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nothing but gray

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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!

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