Thanks for your reply, Hazen!
I tried to image membrane proteins in fixed cell. The microscope I used is Nikon Ti2 TIRF N-STORM with objective of CFI SR HP Apochromat TIRF 100XAC Oil and iXON Ultra 897 EMCCD camera. Under TIRF view, I can successfully get SMLM image using AF647 labeled antibody. My issue is I can not get bright blinking events when I used CF568 and JF549 dye. I’m trying to do a multiple-color STORM, thus I want to use CF568 or JF549 combined with AF647. For CF568 and JF549 dye, I always got a very dim blinking event just above background. I did not test the power of laser, but I usually used 100% power of 561nm laser controlled by an Agilent laser box. The excitation light is reflected by a quad dichroic and the emission is filtered by a quad emission filter (open at 422 – 478 nm, 502 – 549 nm, 581 – 625 nm, and 674 – 786 nm).
I thought it might be due to the buffer. So far, I have tried two kinds of buffer below, but none of them worked.
MEA/OS buffer: 50 mM Tris (pH 8) +10 mM NaCl + 10% glucose + 100mM MEA + 0.56 mg/mL Glucose Oxidase + 0.034 mg/mL Catalase
OxEA buffer: 1xPBS (pH 8) + 20% (v/v) of sodium DL-lactate solution + 50 mM MEA + 3% (v/v) OxyFlour™
It would be highly appreciated if you have any suggestions to solve this problem.