nothing but gray

[hsiao]

Hi,

I was trying to do my first Gcamp experiment.

I planned to record dCA1 in rats. Since the grin lens (#64-519, Edmund optics) is not long enough, I stacked 2 lenses and screwed them in the bottom of Miniscope as an object lens. Then, I injected AAV into the dCA1 around the stratum radiatum and implanted 1 lens on the pyramidal layer (total 3 lenses, including 2 object and 1 relay lenses). After 3 weeks, I saw nothing. I even can’t see the blood vessels. I could only see black and gray under the maximal power and gain. I waited another week but still had no luck. You can check the screenshot below. This is a screenshot after 4 weeks of incubation.

 
I just perfused this rat, no obvious blood clot is noticed. I am still waiting for the histology and want to make sure my Gcamp express well but can’t wait to ask questions here for improving the protocol for my second rat.

 

I am not sure if my setup is correct (2 object lenses plus 1 relay lens) but I did test and see clear signals on a paper with green lines. I used some methods like this http://miniscope.org/index.php/Initial_Testing_of_Assembled_Miniscopes

 

Thank you!

[Daniel Aharoni]

Hi hsiao,
Can you clarify what you mean by stacking 2 objective lenses? Do you mean you put two of the #64-519 Edmund lenses end to end? If so this will create a ~0.5pitch GRIN lens which has very different optical properties than what the Miniscope is expecting as an objective lens. In other words, you have to keep the pitch of the objective lens to ~0.25 + 0.5*N pitch where N can be 0, 1, 2, ... We have imaged in rats by stacking 3 objective lenses together but using only 2 won't work.

 I am guessing the reason you saw clear signals outside the brain with a stack of 2 objective lenses is because you were imaging with a rather large working distance, a working distance much larger than what would work in the brain.

[hsiao]

Hi Daniel,

Thank you for your fast reply

I am currently using a setup like attached file Fig1A.
What you mean is I need to use a setup like Fig2A or 2B?

[Daniel Aharoni]

Thanks for the schematics. This clarifies things a bit. So you actually just want to use 3 of those GRIN lenses stacked end to end with no gaps in between them.  This will effectively make a single GRIN lens with a 0.75 pitch which will work for imaging with a short working distance of ~50um to 250um.

[hsiao]

So I think the setup I was using is fine. However, I saw the histology slice yesterday.
It seems that my problem is the implantation.

The lens was above dCA1 cell body over ~500um. I should lower it further.

But here comes other questions.
1. The working distance of #64-519 is 0, so I should implant the lens as close to the cell bodies of dCA1 as possible (at least < 250 um)? If I choose a lens with a 0.2 mm WD, then it gives me more space so that I can implant the lens like 0.2mm or more above the cell bodies?

2. The medial hippocampus is higher than the lateral. Will you tilt the lens a little bit to fit the hippocampus?

3. In the dual lens system, the wiki page uses the title "Imaging With Thin GRIN Lenses". My question is that is it necessary to use a relay lens that is thinner than the objective lens? Will there be any problems if I use a relay lens with the same diameter with the objective lens?

Thank you!

[Daniel Aharoni]

Hi,

The lens was above dCA1 cell body over ~500um. I should lower it further.

Yea, you should really try to keep this distance to under 300um. Even 300um is a bit large.

But here comes other questions.
1. The working distance of #64-519 is 0, so I should implant the lens as close to the cell bodies of dCA1 as possible (at least < 250 um)? If I choose a lens with a 0.2 mm WD, then it gives me more space so that I can implant the lens like 0.2mm or more above the cell bodies?

You should always implant the lens as close as possible to the cells you want to image. The lenses from Edmund have a design wavelength different than what you will be using. This wavelength affects the actual pitch and working distance of the lens. So the quoted 0 or 0.2mm WD willbe different when imaging green fluorescence. But in general you want the effective pitch of your objective lens to be slightly under 0.25.

2. The medial hippocampus is higher than the lateral. Will you tilt the lens a little bit to fit the hippocampus?

I'm not sure. A little tilt might help with imaging.

3. In the dual lens system, the wiki page uses the title "Imaging With Thin GRIN Lenses". My question is that is it necessary to use a relay lens that is thinner than the objective lens? Will there be any problems if I use a relay lens with the same diameter with the objective lens?

No, it can be the same diameter.

