Re: Cy7-TSA dye

Posted by George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Cy7-TSA-dye-tp7587083p7587089.html

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Hi Sripad,

Molecular Probes can conjugate whatever Alexa Fluor you want onto
tyramide, and yes, there is an AF Cy7 equivalent.Tyramide is off patent,
other 'chemistry' companies could conjugate their/your favorite
fluorophores.

//

At higher plex, you are going to have spectral overlap: learn to love
spectral unmixing. For example:

PLoS One. 2016 Jul 8;11(7):e0158495. doi: 10.1371/journal.pone.0158495.
eCollection 2016.

Multiplexed Spectral Imaging of 120 Different Fluorescent Labels.

Valm AM(1)(2), Oldenbourg R(1)(2), Borisy GG(3).

Author information:
(1)Marine Biological Laboratory, Woods Hole, Massachusetts, United States of
America.
(2)Brown University, Providence, Rhode Island, United States of America.
(3)Forsyth Institute, Cambridge, Massachusetts, United States of America.

The number of fluorescent labels that can unambiguously be distinguished in a
single image when acquired through band pass filters is severely limited by the
spectral overlap of available fluorophores. The recent development of spectral
microscopy and the application of linear unmixing algorithms to spectrally
recorded image data have allowed simultaneous imaging of fluorophores with highly
overlapping spectra. However, the number of distinguishable fluorophores is still
limited by the unavoidable decrease in signal to noise ratio when fluorescence
signals are fractionated over multiple wavelength bins. Here we present a
spectral image analysis algorithm to greatly expand the number of distinguishable
objects labeled with binary combinations of fluorophores. Our algorithm utilizes
a priori knowledge about labeled specimens and imposes a binary label constraint
on the unmixing solution. We have applied our labeling and analysis strategy to
identify microbes labeled by fluorescence in situ hybridization and here
demonstrate the ability to distinguish 120 differently labeled microbes in a
single image.

DOI: 10.1371/journal.pone.0158495
PMCID: PMC4938436
PMID: 27391327



George


On 7/28/2017 2:22 PM, S Ram 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,
> Just wondering if anyone has ever come across (or used) a Cy7 or similar
> TSA dye probe? I checked a few of the vendors that I could think of
> (Molecular Probes/Invitrogen/Fisher, Perkin Elmer) but none of them had any
> probes in that range.
>
> The goal is to do automated staining of FFPE tissues using Cy5 and another
> NIR probe. The dyes need to be TSA conjugates. I do not want to use Cy5.5
> due to spectral overlap. Also, dyes in the visible region are no beuno due
> to too much autofluorescence.
>
> Any help/suggestion is greatly appreciated.
>
> Thanks.
>
> Sripad

--


George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75   (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
http://confocal.jhu.edu (as of May 22, 2017)