I run an imaging core with a Zeiss Elyra 7 doing SIM. I have a new user doing some experiments using fixed, isolated cells. For some reason, he's getting tons of photobleaching with the 642 laser (not present in the other channels). He's using AF647 conjugated antibodies, only .5% laser power/100ms exposure. The only thing I can think of that might help this would be adding mounting medium to his dishes (right now he's just got an imaging buffer). [Also, JUST had my preventative maintenance visit, so I know it's not a problem with our 'scope]
Would this help stabilize his fluorophores? What's the best way to add mounting medium to a 35mm coverslip dish? Is that actually a thing?
Dear Rachel Escue ,
I con not comment on the photo bleaching, but on the mounting of 35 mm glas bottom dishes (I assume that is what you meant with cover slip dishes). I have used WPI FD35-100 extensively and always used round cover slips (Thermo Fisher) with them. You/he should get some 1-2 mm lower than you inner plastic circle. Than I have cleaned them with some paper and applied a drop of mounting medium to the round coverslip. Than I turned them with tweezers, so that the drop is hanging and all bubbles go to the out side of the drop. Afterwards I just put the coverslip onto the glas bottom (without buffer just residual humidity) and it's even possible to push then the rest of the buffer and bubbles to the edges (with the back of the tweezers). He can have some test runs without the actual sample.
Best wishes Soenke