In vivo Imaging of Deep Cortical Layers using a Microprism

Summary

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 μm) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

Abstract

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism. Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light. The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations. Images are collected with a large field-of-view (~900 μm). In addition, we provide details on the non-survival surgical procedure and microscope setup. Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts. Resolution was sufficient to image dendritic spines near the soma of layer V neurons. A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex. Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

Protocol

Part 1: Microprism Optics and Microscope Setup

1. The microprisms we use are 1-mm, right-angle prisms made from BK7 glass purchased from the Optosigma Corporation (Figure 1). Microprisms are handled using forceps whenever possible.
2. The hypotenuse of the microprism is coated with enhanced silver to provide a reflectivity of greater than 97% from 400 – 2000 nm. This provides internal reflection of both the laser excitation light and the emitted fluorescence.
3. Microprisms are always handled using forceps when possible. Gloves are worn at all times to keep the prism clean.
4. We use a custom-built two-photon microscope capable of small-anim....

Discussion

Using a microprism in an imaging experiment is relatively straightforward and offers many advantages for in vivo studies on the neocortex. Representative examples using this technique include images taken from transgenic mice expressing yellow fluorescent protein in layer V cortical neurons. Using microprisms one can see a collection of large pyramidal cell bodies in layer V, nearly 1 mm below the cortical surface. Also seen are the apical dendrites that extend through all the superficial layers before diverging into .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
With enhanced silver reflective coating-(R77)	OptoSigma Corp. (Santa Ana, CA)	055-0010	With enhanced silver reflective coating-(R77)
Fluorescein-dextran	Sigma-Aldrich	FD70	70 kDa
0.28 NA, 4x air objective	Olympus Corporation	XLFLUOR 4x/340
0.60 NA, 40x air objective	Olympus Corporation	LUCPLFLN	With 0-2 mm coverglass correction