Identification of cortical arteries and veins in awake mice using two-photon microscopy.

Distinguishing arteries from veins in the cerebral cortex is critical for studying hemodynamics under pathophysiological conditions, which plays an important role in the diagnosis and treatment of various vessel-related diseases. However, due to the complexity of the cerebral vascular network, it is challenging to identify arteries and veins in vivo. Here, we demonstrate an artery-vein separation method that employs a combination of multiple scanning modes of two-photon microscopy and a custom-designed stereoscopic fixation device for mice. In this process, we propose a novel method for determining the line scanning direction, which allows us to determine the blood flow directions. The vasculature branches have been identified using an optimized z-stack scanning mode, followed by the separation of blood vessel types according to the directions of blood flow and branching patterns. Using this strategy, the penetrating arterioles and penetrating venules in awake mice could be accurately identified and the type of cerebral thrombus has been also successfully isolated without any empirical knowledge or algorithms. Our research presents a new, more accurate, and efficient method for cortical artery-vein separation in awake mice, providing a useful strategy for the application of two-photon microscopy in the study of cerebrovascular pathophysiology.

[In vivo imaging of blood flow using two-photon laser scanning fluorescent microscopy].

OBJECTIVE: To observe the three-dimensional distribution of vessels, and to establish a new method for measurement of blood flow velocity in mice cerebral cortex using two-photon laser scanning microscopy and fluorescence probe labeling technique. METHODS: The mouse was made cranial window surgery and injected Texas-Red through tail vein after anesthetized. The three-dimensional imaging of vessel was obtained through z-stack scanning, and blood flow velocity was quantified through line scanning. RESULTS: We could detect vascular distribution for more than 500 µm depth using two-photon microscopy. The velocity of blood flow was (0.59 ± 0.12) mm/s in capillary. CONCLUSION: The method for observing the brain blood flow by two-photon microscopy was established, which could achieve quantification of single vascular blood flow velocity and provide experimental evidence for basic research and medical applications.