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Hi Doug,
I have had decent success using Atto 390 (https://www.atto-tec.com/attotecshop/product_info.php?language=en&info=p1_ATTO-390.html), both in live brain tissue and fixed cells. One thing to really watch out for, though, when using these near-UV dyes for multicolor imaging, is that they frequently show much stronger chromatic effects than the other color channels. This depends strongly on the optics you are using (especially the tube and objective lenses), as the chromatic correction usually gets worse the closer you get to the UV. In the past I have had severe problems with chromatic shift along the optical axis, as well as much stronger aberrations (especially coma and astigmatism) in the deep blue channel. If you go (deep) into tissue, this can become prohibitive very quickly.
Not to discourage you, just be sure to keep an eye out for potential issues and test your samples, dyes and system thoroughly with this in mind.
Good luck!
Nicolai Urban
>>>>>>>>>><<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<
Dr. Nicolai T. Urban
Max Planck Florida Institute
Jupiter, 33458 FL, USA
-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Frohlich, Victoria
Sent: Dienstag, 21. August 2018 08:33
To: [log in to unmask]
Subject: Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL RESPONSE
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Try CF405 from Biotium.
Bright and robust
Vickie
Sent from my iPhone
> On Aug 21, 2018, at 7:32 AM, Jeff Carmichael <[log in to unmask]> wrote:
>
> *****
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> *****
>
> This has been addressed more than once in the listserv, but below are
> some resources to look into the BrilliantViolet fluorophores which are
> much more than an order of magnitude brighter than AF405. You could
> potentially add two additional channels in this spectral "dead zone".
>
> Chroma Technology has no commercial interest in these fluorophores,
> only in the filter sets we've recommended for detecting them.
>
> https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.j
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> 3D&reserved=0
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>
> Good luck and please share your results!
> Jeff
>
> *Jeff Carmichael*
> *Product & Technical Marketing Manager*
>
> *[log in to unmask] <[log in to unmask]> | 802-428-2528*
>
> * <[log in to unmask]>*
>
> On Mon, Aug 20, 2018 at 7:06 PM, Cromey, Douglas W - (dcromey) <
> [log in to unmask]> wrote:
>
>> *****
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>> Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636704534756076388&sdata=M%2Bo69yJjhaqpGhI46moEzhlfaOP0yPRXPvSyJXsU7%2Fk%3D&reserved=0 and include the link in your posting.
>> *****
>>
>> Is there a better or preferred "blue" fluorochrome? Mostly this is a
>> widefield fluorescence question (using a DAPI cube), but there is the
>> possibility for using a confocal with a 405nm laser line.
>>
>> I'm working with a group that is trying to work around a frustrating
>> autofluorescence issue in liver tissue. What we ended up doing on an
>> earlier project was not using any secondaries that fluoresced in the
>> green channel (no fitc/AF488/GFP) and instead used the green channel
>> on our widefield microscope as a control, ratioing this image so we
>> could subtract it out of the other channels to remove some of the
>> broad autofluorescent background. This worked nicely. In the
>> published images we showed nuclei
>> (blue) and two specific stains using AF568 and AF647, with reduced
>> background autofluorescence.
>>
>> The PI now wants to have labelling with three specific fluorochromes,
>> and I am advocating using the green channel as the generic
>> "everything lights up" channel for context, so we can hopefully skip the Hoechst 3342 or DAPI.
>> Since we won't be using DNA dyes, is there a good blue fluorochrome
>> that can be used as a secondary for specific labelling of a marker
>> found on an intracellular organelle (other than the nucleus)?
>>
>> Thanks,
>> Doug
>>
>> ------------------------------------------------------------
>> ------------------------------
>> Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of
>> Cellular & Molecular Medicine, University of Arizona
>> 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA
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