Hi everyone, I’m new to the forum. We have LSM800 with 4 lasers (405, 488, 561 and 640nm), 3 detectors (one of which is a slider) and built in filters include SP470, SP545, SP620, LP575, LBF 640, LP655. I am trying to multiplex 5 dyes on this machine, thinking of BV421, BV480, AF488, AF594, and AF647. Just wonder if anyone has tried BV421 and BV480 and has insights into BV421 bleeding into BV480.
Thank you.
I do not have a Zeiss 800 (nor 700 or 900), but I’ve tested/demo-ed them. The emission light path to perform multi-wavelength imaging is more difficult to adjust (in my opinion) than the 710/780/880/980 counterpart or other laser scanning emission systems. And 5 fluors will be difficult to separate without blead through, I’d think (even if imaging on another system). I think BV480 and AF488 are more likely to bleed into each other. The best separation would be to image them sequentially one at a time with the prism slider to control emission range. I do not know the controls/adjustments to use multiple track combination - perhaps contact your Zeiss applications rep.; they are usually quite helpful (at least in my experience in US).
-Lisa Cameron (lisa.cameron@duke.edu)
You’re definitely going to have problems with the BV480 and AF488 together. If you can - I would switch it up to BV421, AF488, AF555, AF594, AF647 for best results. You can use sequential spectral imaging to characterize the BV480 and AF488 from controls (along with all your others) - but unless your implementation results in super stable (i.e. no bleaching) conditions - I generally discourage sequential spectral scans in favor of the simultaneous approach on a 880/980 platform.
Thank you all for your comments. Surprisingly I don’t have any problem with BV480 and AF488. Probably because BV480 is barely excited by 488 laser. BV480 was quite dim (or at least because of direct conjugation as CD4 has pretty good antigen density so I don’t think the brightness is due to the antigen). I am switching to indirect to not juggle so much with the bleed through from BV421. I did try AF555 before but could not handle bleaching and the bleed through into AF594. Luckily, it seems like both of these brilliant violet dyes are more photostable.