The functional spatial resolution in most of hemodynamics-based functional neuroimaging techniques is limited by the fineness of hemodynamic control with the active vascular beds likely at submillimeter resolution. This study was designed to visualize changes of cerebral blood flow (CBF) at submillimeter spatial scale on the prolonged isoflurane-anesthetized rats model by using laser speckle contrast imaging (LSCI) technique. Recently, this old method has attracted an increasing interest in studies of brain activities under normal and pathophysiologic conditions. However, some paramount assumptions behind this imaging technique have been kept ignored in this field since 1981 firstly proposed by Fercher and Briers. Most recently, these assumptions are claimed as serious mistakes that made LSCI fail to reproducibly and correctly measure blood flow speed. In our study, these issues are also re-examined theoretically and re-evaluated experimentally based on the results from the classical carbon dioxide challenge model. The detailed distribution of CBF responses to the stimulation induced by different levels of carbon dioxide pressure was obtained with tens of micron spatial resolution. The relative CBF images over the exposed cortical area acquired by LSCI were also compared with laser-Doppler measurements. Our results show that these assumptions would not produce any significant errors on investigating changes of blood flow and also achieve high specificity to assess cerebral microcirculation, as would facilitate its broad application in functional imaging field.