During my experiments doing in vivo 2p imaging I get some odd background fluorescence around cells infected with AAV - Syn- GCAMP6. I am using a 10x objective with a low Na, but it seems as if the tissue is glowing, when imaged through a cranial window? Any thoughts what this might be? I was thinking this could be arbors? It does not seem as if the point spread is bad- I get good contrast elsewhere (but will calibrate it all the same in the coming weeks)
I will try to upload an example.
We would likely need a little more information. 1st, higher, NA (numerical aperture) generally means better resolution, and remember 2p excites in IR. So, using an IR corrected, long-working distance, high NA objective would be ideal for maintaining a tight PSF and assure the excitation and emission focal planes overlap. 2nd, knowing how you are introducing the virus would be helpful. If you are injecting virus, proteins and ECM expressed in the injury tract can introduce auto-fluorescence. Also the effects you see could be from inflammatory proteins in your viral prep. You man need a sham control or ideally, a second virus expressing a junk protein as a control. It is imperative we know you have the proper controls.
Our setup/ imaging area poses issues to getting a higher Na objective in the area. Also it it very difficult to use an immersion objective for our experiment.
The virus is being injected with a 20-30um bevelled glass capillary- pretty standard. No obvious localization to the injury tract- glow is present pretty far away.
As for controls- Gcamp6 is a standard protein these days and so is the virus construct running off Syn promoter. No one I know who uses this construct reports any similar glows.
Our windows are not standard though- they are made of glued pieces of glass (with optical glue). Could thin film interference be responsible for such and effect?
Could you post an example of the background? And perhaps a sketch of the prep? I don't understand where the interference might be happening.
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