Objective · To evaluate the effect of two-photon in vivo imaging on detecting the blood brain barrier (BBB) injury in the ultra-early stage of cerebral ischemic stroke. Methods · Twelve clean grade C57BL/6 healthy male mice aged 8-12 weeks were randomly divided into Sham group and middle cerebral artery occlusion (MCAO) group, which were sham operated or middle cerebral artery occluded, respectively. After 60 min of ischemia, MCAO mice were treated with reperfusion for 30-60 min after the suture being removed. The vessels and the neutrophils of mice in the two groups were labeled intravenously with Alexa-Fluora-488 conjugated dextran and rhodamine 6G, respectively. The integrity of BBB was detectedintravenous injection of tetramethylrhodamine-5-maleimide (TMR). Before and after the stroke, the real-time changes of the fluorescence intensity of the inside and outside cerebral vessels of mice in the MCAO group were observedtwo-photon fluorescence microscopy. Results · The fluorescence intensity of TMR in the external cerebrovascular of mice in the MACO group was significantly increased within 30-60 min after stroke (P0.000), suggesting there existed tracer leakage. Compared with the Sham group, the movement of neutrophils in the blood vessels of mice in the MACO group was significantly slowed down (P0.000). Conclusion · Two-photon in vivo imaging can be used to detect the BBB injury in the ultra-early stage of cerebral ischemic stroke, which provides a certain reference value for clinical application.