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Does the polymer chain affect cell biochemistry? It looks pretty big.
I always understood it to be beneficial if the antibody / fluorophore was as small as possible to avoid influencing molecular behaviour.
James
James Wainwright
Global Applications Specialist - Microscopy Systems
Tel: +44 (0) 2890 237 126 ext. 2130
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Carol J. Bayles
Sent: 10 January 2018 18:09
To: [log in to unmask]
Subject: -|EXT|- Re: additional probes to standard lineup, Brilliant Violet?
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A little late, but check out the interesting chemistry behind the Brilliant Violet dyes:
http://www.bdbiosciences.com/us/applications/multicolor/s/brilliantdyes
http://www.sirigen.com/sirigen_technology.html
Carol
<><><><><><><><>
Carol Bayles
BRC-Imaging-FACS
B46 Weill Hall
Cornell University
Ithaca, NY 14853
607-275-9090 cell
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Jeffrey Carmichael
Sent: Friday, December 29, 2017 1:00 PM
To: [log in to unmask]
Subject: Re: additional probes to standard lineup, Brilliant Violet?
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*****COMMERCIAL RESPONSE*****
Hi Michael,
Like George, I also have no commercial interest in BD Biosciences and also recommend BV421 as well as BV480. The link below does refer to Chroma's optimized filter sets, hence the "COMMERCIAL" notice....
If workers can free up the channel normally used by DAPI by using one of the far red DNA probes, that opens up two channels: both BV421 & BV480.
Even without giving up DAPI, one can fit in the BV480. Obviously, the caveat is that all of these filters sets should be more narrow in bandwidth than the more conventional filter sets for things like GFP or AF 488, AF546, etc.
BV480 is very bright whereas DEAC is useless. Both of these BV dyes (and most others) have exceptionally large extinction coefficients. It is adequately separable from AF488, unlike CFP and related FPs.
If workers are using DAPI, then the cells are assumed to be dead (fixed) so that allows for UV excitation, and whole breadth of the BUV tandems from BD. These excite with a 350-375nm source and are visible as various tandems all the way to 800nm. The native BUV395 is just that - it emits at approx. 395nm, which is also unique.
George provided some very useful info, here's a link to an admittedly "lightweight" but very illustrative example of a simple 5 color scheme for the non-specialist which gives you the gist of how this could be expanded upon by more ambitious workers:
https://www.chroma.com/5-channel-fluorescence-imaging-simplified
<https://www.chroma.com/5-channel-fluorescence-imaging-simplified>
Slides #7, 12 & 16 lay out possible labeling schemes with accompanying filter recommendations. I've personally seen the recommended 5 colors/set combinations on samples and was impressed by the ability to separate BV480 from BV488. The rest are a bit less challenging.
Finally, I think the best thing about these BV dyes is that they're bright enough to use as directly conjugated primary antibodies.....if one can conquer the chemistry. BD has been very helpful with some of my customers with custom probes, but it's not easy.
Happy staining,
Jeff
*Jeff Carmichael*
*Technical and Product Marketing Manager**[log in to unmask]
<[log in to unmask]>*
On Fri, Dec 29, 2017 at 11:09 AM, Cammer, Michael < [log in to unmask]> 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.
> *****
>
> One of our clients wants to add a fifth or sixth probe to the standard
> Dapi, AF488, AF568, AF647 lineup.
>
> One of our confocals has a 594 nm laser and we also have true spectral
> detection, but rather than sandwich in overlapping probes, thought we
> would recommend
>
> Brilliant Violet 570(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 570 nm.
>
> Brilliant Violet 605(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 605 nm.
>
> Brilliant Violet 650(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 650 nm.
>
> Brilliant Violet 711(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 711 nm.
> We'd also recommend multiple controls (although if signals are truly
> spatially exclusive, controls built in).
>
> Any recommendations which of these is best? Are they all good? Any
> feedback appreciated. (But not the question, what are you doing at work
> between Christmas and New Year?)
>
> Cheers-
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU
> Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
> C: 914-309-3270 [log in to unmask]<mailto:[log in to unmask]
> edu> http://nyulmc.org/micros http://microscopynotes.com/
>
>
>
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