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello All,
>
> Does anyone have any experience imaging Metasystems 24XCyte DNA Probe Kit (or any other Metasystems kit) using microscopes other than the company's own Isis imaging system? The kit allows identification of the 24 human chromosomes by painting them with combinations of 5 different fluorophores. There is significant spectral overlap so the recommended filter sets have narrow bands. I would like to know whether anyone has tried imaging these (or other similar chromosome painting products and protocols) using other filter sets or confocal microscopes and perhaps using spectral imaging and un-mixing to separate the fluorophores.
>
> Here's the link to Metasystems page:
>
> http://www.metasystems-international.com/index.php?option=com_joodb&view=article&joobase=5&id=1%3Ad-0125-060-di&Itemid=242
>
> Any advice would be much appreciated.
>
> Thanks,
>
> Andrew Vaughan.
>

--



George McNamara, Ph.D.
Houston, TX 77054
[hidden email]
713-239-0365 home
305-764-2081 cell
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/   Tattletales and T-Bow