Evaluation of cranial window types for in vivo two-photon imaging of brain microstructures

In vivo two-photon laser scanning microscopy (2PLSM) of neurons and glia in the mouse neocortex can be achieved using two distinct surgical methods: the thin-skull and open-skull window techniques. The open-skull window technique is more invasive, but provides more flexibility in imaging periods and frequencies. Critical evaluations of the two surgical methods are required, particularly their ability to image submicron structures such as dendritic filopodia and spines. Here, we used multiple approaches to evaluate the optical resolution of 2PLSM through two types of cranial windows. Two-photon point spread function (PSF) on the surface of the neocortex in vivo was studied by imaging the fluorescent microspheres attached to the pial surface. Comparable PSF widths indicated that the optical resolution of 2PLSM on the brain surface was similar between the two types of cranial window techniques. Next, we evaluated the effects of laser beam propagation through the thinned bone by imaging the underlying gel samples containing fluorescent microspheres as phantoms mimicking the brain tissue. We observed deterioration of PSF with increase in the imaging depths through the thinned bone. Reduction in image resolution through the thin-skull window in the deep cortical layer was confirmed by repetitive 2PLSM of the same GFP-positive dendritic spines with two techniques. In summary, the resolution of 2PLSM is comparable with those of two cranial window preparations in the superficial layer of the neocortex, but the quality of the image through the thin-skull windows degrades at points deeper than 50 µm.