4D functional imaging in the freely moving rat.

We describe a two-frequency diffuse optical tomographic (DOT) imaging and EEG recording system suitable for the study of real-time hemodynamic and neural activities in freely moving rats. The system uses a bundle of 16 optical fibers that both deliver light and capture its reemission. This bundle runs in parallel with a cable carrying EEG signals from 16 microelectrodes. Both data collection arrays terminate in a precision-machined cap that is surgically attached to the skull. Free movement is enabled by suspending the cables with an elastic cord. Rats are also tracked with a video system so their behavior can be compared to hemodynamic and neural activity. Optical measurements are done with 760 and 830 nm laser diodes using a time-multiplexed, frequency-encoded illumination scheme at a source-switching speed of 68 Hz. EEG, optical and video data are all timestamped with the same clock, ensuring information synchrony. Automated optical system set-up and control is done with a LabVIEW interface that allows on-the-fly adjustment of gain, data integrity checks and system calibration, among other functionalities. EEG recording is done with a Neuralynx (Tucson, AZ) recording system. Collected optical data are converted to volumetric images either in real-time or offline. The integrated system includes comprehensive image formation, display and time-series analysis software suitable for processing data independently or in combination.