Great questions. Firstly, transgenic mice can be a bit on the dim side so there is a chance you need to boost the gain or excitation power or drop the FPS to see something... especially if the animal is anesthetized.
- How do you accurately determine the depth at which you are implanting the GRIN lens? At the moment I am zeroing the base of the lens on bregma then lowering it to a depth of 1.5 mm into the aspiration channel. However as I cannot judge where the base of the lens is once it goes into the channel I can't be certain that it is positioned where I think it is
This is roughly how we do it as well... but 1.5mm might be a little deep. The surgery powerpoint here, http://miniscope.org/index.php/Online_Workshop
, outlines the approach we often us. For the specific researcher and specific animal and age it can take quite a bit of trial and error before you find the depth that gives you consistent results. You might want to try a batch of surgeries with different lens implant depths to see what works best for you.
- How close to the surface of the CA1 should the base of the GRIN lens be and what is the margin of error?
- Does the top of the GRIN lens need to be flush with the dental cement or can it protrude out?
It is a good idea to build up the dental cement around the GRIN lens to help protect it. While this isn't necessary for image quality, it is a good idea to extend the lifetime of the lens.
- Does the objective lens of the miniscope need to be in contact with the GRIN lens or sitting just above it during imaging?
It shouldn't be in contact with the relay GRIN lens. For the V4 Miniscope, you generally will have around a 900um gap between the top of the relay GRIN and the bottom of the Miniscope. Once you have a lot of experience with this it is easy to get it in position but to start, have the Miniscope focus on the top surface of the GRIN lens (you will be able to see the edges of the GRIN lens and possibly the actual glass surface) and then move the Miniscope up by around 150um to 250um and you should see the brain come into focus.
When baseplating, you will want to set the electrowetting lens position right in the middle and physically adjust the gap between the GRIN and Miniscope until you see cells. Then cement the baseplate. Now you still have around +/-150um of focus adjustment using the EWL if things move on you a bit.
- What method do you use to correctly align the miniscope with the GRIN lens when attempting to image through it?
With the V4 Miniscope and relay GRIN lens, it is very important to have the GRIN and Miniscope's optical axis aligned parallel with each other. As the angle of the Miniscope relative to the GRIN gets out of parallel, you will loose focus of the brain. Search in this google groups information about 3D printable Miniscope mounts which can be attached to a stereotax for fine tuned placement.