2.1. Mouse Holder Design

The newly designed mouse holder was inspired by the holder developed by Stenroos and colleagues [17]. Additions to this original version of the mouse holder were added because the Stenroos version was compatible for rats, rather than for mice. The final designed holder, (Supplemental Figures S1–S6), is interfaceable with, and detachable from, the ¹H and heteronuclear surface coils and as well as with volume resonators. The newly designed mouse holder was designed with several key elements to enable awake imaging. This includes a reduced inner volume of the nose cone that includes a trimmed top to allow the surface coil to fit (Figure S2), and a mouse bed that has an indented inner surface for a mouse body to firmly fit, a longer bed for a mouse to lay on, and the incorporation of an oxygen hose through which a tooth bar was placed to reduce head motion during imaging (Figure S3). Additionally, an altered neck bridge, with a larger inner cutout (Figure S4), and the addition of a cheek supports, in order to further reduce head motion (Figure S6), are included. STL files are included in the Supplemental Materials in order to provide easy access to this design.

To properly secure the mice in the holder, mice are placed in the dorsal, anatomical position with their heads fitted into the built-in head brace. They are placed on a tooth bar and their nose fitted snuggly into the nose cone, with pieces of gauze placed on each side of the head for increased head stability. Artificial tears are administered, a temperature probe is inserted, and a respiration monitor is placed under the mice. To ensure the mice remain properly restrained in the holder, all four feet and the tail are taped down. Additionally, a piece of gauze is placed over the animal’s body and head and tape is firmly secured around the animal to prevent any movement (Figure 1). This mouse holder was tested with mice ranging in weight from 20 g to 50 g. The STL files have been provided in the Supplementary Material. The size of the nose cone can be adjusted as needed to fit a variety of mouse sizes. The body of the holder and restraint methods allow for the use of the holder for any size mouse.

Awake Animal Imaging Holder. A custom mouse holder with nose cone and head brace (A), and mouse holder with tooth bar, respiration monitor, and heat source (B). Image of mouse placed in holder, with nose in nose cone and on tooth bar (C). Image of mouse with limbs taped down and temperature probe inserted (D). Image of mouse fully secured with gauze and tape to reduce all movement during imaging (E). Oxygen was supplied to the subjects through the oxygen hose during imaging and acclimation.