Hi,
I am a PhD student, and my project heavily depends on imaging. I’ve been using LSM880 for my confocal images; however, recently, my PI suggested I start using airyscan for some of my images. I am trying to visualize nuclear pore complexes, similarly as in this paper (Fig. 1h), so my images require using 63x objective and max zoom. I recently tried using airyscan on my samples, but my fluorescent signal photobleaches so fast that I can’t even find a field of focus before taking the picture (I used gain at 800 and laser power at 0.5; otherwise, I didn’t see any signal at all). In my staining, I used Hoechst stain, a secondary antibody conjugated with Alexa Fluor 568 (Invitrogen; # A10042) and ibidi µ-Slide 8 Well (#80826). I have successfully used this combination for confocal microscopy, but I started questioning this method since the airyscan imaging didn’t work. What kind of more stable fluorophores would you recommend for this technique? Could it be the fault of the ibidi slide, and should I switch to the glass slide/glass coverslip?
Thank you so much for your help!