Dear Soledad, I have been using STED regularly for more than 5 years at our BioImaging facility and tested a lot of fluorophores. The best by far and large in my opinion is AF 594 (depleted with 775). You can couple it to ATTO647N that works very nicely and would use the 775 too as added bonus.

As 3rd dye for 3 colour STED we use here AF 532 (depleted at 660). We nearly never use the 592 depletion laser since (A) we always get good results with the 3 dyes mentioned above and (B) there are not "good" "green" dyes. However, if you really need the green, then go with OG488. An alternative to ATTO647N that is very lipophilic and sometimes interacts too much with membranes is Abberior 635P. PS: remember to use 2x primary and 2x secondary amounts compared to standard confocal doses. AF488 is pretty bad in my hands but can work for a single XY image

Rolando Berlinguer Palmini Great advice. I really appreciate you taking the time to answer. One more question if I may: My brain tissue already expresses YFP or GFP, but I want to increase the signal by using an anti-GFP antibody, that is why we thought to use a green dye. What would you do in this case?. (Also note that our Leica microscope can only depleted at 775 and 592).

Hi Soledad Miranda-Rottmann . I (personally) wouldn't care if you already have GFP in your sample, you just need a signal and whatever colour looks to your human eyes is not important. Also......the detector is colour blind anyway :-). I would (if exist) use AF594 anti-GFP. This will give you a very good signal. ATTO647N would work too but is not as good as AF594. AF532 is not an option anymore since you don't have the 660 laser. All the above is valid assuming that you have no other "dyes" in your sample (apart the mentioned GFP and DAPI)

I also did a lot of STED images, and I totally agreed with Rolando Berlinuer Palmini, AF 594 works very well with STED. We have been using Abberior Start 635P as our second color, and it is as good as 594 on my hand. We haven't use 647N too much, didn't know which one is better.

Does anyone know or can recommend how Alexa Fluor 647 works for STED using the Abberior / 775 nm STED laser? We are using STAR Red now but would like to compare with AF647. Also, has anyone tried the new STAR Green from Abberior to work with the 775 nm laser? We've tried ATTO 490 LS and it works...

Hi Dion, AF647 is, in my hands, one of the worse, dye for STED. It is however one of the best for STORM. Basically bleaches immediately using the 775 laser (Leica).....but of course it also depends on the laser power.

There are a lot of good alternatives such ad ATTO647N, Abberior star 635P, Abberior Red. All of these are way better than AF647 (in my opinion). I have not tried STAR Green from Abberior.

Hi,

I am new on STED microscopy.

I don't think dyes that I use create any issue ( AF555, AF594n and SiRDNA on 775 laser.

However I feel my STED images are not sharply different than deconvolved confocal images.

I was wondering my imaging technique may not be good. I wonder how I can improve resolution of my images. Can anyone suggest me any techniques in terms of preference of pixel size, what percentage STED laser should be used, what about 3D STED? I also wonder what exactly Gated-STED mean.

Thanks.

Hi Cagla,

AF555 is OK for STED but is the combination with AF594 that is problematic since it leave you a very narrow acquisition window before you get bleed through (from 594 to 555), hence very few photons to collect. If you STED images do not have more resolution than a deconvolved confocal image then your setting are probably not optimal. You should get something like this (left, confocal; middle confocal deconvolved; right STED - 100nm beads). STED laser power? As much as your sample can stand.