Imaging Cortex with V4

[Chelsea Pernici]

Hi. I am planning to image cortex using the V4 and am trying to avoid using a GRIN lens. How deep can we image into the cortex with just a cranial window before light scattering will be an issue?

Thanks in advance!

[Daniel Aharoni]

The standard lens configuration for the V4 gives you a working distance of around 600 to 900um. This should be enough to image through a cranial window and into layer 2/3. Generally speaking, imaging any deeper will be difficult due to scattering of GCaMP fluorescence through tissue between your cells and the surface of the brain.

[Jyun-you Liou]

Hello,

It seems to me that both of us are trying to use V4 to image cortex.  I have not been able to get single cells yet.  However, I found it pretty important to fully press down the V4 scope down to the baseplate. With a standard 0.17 mm thick coverslip, making sure the scope contact with the baseplate tightly gives me a view at the brain surface with my EWL at +127 position. 

Are you using a transgenic animal? I tried Thy1-GCaMP6f and PV-GCaMP6f. I couldn't see individual cells (perhaps just too much background?).  Could you share what animals you use to obtain a single-cell resolution while using Miniscope to image the cortex? 

Sincerely appreciate any suggestion,

[Ana Carolina Bottura de Barros]

Hi,

I also tried using transgenic animal and didn’t have success with v4 through a cranial window (I think there was a lot of background). I know some people are able to image Thy1, but I am now injecting GCaMP virus for expression to get single cells. It takes some tweaking to get the proper dilution for a specific area but you will get there at some point.

Ana

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[Richardson Leão]

we have some animals w camk2a in layer 2 that i imaged w the v3 w the dura intact. and the 1.8mm lens (edmund). I guess that would suffer from the same scatter problem. we have more injected animals without the plate that we could try buty i dont have a clue on how to put a coverslip in the skull. Do you know of any published protocol for that?

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Richardson N Leao, MD PhD,

Neurodynamics Lab, Head

Brain Institute, UFRN/Brazil

[Chelsea Pernici]

Hi all, 

I am using thy1-gcampf for my experiments. I have yet to see cells as well but I am still in the infancy of the project so I have only imaged a few times and have not gone through many troubleshooting steps. Below is a link to the expression data for the Thy1-Gcamp lines: 

http://jackson.jax.org/rs/444-BUH-304/images/image_summaries_24275_24276_24339.pdf 

Cells should be evident in layers 2/3. I haven't changed the lens configuration of the V4, so plan on doing that to make the WD longer and minimize error from not having the V4 directly on the coverslip for the cranial window. I also need to a good control to see if the V4 is able to image this transgenic line. I will probably fix the tissue and just image native fluorescence to check. If this checks out, it may be that trying to image layer 2/3 is causing too much scattering and I will need to implant a lens of some sort. I don't really want to implant a GRIN lens as this will decrease my FOV unless I use a large GRIN lens. I know Bern Optics make glass windows which would be useful for this application (https://www.bernoptics.com/). You can have glass windows custom made to the length and diameter you want. I think a relatively short lens would get you close enough to layer 2/3 and minimize the amount of tissue you have to image through. They only caveat is you will need a larger WD with the V4...probably lens configuration 2 or even 3. I need to do some math. 

As for the coverslip on the skull...do you mean just a cranial window if so here is a protocol I use: https://www.jove.com/t/680/a-craniotomy-surgery-procedure-for-chronic-brain-imaging. It's an older protocol but works. 

I will update this conversation as I troubleshoot my way through this problem. 

[Zahra Laouby]

Hello everyone,

I am doing cortical imaging (layer 2/3) in the SD rat model. I proceed by injecting GCAMP-7 in the cortex, and then I place a grin lens on top without compression (during the same surgery session). After 3 to 5 weeks, I baseplate. Unfortunately, although I see individual cells with the V3, the cells don't look that happy. Indeed, I  see massive damage within the site of the injection. I've already tried different virus concentrations and injected at different rates, but I get inconsistent results. Does anyone experience the same issue with rats?  I would appreciate any suggestion.

I really appreciate any help you can provide.

Best regards,

Zahra

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[JIAXIN FU]

Hi all, 

We've tried injecting AAV9-hsyn-jfCaMP8m with various dilutions and failed to visualise individual neurons in layer 2/3. The expression was there, but it was centred around the vessels.

Now we're going to try GCaMP6f, and several questions are in mind: 

1. Is it better to remove the dura or leave it intact? Sometimes we see the brain was pressed down and covered with blood after removal, but regrowth can happen without removal

2. Through a cranial window, has anyone figured out suitable injection depths? We've tried values between 200 - 300 

3. I know dilution is an issue, any recommendations on dilution ratio? 

Thanks!