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blue dye (ex 405nm) suggestions

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Graham Wright

blue dye (ex 405nm) suggestions

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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

--
*Dr Graham Wright*
Microscopy Unit Manager

Institute of Medical Biology
8A Biomedical Grove, #06-06 Immunos, Singapore 138648

E: [hidden email]
W: http://www.imb.a-star.edu.sg/imu/

Steffen Dietzel

Re: blue dye (ex 405nm) suggestions

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How about Quantum dots? Should be excitable with many wavelengths and
emit in a rather narrow band. Depends on your experiment whether it is a
problem to have, for example, only Qdots in the green channel when
excited with 405 and Qdots plus green fluor when excited with 488.

Steffen

On 28.06.2012 12:00, 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
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
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Kurt Thorn

Re: blue dye (ex 405nm) suggestions

In reply to this post by Graham Wright

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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:

Eric Griffis

Re: blue dye (ex 405nm) suggestions

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Just anecdotally, we've found that Atto425 works very well with the 405nm on our OMX, outperforming Pacific Blue, and Alexa 405 when conjugated to phalloidin.

Best,

Eric

On 28 Jun 2012, at 17:34, Kurt Thorn wrote:

> *****
> 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
>>
>

The University of Dundee is a registered Scottish Charity, No: SC015096

lechristophe

Re: blue dye (ex 405nm) suggestions

In reply to this post by Kurt Thorn

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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 <[hidden email]> a écrit :

Kelly Lundsten-2

Re: blue dye (ex 405nm) suggestions ** commercial interest!

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." <[hidden email]> 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 <[hidden email]> 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
>>>

Martin Wessendorf-2

Re: blue dye (ex 405nm) suggestions ** commercial interest!

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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
Assoc Prof, Dept Neuroscience lab: (612) 624-2991
University of Minnesota Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
Minneapolis, MN 55455 e-mail: [hidden email]

Kelly Lundsten-2

Re: blue dye (ex 405nm) suggestions ** commercial interest!

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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" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> Assoc Prof, Dept Neuroscience lab: (612) 624-2991
> University of Minnesota Preferred FAX: (612) 624-8118
> 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
> Minneapolis, MN 55455 e-mail: [hidden email]