Obtaining high signal to noise ratio is challenging in wide-field two photon microscopy and one must ensure the mouse brain can be imaged safely under high laser power. Here, we demonstrated a simultaneous thermal imaging and two photon imaging technique. The maximum temperature of the cortex was below 39°C using 400mW laser power with a 5 x 5mm field of view. Together with the brain activities under hind paw stimulation and at rest, we argued that high laser power for wide-field two-photon imaging can potentially be used while keeping the temperature under safety limit.
Scattering in the skull limits spatial resolution in optical neuroimaging experiments in mice, so it is necessary to excise a portion of the skull. Such craniotomy procedures have traditionally been done manually, but automating the process provides greater control of the cutting path and depth. A computer numerical controlled (CNC) milling machine can perform craniotomies of arbitrary shape and position with high precision. Automating the procedure improves repeatability and consistency, poses fewer risks for damaging the brain, and makes the procedure easier to learn. We have developed an automated craniotomy procedure which utilizes a CNC machine to obtain a curvature profile of the skull with an impedance-sensitive probing circuit and to interpolate a cut path along this profile to remove a portion of a mouse’s skull. This procedure does not damage the underlying brain tissue and can be performed in under two hours.
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