Imaging cortical layer 2/3 and 5 with miniscope V4

[Vanessa Kan]

Hi everyone,

I am trying to image cortical layer 2/3 and 5 with the miniscope V4, and I still have some questions after reading the tips shared by others in this forum. 

Layer 2/3: 

(1) I have seen both (i) removing the dura, and (ii) leaving the dura intact - did anyone test both, and what were the outcomes?

(2) How deep and dense should the injections be? Right now, I am doing 2 injections at ~300um depth. Should I do even shallower injections to label layer 2/3 neurons?

Layer 5:

(3) What GRIN lens (diameter and length) do you use to image cortical layer 5? 

(4) How deep should the GRIN lens be implanted, without aspiring tissue, to access layer 5?

(5) How should the baseplate be positioned, as the GRIN lens will be sticking out, and if you mount the baseplate close to the surface, you won't be able to attach the miniscope properly. Should the miniscope be touching the surface of the GRIN lens?

Thank you in advance for reading this, I look forward to discussing further :)

Vanessa