Using Baseplating and a Miniscope Preanchored with an Objective Lens for Calcium Transient Research in Mice
June 5th, 2021
The shrinkage of dental cement during curing displaces the baseplate. This protocol minimizes the problem by creating an initial foundation of the dental cement that leaves space to cement the baseplate. Weeks later, the baseplate can be cemented in position on this scaffold using little new cement, therebyreducing shrinkage.
This miniscope can reduce the shrinkage problem with dental cement during the baseplate procedure and increase the success rate of miniscope recordings of fluorescence signals in two length configuration. If the fluorescence transient cannot be observed during baseplating, check the expression of calcium indicator and location of a relay lens on the brain slice. Start with mounting the inner needle of a 20 gauge intravenous catheter with a diameter of one millimeter onto the stereotaxic arm.
Lower the catheter into the ventral corn ammonis 1 into the target region. Then, lower the microinjection needle at a speed of 100 to 200 micrometers per minute into the ventral cornu ammonis 1, and infuse 200 nanoliters of the viral vector into the target region at a speed of 25 nanoliters per minute. After allowing the virus to diffuse for 10 minutes, withdraw the microinjection needle at a speed of 100 to 200 micrometers per minute.
After disinfecting the relay lens, soak the lens in cold pyrogen-free saline until implantation. Using a micro bulldog clamp with heat shrink tubing, hold the relay lens and place it on top of the target region at a speed of 100 to 200 micrometers per minute. Then, stabilize the lens with dental cement.
To set a miniscope, fasten the objective lens on the bottom and assemble the base plate onto the miniscope. Wrap 10 centimeters of paraffin film around the outside of the baseplate. Using reusable adhesive clay, hold the miniscope with the stereotaxic arm probe.
Align the objective lens on top of the relay lens with as little space between the lenses as possible. Use dental cement to secure the positioning of the baseplate, such that the cement touches only the paraffin film. When the dental cement has dried, remove the paraffin film, the base plate, and the miniscope, keeping the dental cement base hollow.
Seal the relay lens with molding silicone rubber and cover the silicone rubber with a thin layer of dental cement. Disengage the mouse from the stereotaxic instruments and place the mouse into a recovery chamber. For baseplating, cut the thin layer of the dental cement roof carefully with a bone rongeur and remove the silicone rubber.
Clean the surface of the relay lens with 75%alcohol. Fasten the set screw beside the baseplate to fix it to the bottom of the miniscope. Adjust the focus slide to be approximately 2.7 to 3 millimeters from the main housing.
When the setting is done, connect the miniscope to the data acquisition board and plug the miniscope into a USB 3.0 port on a computer. Run the data acquisition software developed by the UCLA team. Adjust the exposure to 255, the gain to 64x and the excitation LED to 5%To connect the miniscope to the data acquisition software, click the connect button and watch the livestream of the data acquisition software considering the margin of the relay lens as a landmark.
Once the relay lens is found, align the miniscope objective lens to the relay lens by hand. Adjusting the various angles and distances of the miniscope, begin searching for the fluorescence signals. Holding the miniscope at the optimal position, move the stereotaxic arm towards the mini scope and adhere the miniscope to the stereotaxic arm with reusable adhesive clay.
Search for the best view by adjusting the X, Y, and Z arms slightly. Adjust the angle between the surface of the objective lens and relay lens by turning the ear bar, tooth bar, or reusable adhesive clay on the Z axis. Viral expression and lens implantation are successful when at least one cell displaced fluorescence transients during baseplating.
Fix the base plate firmly with the smallest possible amount of dental cement while monitoring the region of interest to ensure that the optimal position does not change. In the representative study, the procedure of dummy baseplating proved advantageous as it resulted in a smaller shift in the baseplate location than the original procedure. The shift of the baseplate was also influenced by the height of the dental cement.
The baseplate that was anchored 0.5 centimeters above the skull shifted significantly less than the baseplate that was fixed one centimeter above the skull. In an example of successful imaging during the base plating procedure fluorescence transients along with blood vessels were noticed in the anesthetized animal. Fluorescence transients were observed five days after baseplating even when the mouse was freely behaving in its home cage with only a slight shift in a position.
Subsequent calcium imaging of mice was carried out. In a few instances, problems occurred during the baseplating procedure, such as the absence of anything except a uniform background or visibility of white cell margins. without fluorescence transients.
It is important to fix the baseplate firmly on the target region with the smallest possible amount of dental cement. With slate technique, researchers can longitudinally observe neural activity across this. With the proper license, this procedure can also apply to observe the neural network in the field of view.
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Overview
This article presents a detailed protocol for minimizing misalignment issues during baseplating in UCLA V3 miniscopes used for in vivo calcium imaging in mice. The method addresses the problem of dental cement shrinkage, which can alter the distance between the objective and relay lenses, leading to poor image quality. By establishing a cured dental cement foundation before final baseplate attachment, the protocol enhances the stability and success rate of miniscope recordings, particularly in two-lens configurations for deep brain imaging.
Key Study Components
Area of Science
- Neuroscience
- In vivo imaging
- Calcium imaging
Background
- Miniscopes enable observation of neuronal activity in freely behaving animals.
- The UCLA V3 Miniscope is a widely used open-source device for fluorescence imaging.
- Imaging deep brain regions requires precise alignment of objective and relay lenses.
- Shrinkage of dental cement during curing can cause misalignment and degrade image quality.
Purpose of Study
- To provide a protocol that reduces misalignment caused by dental cement shrinkage during baseplating.
- To improve the reliability and quality of miniscope-based fluorescence recordings in mice.
- To enable stable, longitudinal imaging of neuronal activity in deep brain regions.
Methods Used
- Viral vector injection for expression of calcium indicators in target brain regions.
- Implantation of a relay lens into the ventral cornu ammonis 1 (CA1) region.
- Stabilization of the relay lens with dental cement and molding silicone rubber.
- Preparation of a hollow dental cement base using paraffin film to minimize new cement volume during final baseplating.
- Alignment and fixation of the miniscope baseplate with minimal dental cement while monitoring fluorescence signals.
- Use of data acquisition software for real-time imaging and alignment verification.
Main Results
- The dummy baseplating procedure resulted in smaller shifts in baseplate location compared to the original method.
- Baseplates anchored closer to the skull (0.5 cm) exhibited less positional shift than those fixed further away (1 cm).
- Successful imaging showed clear fluorescence transients and blood vessels in anesthetized and freely behaving mice.
- Stable fluorescence signals were observed up to five days post-baseplating with minimal positional drift.
- Occasional issues included absence of fluorescence transients or only background signals, highlighting the importance of precise alignment and viral expression.
Conclusions
- The described protocol effectively minimizes misalignment due to dental cement shrinkage during miniscope baseplating.
- Using a pre-cured dental cement foundation allows for stable, high-quality imaging in deep brain regions.
- This approach supports longitudinal studies of neural activity in freely behaving mice.
