Membrane Chamber for submerged, enhanced and long duration dual surface perfusion of slices
The MC is a new design of in vitro slice recording chamber (Hill & Greenfield 2010). Brain slices are submerged on a completely flat, transparent semi-permeable membrane. This offers for the first time ideal conditions for inverted microscopy of in vitro preparations. The utilisation of a membrane with a high molecular weight cut-off ensures all nutrients and oxygen can flow freely for optimum perfusion. The design of the flow path is such that rates up to 20ml/min can be used without inducing mechanical noise. A further advantage is that the high speed flow of oxygenated aCSF directly underneath and across the semi-permeable membrane exerts a Bernoulli effect, resulting in a pressure difference between the upper and lower surfaces of the membrane. This in turn causes a significant movement of nutrients and oxygen downwards through the semipermeable membrane, increasing the availability to the slice especially on the underside and thereby surpassing passive slice perfusion techniques. The active flow downward through the membrane keeps the slice from floating and makes it mechanically stable. The transparent membrane offers ideal conditions for imaging through a glass coverslip window on the underside of the chamber with an inverted microscope whilst the upper chamber is exposed for microelectrode access. Alternatively there is sufficient access from above for upright microscopes to allow immersion objectives to be used and allow access with microelectrodes from the front and sides. This highly stable preparation with enhanced oxygenation has allowed long duration patch clamp recordings to be made (Morris, Jiruska, Jefferys & Powell, 2016).
- Upright or inverted microscope access to a well perfused, perfectly flat and transparent in vitro slice preparation
- Slice viability increased beyond 16 hours as demonstrated with LFP recordings
- High mechanical stability, high flow rate and Bernoulli effect to optimize nutrient access for slices
- Quick replacement of membrane discs and chamber designed for easy cleaning
A peristaltic pump delivers pulsatile flow at high speed in the range 15ml / min (A) into a reservoir containing a buffer (B) which induces turbulence and reduces pulsation. Smoothed laminar flow passes through the infra chamber (C) directly below the surface of the semipermeable membrane (E)carrying the slice preparation. A Bernoulli effect produces a pressure difference between the upper and lower surfaces resulting in a net movement downwards (arrows) through the membrane and also increases access to slice preparation. A glass coverslip window allows for optical access (D) from below. Fluid exits into the supra chamber (F) and leaves from the chamber through a buffered exit for smooth outflow with a pump or vacuum line.
Use Instruction Video
Below is a short video on the preparation and set up of MC chamber