Thy1-GCamp6 cortical imaging with miniscope V4

[fro...@gmail.com]

Hi all!

 We are trying to use the Miniscope V4 with Thy1-GCaMP6sGP4.3 transgenic mice. We tried to image from the visual cortex of 2 different mice over a 4mm cranial window with a glass coverslip. We can clearly see the vessels at the top of the cortex, and we have tried to image at different depths from L1 to L2/3.  However, we can never see cells in either the raw image or the processed df/f denoised image. There is calcium activity though and when we use the Minian (Dong et al. 2021) processing package, there algorithm can separate some cells. These are never clearly seen in the images (example attached). 

We are wondering if this could be because of the mouse line and cortical imaging (such as dense expression of GCamp6 in neuropil) or if is a miniscope issue. 

Any suggestions would be greatly appreciated. Thank you in advance!

Cheers,

Emmanouil

[Federico Sangiuliano]

Hello Emmanouil. It looks like the FOV is very out of focus. At which value is the electrotunable lens set?

[fro...@gmail.com]

We have tried several values of the electrotunable lens spanning the whole range of it. Adjusting the miniscope to the base plate we can see that at least a couple of different values can produce a sharp image of the vessel pattern at the surface of the cortex. 

The attached image is from an imaging depth of ~200 bellow the surface of the cortex. I believe that it is expected to be blurry if there is nothing with a lot of contrast at the imaging depth. Isn't that the case?

Thanks!

E.

[Federico Sangiuliano]

From that image it will be very hard to see any neuron, so I suggest redoing the recording with a better focus. Here is an example of one of my cranial window recording done with my colleague Megha from Alcino Silva Lab.

In this screenshot you can clearly see the blood vessels and a few neurons that we active in that frame. I hope this helps.

[fro...@gmail.com]

Thanks!

Is this with the Thy1-GCaMP6sGP4.3? 

So you think we were too deep and should get closer to the surface as you are? 

Also your line noise levels are much lower. How long is your cable between the miniscope and the DAQ?

Best,

E.

[Alan]

Hi all,

Can anyone share a good protocol for making a reliable cranial window? 

Best,

Alan

[Daniel Aharoni]

Hi  Emmanouil,

I can answer some of these questions you had for Federico:

• Is this with the Thy1-GCaMP6sGP4.3? 

No, this is with a viral GCaMP. Pretty sure it is AAV1 GCaMP6f. Thy1 mice are generally quite a bit dimmer than AAV mice. From the image you posted though, it looks like you are getting a decent amount of fluorescence... it just isn't in focus.

• So you think we were too deep and should get closer to the surface as you are? 

Not sure. Layer 2/3 is generally down around 200um below the surface of cortex. You might even want to push down slightly with your cranial window implant to compress cortex by ~50um... This can sometime help reach a bit deeper and stabilize the brain. Also, cortex can be very quite when the animal is athetized. You might not see any fluorescent dynamics until the mouse is up and about. Some people will add a headbar when doing cranial window implants to keep the mouse awake during baseplating.

• Also your line noise levels are much lower. How long is your cable between the miniscope and the DAQ?

This shouldn't be a function of the length of cable as long as you aren't going over 10 ft in length or using some crazy amount of excitation power. This might be from you using a higher gain than what was used in Federico's image. It could also be that you are using a slightly older version of the V4 Miniscope which can have a tiny bit of row noise. Here is a Jupyter Notebook script to remove this noise from recordings: https://github.com/Aharoni-Lab/Miniscope-v4/tree/master/Miniscope-v4-Denoising-Notebook

[Rob Graham]

Hi Emmanouil, 

I'm not sure about other people's experience of using the miniscope just with a cranial window (please share them if they are different!), but in my experience (UCLA, V4) it is very difficult to get good snr and clear neurons with a simply GCaMP injection. It reliably created a field of expression in the L1 neurites which made L2 invisible. Instead, we had success driving sparser expression with an injection of AAV1-hSyn-Cre (10^10) and flex_GCaMP6f (10^12). This has given us much better cell labeling against the neuropil background. Further, it's possible to get close enough with #0 thickness glass windows, but we have had much more success with pdms optical silicone windows INSIDE the craniotomy, so the top of the window is flush with the top of the skull, this allows us to have pial vasculature in focus in the upper quartile of the focal range, and plenty of depth for imaging. 

Hope this helps!

Rob

[Георгий Буков]

Hi all. 

I'm trying calcium imaging of the mouse somatosensory cortex through cranial window with V4 Miniscope with viral injections and it's still not successful. 

Can anyone give some advises or protocol for cortical imaging? I think we should try retrosplenial cortex with dura mater removing - does it makes sense?

Thank you for answers.  

четверг, 7 сентября 2023 г. в 22:32:33 UTC+3, rgrah...@gmail.com: