Sequences of images of the cortical surface can be processed to reveal information about the cortical microcirculation, regional cerebral blood flow (rCBF), and changes induced by neuronal activity. This study examined the use of different analysis methodologies on intrinsic optical images taken from rat sensory motor cortex and testes. Generalized linear model (GLM) analysis was used and compared with standard signal processing methods including principal component analysis. The GLM method has been used by Friston et al. (1994, Hum. Brain Map., 1: 214-220) in the analysis of functional magnetic resonance imagery to identify regions of focal activity. We investigated the use of this method to analyze video image data of the modulation of rCBF from rat cortex. The results revealed spatiotemporal variations in rCBF in response to stimulation within local regions of cortex. The advantage of the GLM method is that it augments ordinary signal processing methods with an estimate of statistical reliability. The use of different wavelengths of illumination reveals spatial structures with different temporal relationships. In image time series data collected under green and red illumination a phase difference was found in the low frequency approximately 0.1 Hz vasomotion oscillation. This phase difference occurred in data from both cortex and testes. A possible explanation of these differences is that the spectral absorption characteristics of the tissue reflect changes in the volume proportions of the different hemoglobin derivatives in interacting with the modulation of the volume of blood. It is suggested that the combination of these effects produces the phase differences we detect.