I wondered if anyone would be able to share fluorophore combinations that are appropriate for making the most of the Tausense module in multi-channel immunofluorescence experiments for imaging on a (new!) Leica Stellaris system. I’ve had a look for values of lifetime for common IF fluorophores (e.g. ISS Data Tables | Lifetime Data of Selected Fluorophores),
but since these are dependent on solvent etc and measured in vitro, how useful are they for work in tissue sections?
Can you, for example, stain Cy3 and AF555 together?
I’m obviously going to ask Leica for their suggestions on this, and see if they publish any materials to help design for Tausense, but I’d be grateful to hear from anyone with experience of using this.
Presumably this could look quite different from the way you would conventionally go about designing antibody panels for systems using spectral detector or unmixing-based methods of dye separation.
Or is it not sensible to use Tausense in this way at all - i.e. should you only use it for removing autofluorescent components, and try to keep a more conventional wavelength separation between your fluors (especially now with all that space out in near-IR on Stellaris ?
I’d be grateful for any advice and examples. Thanks!
I don’t really have an answer for you but we also have a Stellaris and also considering its usefulness in this regard, but practically we haven’t used it for dye separation. I had someone looking at collagen and it appeared to show nice lifetime differences between the collagen types so the user told me although they didn’t need this information it turned out. The issue for me is that you end up with a mixed dataset some of it based on intensity and other channels lifetime and i’m not sure yet how to marry the two together especially if you intend to do image analysis afterwards. Whereas if you stick to spectral unmixing or intensity based data it would seem less complicated but i’m new to FLIM myself. Some isolation of autofluorescence would seem useful though.
Hi Matt, thanks for replying. Yes, it does seem very easy to use, which is part of the appeal!
Leica seemed to tell us that for a Tau-separation approach to work well for unmixing, the difference in the lifetimes of the two fluorophores needs to be quite large. Like the example shown in the video (thanks for posting it here!), there’s no overlap at all there in the spread of arrival times. I suspect that for most potential combinations of fluors it will be less clean than that? But I don’t really know.
I’ll try to get some trials done on this, and if I can I’ll add the outcome to this thread.
From experience, I would advise you to choose your fluorophores carefully. It is best to select the second fluorophore with a 2x longer delayed lifetime. This will help optimize your results.
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