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Hi Martin,

No, it's not a stain.  It's basically just a whole bunch (70kD) worth of PacB-like monomers that are tuning the spectrum, but polymerized into a long chain (actually embedded in the polymer similar to being encased in a shell).  In this way, more individual fluors are able to "report" the ID of the antibody without self-quenching or shifting spectra.  That's the only reason the EC is so high, simple abundance of monomers to absorb the energy.  It's a decentralized electronic conductance, so the energy can move up and down the polymer to increase efficiency.  On a single antibody there are 2-3 polymers conjugated.

I always said in the past "blue can't be bright, it's a physical limitation of the chemical structure required to tune it blue... Which is why we use DAPI and Hoecsht" but then this exception was made.  

Anyway, I hope microscopists can find it useful.  The photobleaching curve and full spectra are on BioLegend's website.

Kelly 


On Jun 29, 2012, at 8:20 AM, "Martin Wessendorf" <[log in to unmask]> wrote:

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> To join, leave or search the confocal microscopy listserv, go to:
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> 
> Dear Dr. Lundsten--
> 
> Is Brilliant Violet 421 a tinctorial stain?  I.e. does the polymer stain (for instance) DNA or RNA?  Or does the polymer need to be conjugated to antibodies for immunocytochemistry?
> 
> Thank you for offering samples!  It'll be interesting to hear from List how well it works.
> 
> Martin Wessendorf
> 
> 
> On 6/29/2012 9:51 AM, Kelly Lundsten wrote:
>> To be fair, there is commercial interest in my reply.  However, I have a lot of experience with the blue emitting fluors from years with Molecular Probes.  So, with that said, there is a fluorescent polymer called Brilliant Violet 421 that can be very useful for confocal, but I haven't had nearly as many people testing it out as I'd like, though.  Right now people use it mostly for flow cytometry.  It's sold by BioLegend.
>> 
>> It's about 70-75kD, entirely organic (no protein component to the fluor), excites at ~405nm and emits at a peak at 420nm (but in flow people use a 450/50 emission filter typical for PacB and AF405).  Its been validated multiple times in confocal but DOES NOT fit ideally into most commercial DAPI widefield filters.  The DAPI filters usually block the ideal excitation and the  dichroic in AF405 filter sets often block part of the emission.  Like any other blue fluor, it needs anti fade.  I used prolong to compare the photobleaching curves.  Signal persists at a detectable level WAY longer than PacB or AF405 because as a single molecule it has an extinction coefficient of over 2 million and a QY of 65% in water.  It's super "bright".  Also, it's great for multiphoton.  I have the 2P cross-section for anyone who wants it and the only issue in MP is that it's sweet spot is similar to collagen and they both emit blue.  There's a second polymer that'll be released in a few months
> called Brilliant Violet 510 that excites at 405nm and emits at 510nm or so that would be interesting to see on a spectral scope:one scan, two open windows of emission.  In flow cytometry, the BV510 doesn't excite much at 488nm, so it may be an easy way to insert in a color into a mix without increasing the bleed through/background spectral overlap area between AF488/AF555/AF594.
>> 
>> I'm interested in collecting images and demonstrating applications for these fluors in microscopy, so please let me know offline if and how you'd like to test it.
>> 
>> Kelly
> 
> -- 
> Martin Wessendorf, Ph.D.                   office: (612) 626-0145
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