You might want to contact Walter Boyles at GoFoton, walter.boyles_at_gofoton(dot)com, and request a custom 2mm diameter GRIN lens made by them. If you give him the working distance you want (something like 200um on both ends of the GRIN lens) and the overall length you need he can maybe make you a single lens that will work for you. This would result in a single GRIN lens instead of stacking multiple lenses.

[hsiao]

I am really appreciated your inputs.
 
Good luck on my second rat

[kjlawlor]

Hi,

I am also trying to get my imaging in Rats to work. I have implanted 10 rats using two different viruses, and have yet to see anything. I am using the two lens relay system, with the implanted lens 8.4mm long for a region that is 5.5mm deep. I am wondering if there are any changes in the protocol for rats. Also, do you know people who have successfully imaged rats using the miniscope in such deep regions?

Does the miniscope team have a list of publications that are using the miniscope system?

Thanks for the help!

[hsiao]

Hi, kjlawlor

Would you mind to share your experience? Like where did you get your viruses and what kind of gene did they express?
How is the histology going?
Which lens did you use as objective and relay lens?

I had my second surgery done and would love to report the outcome 3~4 weeks later
This time I used addgene aav1 https://www.addgene.org/100843/

Thanks

[Daniel Aharoni]

Hi kjlawlor,
There are a few labs I know of successfully imaging in rats. We work most closely with Tad Blair's Lab at UCLA and they are getting really nice data sets. Surgeries are a bit trickier in rats since their brains are larger than mice's but you still need to get the implanted less withing a couple 100um of the cells you want to image. Also, I know getting the correct GCaMP virus was very important. I think they had the most luck using AAV9 but you should double check with them.

Bastijn van den Boom at NIN has also had success imaging in rats.

[kjlawlor]

Hi Daniel,

Thank you so much for the reply. I have reached out to Tad Blair's lab and they have been very helpful regarding the viruses and other information. They did indeed use AAV9.

Hsiao,

I bought the virus from addgene as well, https://www.addgene.org/51085/
So far, the histology shows that the virus expresses, however we have had to dilute it 1:10 but we are still experiencing over-expression so we may need to dilute even further.
We are using the relay lens from inscopix (8.4mm), and the objective lens from Edmund Optics, 5mm Dia. x 12.5mm FL, MgF2 Coated, Achromatic Doublet Lens.
We have not successfully seen any dynamic cells, and I believe that may be either due to the virus or possibly the lens implantation process. We are still troubleshooting to figure out how to improve. Hope your second surgery turns out well!

Kristen

[hsiao]

Hi Kristen,

Do you mind to tell me the information from Tad Blair's lab?

Thanks!

Hsiao

[TBNentwig]

Kristen,

What brain region are you targeting?

From what I have heard from people trying to image deep brain regions in rats (here at MUSC) using the Inscopix system is that it is very difficult and they have been unsuccessful. They have got good cells in mPFC but it seems like deep targets like NAc and VP don't yield good cells, not sure of the theory behind it, perhaps due to more inflammation in rats.

[hsiao]

Still have no luck on my second rat ,

3 weeks after virus injection, I check the image today.

This time, I did not use the objective lens.
The relay lens just sticks out the skull about 1mm.

I think I spotted a thin blood vessel (not sure since it is not clear either) but could not find any white neuron.
I cemented the baseplate anyway because I think maybe the cells will appear next week.


Maybe it is something wrong during the surgery...

I read the instruction again and found one step that I didn't do: Very slowly and carefully remove the horizontal white striations of the corpus callosum until you reach the striations that go vertical. 
Several questions I want to ask about it.

1. How do you remove the corpus callosum? by tweezer, needle or aspiration?
2. How to tell the horizontal and vertical corpus callosum?
3.
During the implantation surgery, will you see blood vessels through the relay lens?
I didn't see any blood vessel on the corpus callosum during the surgery. Is that normal?
Maybe I can't see the blood vessels during implantation and baseplating it is due to lack of removing corpus callosum?
I think it may be good to have a same blood vessel landmark during surgery and baseplating.

Thank you

[Daniel Aharoni]

Hi hsiao,
You need to always use the objective GRIN lens in conjunction with the relay lens. If you try to image with just the relay lens you will never see anything in focus.

1. You remove the corpus callosum with aspiration, never forceps. I think we should have videos in the surgery power point presentation on miniscope.org under Workshop Resources.
2. You should be doing all your aspiration under a steroscope. You will visually see the fibers turn direction as you aspirate deeper.
3. We generally do not see much during implantation. It takes a could weeks for the imaging field to clear up to see a decent image.