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 


On Jun 28, 2012, at 3:38 PM, "Christophe L." <[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
> *****
> 
> I have switched from Alexa 405 to DyLight 405 coupled secondary antibodies, as it is much more stable,  even as stable as other channels (Alexa488 / 555 / 647 usually). Coupled with Prolong Gold as a mountant I barely see any photobleaching when imaging on a widefield microscope. Also on a widefield system, getting a true 405 filter cube rather than a standard DAPI one significantly enhances the signal. 
> 
> Christophe
> 
> 
> Le 28 juin 2012 à 18:34, Kurt Thorn <[log in to unmask]> a écrit :
> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> I haven't used any of these myself, but I've compiled a list of commercially 405nm excitable dyes for our users here:
>> http://nic.ucsf.edu/dokuwiki/doku.php?id=dyes:405nm
>> 
>> If you try any it would be great to get some feedback about how they perform.
>> 
>> Best,
>> Kurt
>> 
>> On 6/28/2012 3:00 AM, Graham Wright wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>> 
>>> Dear all,
>>> 
>>> I'm looking for recommendations for a blue dye (ex. 405nm) to be used on a
>>> secondary antibody. Unfortunately, the other wavelengths are already taken,
>>> which would normally be my suggestion.
>>> 
>>> I've had a look through the archives of this list and there is confirmation
>>> that they are not ideal and tend to bleach easily, but are there any
>>> updates on this since 2008/2010?
>>> 
>>> Any comments and recommendations will be appreciated about the options:
>>> Alexa 405, Pacific Blue, Cascade Blue...
>>> 
>>> Much obliged,
>>> Graham
>>>