V4 imaging through cranial window

[Alan]

Has anyone tried this? I am wondering whether I have to place a coverslip on top of Dura. Then should I thin the skull (on which the baseplate will be placed) so that the lens directly contacts the coverslip?

Thanks for your help!!

Alan

[Daniel Aharoni]

Hi Alan,

We do V4 Miniscope imaging through a cranial window all the time. In mice you can leave Dura intact but in larger animals you usually have to remove it. The key here is you need to be able to get the bottom of the Miniscope within its working distance to the cells you want to image (also remember that you generally are limited to imaging through ~300um of tissue before scattering of fluorescence degrades your spatial resolution too much to resolve cell bodies). With the standard V4 Miniscope lenses (https://github.com/Aharoni-Lab/Miniscope-v4/wiki/Lens-Configurations) this means the bottom of the Miniscope will need to be ~700um from the cells you want to image. In this space you need to fix a cranial window and the cement for the Miniscope baseplate. It can sometimes be challenging to fit this all within ~700um so you might want to swap out one or both of the Miniscope objective lenses to give you a larger working distance.

When baseplating, you will want to first attach the baseplate to the bottom of your Miniscope and watch the live view of the Miniscope while baseplating. You will want to adjust the x,y,z position of the Miniscope + baseplate until you see cells (or other landmarks that denote the layer you want to image) and then cement the baseplate in this exact spot. Generally this will still leave a small gap between the bottom of the Miniscope and the cranial window but this will depend on the lenses used, the thickness of your window, and the depth you are trying to image.

[Alan Jung Park]

Thank you Daniel for providing a detailed explanation!

So you don't need to place a coverslip onto the Dura or thin the skull? I have the standard lens configuration (config 1).

Thanks,

Alan Jung Park, PhD

Postdoctoral Fellow 

Gogos Lab

Columbia University

Mortimer B. Zuckerman Mind Brain Behavior Institute

Jerome L. Greene Science Center, L5-053

3227 Broadway,

New York, NY 10027

Neural Circuits laboratory

Department of Psychiatry

Columbia University and the
New York State Psychiatric Institute
1051 Riverside Drive, Unit 87
Kolb Annex, Room 136
New York, NY  10032

alanjp...@gmail.com
Phone: (646) 774-7116
Fax:   (646) 774-7101

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[Daniel Aharoni]

You will need to do some sort of standard cranial window implant for imaging applications. This often involves cementing a small piece of cover glass to the skull where it is also directly in contact with Dura. Some people will used a custom stepped glass window where it both extends into the craniotomy as well as over the skull near the craniotomy. I bet you can find some decent methods/protocols in miniature microscope and 2P imaging publications.