Microprisms for in vivo multilayer cortical imaging.

Cortical slices allow for simultaneous imaging of multiple cortical layers. However, slices lack native physiological inputs and outputs. Although in vivo, two-photon imaging preserves the native context, it is typically limited to a depth of <500 microm. In addition, simultaneous imaging of multiple cortical layers is difficult due to the stratified organization of the cortex. We demonstrate the use of 1-mm microprisms for in vivo, two-photon neocortical imaging. These prisms enable simultaneous imaging of multiple cortical layers, including layer V, at an angle typical of slice preparations. Images were collected from the mouse motor and somatosensory cortex and show a nearly 900-microm-wide field of view. At high-magnification imaging using an objective with 1-mm of coverglass correction, resolution is sufficient to resolve dendritic spines on layer V neurons. Images collected using the microprism are comparable to images collected from a traditional slice preparation. Functional imaging of blood flow at various neocortical depths is also presented, allowing for quantification of red blood cell flux and velocity. H&E staining shows the surrounding tissue remains in its native, stratified organization. Estimation of neuronal damage using propidium iodide and a fluorescent Nissl stain reveals cell damage is limited to <100 microm from the tissue-glass interface. Microprisms are a straightforward tool offering numerous advantages for into neocortical tissue.