Multi-Layer In Vivo Imaging of Neocortex Using a Microprism

INTRODUCTION

Two-photon fluorescence microscopy is an integral tool in the field of neuroscience research. Applications of two-photon microscopy to in vivo deep-brain imaging are limited due to substantial light scattering by dense neural tissue. Conventional in vivo imaging can only access the superficial 300-400 μm of neocortex and suffers from nonuniform intensity and poor signal-to-noise as the depth increases. This protocol presents a technique to produce high-quality images deep into the mouse neocortex by inserting a 1-mm right-angle glass microprism into the neocortex. The microprism serves to reflect the laser excitation light and the fluorescence emission off a high-reflective coating on the prism’s hypotenuse to provide a side-on imaging perspective of multiple neocortical layers simultaneously. This “inverted periscope” technique allows for wide field-of-view imaging of layer V green fluorescent protein (GFP) cell bodies with their apical dendrites extending through the superficial layers. Microprisms maintain spatial resolutions capable of resolving dendritic spines. Furthermore, microprisms can image the network of blood vessels in the neocortex and blood flow through microcapillaries to obtain information such as red blood cell (RBC) flux and velocity.