I have heard that the new generation of Alexa-Plus dyes from Thermo may be superior for use with STED applications. We do STED @ 775nm - so we are mostly interested if any of you may have tested Alexas 594, 647, 680. We are always concerned on: STEDability at 775nm and cross-excitation with the STED laser.
Thx for help,
As far as I have heard, Alexa 647 works well with STED, although I haven’t used it myself. Whether there is any difference in performance with the Alexa Plus dyes, I can’t answer.
On a related note, does anyone know what is the structural difference between the standard Alexa dyes and the plus dyes? Has any testing been done in SMLM? @suliana?
Or maybe @melikel?
I emailed the link to this discussion to one of the probe R&D scientists at Thermo and requested some insight.
Unfortunately I haven’t tried these dyes. Would be interested to know how they work for superres
I am also very curious about this! Haven’t tested it yet but have some SMLM users that might be willing to test. Will report here if that test ever happens.
I got the following info from Thermo Fisher:
“This is a becoming a more common question when I’m out visiting with customers, but unfortunately we don’t have any data on the AF Plus dyes with STED or SMLM systems. We don’t have any super resolution platforms on campus (I’m working on it) to test these dyes with, so we rely on external users to give us feedback. I know that we’ve received positive feedback regarding 647+ for use in STORM, and based off the structural differences between 594 and 594+ I would expect 594+ to continue being a great STED dye, but I don’t have data to share with you regarding either use case. The structural differences between the AF+ dyes are all slightly different too, the goal was to make our AF dyes brighter and more photostable than the original, so each dye has a unique modification and/or purification method that allowed us to achieve that goal, which means that each AF+ dye may perform differently on super res platforms that require specific photophysical properties.”
Sorry for the super late reply. Am digging my way out from under a crazy year of teaching 84 hours in French, 126 hours in English, 600 students in total. Never again.
Christian Sieben tried AlexaPlus 647 for STORM, and didn’t notice any significant difference in performance from Alexa647. Nobody in my group has tried the others.
Thanks all of you for answering and taking care. In the meantime we have compared the normal to the “plus” versions of antibodies and could not experience a significant difference in performance - at least true for our settings.
Have used Alexa 674 in a STED demo (Leica) and it worked great, had to be a bit heavy handed with the excitation and STED lasers to get the signal on my particular sample, but it did work.