Re: Long shift dyes ** vendor reply **

[Kilgore, Jason A.]

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** vendor reply **

I agree, Timothy, that this is a neglected area and becoming more so. Yes, there are some flow dye options, but unfortunately many of them rely on phycobiliproteins (mainly RPE and APC) which photobleach badly.

One option is quantum dots, such as Qdot products. All the Qdots excite optimally down in the UV range, but emit much higher (we have options at 525 nm all the way up to 800) in reasonable tight emission curves. Though Qdots have their drawbacks, too (mainly size, being 15-20 nm in diameter, which can lead to a loss of fine resolution compared to organic dyes and a little harder to get into the nuclear compartment unless well-permeabilized. They also blink under many conditions, and they are picky about mounting media).

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Life Sciences Solutions
 

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Feinstein, Timothy N
Sent: Wednesday, April 7, 2021 6:57 AM
To: [hidden email]
Subject: Long shift dyes

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Hi everyone,

A while ago I noted that long shift dyes like the Atto-LS are disappearing from the American market.  Some folks responded with dyes used in flow cytometry (thanks again George!), but I couldn't find any that are currently marketed for fluorescence imaging.

Before I start investing in trial and error, is anyone currently using long-shift dyes for fluorescence multiplex imaging?  If you do, I'd gratefully appreciate any advice you have re their use.

Thanks and all the best,


T

Timothy Feinstein, Ph.D.
Research Scientist, University of Pittsburgh Department of Developmental Biology

[George McNamara]

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SuperBrights (Thermo, but only 5plex), Phitonex (now Thermo, 20plex FRET
constructs), Brilliant Ultraviolets Violets Blues Yellow-Green (BD
Biosciences, see also BioLgend, Jackson Immunoresearch).

Also, Beckman Coulter (Danaher) SuperNova's,
https://www.beckman.com/reagents/coulter-flow-cytometry/supernova-fluorescent-polymer-dyes 
(SuperNova v428 spectra looks a lot like BV421, and first two tandems
similar looking to BV's ... "proprietary" to me means just that someone
in marketing is into big words).

BV421 worked well for us anti-CD11c mouse dendritic cells in PBS,
63x/1.4NA Leica SP8 confocal, 405nm excitation.

StreamBio UK (yes, UK, not America)
https://www.streambio.co.uk/products/   ... 9plex (really 7plex ignoring
the two way-out NIR) Conjugated Polymer Nanoparticles (sub-resolution).

George

p.s. SuperBright and Brilliant tandems (and SuperNova's, if not simply
rebranded BVs) ... should be a lot of fun on fast TCSPC FLIM confocal
microscopes, donore and EACH acceptor channel. If the donor (ex. BV421)
is a lot brighter, could put two (or more) detectors on to that
fluorophore. I am especially curious what fast FLIM confocal microscopes
can distinguish BV421 and SB436 by 'lifetime only'.

On 4/7/2021 2:00 PM, Kilgore, Jason A. 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.
> *****
>
> ** vendor reply **
>
> I agree, Timothy, that this is a neglected area and becoming more so. Yes, there are some flow dye options, but unfortunately many of them rely on phycobiliproteins (mainly RPE and APC) which photobleach badly.
>
> One option is quantum dots, such as Qdot products. All the Qdots excite optimally down in the UV range, but emit much higher (we have options at 525 nm all the way up to 800) in reasonable tight emission curves. Though Qdots have their drawbacks, too (mainly size, being 15-20 nm in diameter, which can lead to a loss of fine resolution compared to organic dyes and a little harder to get into the nuclear compartment unless well-permeabilized. They also blink under many conditions, and they are picky about mounting media).
>
> Jason
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes / EVOS Tech Support
> Life Sciences Solutions
>  
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Feinstein, Timothy N
> Sent: Wednesday, April 7, 2021 6:57 AM
> To: [hidden email]
> Subject: Long shift dyes
>
> *****
>
> Hi everyone,
>
> A while ago I noted that long shift dyes like the Atto-LS are disappearing from the American market.  Some folks responded with dyes used in flow cytometry (thanks again George!), but I couldn't find any that are currently marketed for fluorescence imaging.
>
> Before I start investing in trial and error, is anyone currently using long-shift dyes for fluorescence multiplex imaging?  If you do, I'd gratefully appreciate any advice you have re their use.
>
> Thanks and all the best,
>
>
> T
>
> Timothy Feinstein, Ph.D.
> Research Scientist, University of Pittsburgh Department of Developmental Biology