Fluorescence and DIC microscopy for patch-clamping
Sandrine Humez

The goal is to visualise individual neurones with a upright microscope from a life brain slice maintained in vitro, in order to realize patch-clamp recordings with direct observation of the target neurone, or to perform neuroanatomical investigations within the slice.

This is achieved by using a Leica DM LFSA microscope optimised for fluorescence and for Difference Interference Contrast (DIC). Fluorescent microscopy mainly allows us to visualise Green-Fluorescent-Protein- labelled neurones from transgenic animals. Observation in DIC requires infrared illumination, due to the thickness of the slice (120 to 400 µm). Neurone relief is optimally imaged and patch-clamp micropipettes or other kinds of micropipettes can be accurately positioned. Fluorescence and DIC are easily switched from one to the other.

Pictures are transmitted through a Cohu video camera and observed on a computer equipped with a Leica FW4000 software for imaging.

Slices are perfused in a chamber (Warner) under the water immersion objectives. The perfusion chamber is anchored on a fixed stage (Gibraltar) that supports micromanipulators to guide pipettes within the slice.

A complete electrophysiological platform is fitted tothe microscope equipment.

micro electrophysio

The equipment components are a microscope, a fixed stage that supports micromanipulator(s), a video camera linked with a image capture system. Brain slices are in a bath chamber located under the objective.