Figure 2.

Setup for diffusion measurements using the real-time iontophoretic or pressure (RTI/RTP) methods and integrative optical imaging (IOI) method. A brain slice or dilute agarose gel is placed in a chamber that is perfused with oxygenated physiological saline solution on the stage of a compound microscope. For RTI/RTP, an extracellular probe ion, most commonly TMA⁺, is released from a glass micropipette and detected with an ion-selective microelectrode (TMA⁺-ISM), positioned about 100 μm away. The resulting diffusion curves (concentration versus time) are amplified, digitized and stored on a computer enabling an appropriate diffusion equation to be fitted to the data. In dilute agarose gel, the free diffusion coefficient, D, and transport number, n_t, are measured. In the brain slice, the effective diffusion coefficient, D^*, volume fraction, α and loss factor, k′, are measured. For IOI, a fluorescent molecule, here dextran (3000 M_r, i.e. 3 kDa) labeled with a fluorescent dye, is briefly pressure injected and a time-series of images captured using a CCD-equipped camera. An appropriate diffusion equation is fitted to the intensity profiles measured along selected image axes enabling D or D^*, to be extracted in agarose gel or brain slice, respectively. Also note that the methods are not confined to brain slices but also may be used in vivo (modified from ref. 10).