FIGURE 10.6. Recording setup for diffusion measurements using real-time iontophoretic (RTI) and integrative optical imaging (IOI) methods.

FIGURE 10.6

Recording setup for diffusion measurements using real-time iontophoretic (RTI) and integrative optical imaging (IOI) methods. The specimen (dilute agarose gel, brain slice, or living animal) is placed on the stage of a compound microscope. For RTI, an extracellular probe, most commonly TMA+, is released from a glass micropipette (Injection pipette) by iontophoresis and detected with an ion-selective microelectrode (TMA+-ISM) positioned about 100 μm away. The resulting voltage versus time curve is amplified, digitized and converted to a diffusion curve (concentration versus time) on a computer enabling an appropriate diffusion equation to be fitted to the data (Nicholson, 2001). In diluted agarose gel, the free diffusion coefficient, D (cm2 s−1), and the transport number, nt, are measured. In brain, effective diffusion coefficient, D* (cm2 s−1), volume fraction, α, and non-specific clearance (uptake), k′(s−1), are measured. The tortuosity, λ, is calculated as (D/D*)1/2. For IOI, a fluorescent molecule, here 3000 MW dextran labeled with Texas Red fluorophore, is pressure injected and a time series of images captured using a charge-coupled device (CCD) camera. The appropriate solution of the diffusion equation is fitted to the intensity profiles extracted along selected image axes (Nicholson, 2001). The diffusion coefficients, D and D*, are extracted from measurements in dilute agarose gel and brain, respectively. The tortuosity, λ, is calculated as (D/D*)1/2. As shown in this figure, RTI and IOI measurements can be performed simultaneously.

From: Chapter 10, Biophysical Properties of Brain Extracellular Space Explored with Ion-Selective Microelectrodes, Integrative Optical Imaging and Related Techniques

Cover of Electrochemical Methods for Neuroscience
Electrochemical Methods for Neuroscience.
Michael AC, Borland LM, editors.
Boca Raton (FL): CRC Press/Taylor & Francis; 2007.
Copyright © 2007, Taylor & Francis Group, LLC.

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