Imaging PFC with V4

[Kayla Howell]

Hi everyone,

I'm relatively new to miniscope imaging and was hoping to get some troubleshooting advice.

I'm using a UCLA Miniscope V4 with a 1 mm Inscopix GRIN lens and AAV-hSyn-GCaMP8f in the prefrontal cortex. I allow approximately 3 weeks of expression before implanting the lens.

I've now imaged several mice but have not been able to consistently identify clear neuronal cell bodies or robust calcium transients. Occasionally I see faint fluctuations or small flashes, but nothing that looks like the examples typically shown in publications.

A few questions:

1. What are the most common reasons for seeing faint fluctuations but no obvious neurons?

2. How can you distinguish between poor viral expression, incorrect lens placement, and an optical issue with the miniscope system?

3. Has anyone experienced a situation where the miniscope only appears sharply focused when extremely close to an object? If so, what ended up being the cause?

4. If a GRIN lens is slightly above the target region, what would you typically expect to see during imaging?

For additional context, I target the PFC and implant the lens to approximately 1.8 mm depth. I've recently been questioning whether my issue is surgical targeting or a problem somewhere in the optical path. I've added a photo for reference of what I am picking up. 

Any advice or troubleshooting suggestions would be greatly appreciated.

Thank you!

[Daniel Aharoni]

Hi! Thanks for the info and these are all great questions.

1. What are the most common reasons for seeing faint fluctuations but no obvious neurons?

This is very often due to too much tissue between the bottom of the GRIN lens and the cells your want to image. 1 photon imaging is affect strongly by fluorescence scattering through tissue. This ends up degrading spatial resolution and at some point results in not being able to resolve single cells. Usually this limit is around 250 to 300 um of tissue between the GRIN lens (or cranial window) and the expressing cells. To address this you might want to aspirate a bit more tissue and implant the GRIN lens slightly deeper.

1. How can you distinguish between poor viral expression, incorrect lens placement, and an optical issue with the miniscope system?

Poor viral expression will often result in just a really dim FOV. If you are seeing decent fluorescence brightness, even if single cells aren't resolvable, you likely have ok expression. From the region, GCaMP,  and image you shared, I would expect to see the type of brightness your example image shows with a Miniscope V4 LED value of between 5 and 40... if you have to push much past 40, you might need to dial up expression.

1. Has anyone experienced a situation where the miniscope only appears sharply focused when extremely close to an object? If so, what ended up being the cause?

The Miniscope V4 should have a front working distance (the distance between the bottom of the Miniscope and the thing you want to image in focus) of around 700 um. If your scope significantly deviates from this number, there might be an optics problem.

1. If a GRIN lens is slightly above the target region, what would you typically expect to see during imaging?

Your example image actually looks quite nice and clean. I think your surgery and GRIN implant are likely totally fine... just need to maybe for a little deeper. Take a lot at our protocol paper, https://pubmed.ncbi.nlm.nih.gov/40354432/, that might provide some additional help.

[Kayla Howell]

Thank you so much for the detailed response! This is really reassuring and very helpful.

One thing I noticed is that I typically need LED values >50 to see anything, and I only really see these faint fluctuations when the focus is near +100 or -100. During bench testing, the Miniscope also seems to come into focus only when it's almost touching the object. Does that sound consistent with an optics issue, given the expected ~700 μm working distance?

Also, I currently implant the 1 mm GRIN lens to ~1.8 mm in the PFC. Since you think I may just be too far above the cells, how much deeper would you typically recommend going? Would another 100–200 μm be reasonable, or would you expect a larger adjustment?

Thanks again for taking the time to help, I really appreciate it!

[Daniel Aharoni]

>50 LED is a bit high but ok. You might also want to increase the gain of the Miniscope from 1 to 2 or 3x so you can drop the excitation lower. This might also hint at needing to adjust expression of your GCaMP.

When you say " the Miniscope also seems to come into focus only when it's almost touching the object", 700 um is quite a short distance already. Do you have a feel for it there is a little less than a mm between the scope and the object when it comes into focus or much much less?

[Paul Vander]

At >50 LED you might just be imaging the autofluorescence of the brain, and any changes in signal could be due to blood flow/motion artifacts. I've also found that there can be small light leaks within the miniscope when LED and/or gain values are turned up very high (which are negligible when used at lower values with a strong GCaMP signal).

I agree with Daniel that you probably need to check the GCaMP expression. Can you see GCaMP-expressing cells in histological sections under a regular microscope (if you have brain tissue from GCaMP-injected mice)?

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Paul Vander

Pronouns: he/him/his

PhD Candidate, NIA F99 fellow

Correa lab | https://sites.lifesci.ucla.edu/ibp-correalab/

Molecular, Cellular & Integrative Physiology PhD Program

University of California, Los Angeles