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In the mammalian brain, blood flow is controlled by a specialized, highly evolved system. We studied the regulation and failure of the cortical vascular system on the size scale of individual microvessels. We developed optical methods for lesioning single microvessels in the cortex of anesthetized rat. Photochemically-triggered thrombosis was used to occlude surface and penetrating arterioles. Femtosecond laser ablation, using nonlinear optical absorption, was used to produce occlusions, and also leakages and hemorrhages, in the microvessels up to ̃500[mu]m below the cortical surface. Blood flow changes following these lesions were measured in individual microvessels using two-photon laser scanning microscopy of fluorescently-labeled blood. We compared the degree of ischemia that resulted from blockages in the different categories of microvasculature. We found that surface arterioles are highly resistant to ischemia, while occlusions in deep microvessels resulted in more dramatic drops in red blood cell speed in the immediately downstream vessels (10% of the initial value). Ischemia was most severe after occlusions in the penetrating arterioles. Immediately downstream vessels dropped to 3% of initial speed and the blood flow was significantly reduced in a 350-[mu]m radius area. The decrements in flow are consistent with the vascular topology of the occluded vessels. The surface arterioles form a network with many obvious loops, but the deep vessels are less redundant, forming fewer, longer loops within the capillary bed. The penetrating arterioles have no anastomoses and are only connected to other arterioles through the capillaries. Ischemia resulting from occlusions of deep microvessels or penetrating arterioles may be a cause of small lesions that are found in the brains of elderly humans. In addition, leakages and hemorrhages, similar to those we produced with femtosecond laser ablation, can also cause or exacerbate neurodegeneration. We also studied the regulatory function of vasculature in the healthy brain. We developed analysis tools to improve optical intrinsic imaging, and also used two-photon laser scanning microscopy in a preliminary study of dilation of and blood flow through individual arterioles in response to neural stimulation. In conclusion, both healthy and lesioned microvasculature were studied in vivo using optical tools