Fast volumetric imaging of oxygen delivery in the mouse brain at single red blood cell resolution

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PROJECT SUMMARY/ABSTRACT The human brain represents 2% of the weight of the body, but consumes ~20% of the total energy at rest. All that energy – mostly in the form of glucose and oxygen - has to be delivered to the brain cells through an intricate network of microvessels, the majority of which are capillaries. Although it is well-known that increased neuronal activity in a region of the brain is associated with an increase in cerebral blood flow (functional hyperemia), the role of capillaries in blood flow regulation and oxygen delivery remains controversial. Nevertheless, capillary dysfunction has been suggested to play a crucial role in a wide variety of brain diseases and conditions, especially Alzheimer’s disease. To better understand capillary function and how it meets the widely varying energy demand of neurons, it is crucial to be able to measure oxygen delivery at high spatial and temporal resolution. Unfortunately, current technologies for imaging oxygen delivery, including intrinsic optical imaging, optical coherence tomography, and photoacoustic microscopy, have either limited spatial resolution or are incompatible with existing neuronal activity imaging. These limitations prevent a detailed understanding of the interaction between capillaries and neurons and how capillary dysfunction impacts brain function. To address this critical technological gap, we aim to develop a novel transient absorption microscopy technique that is capable of oxygen saturation (sO2) imaging at single red blood cell (RBC) resolution using intrinsic hemoglobin contrasts. This technique exploits a fundamentally different contrast mechanism from other optical approaches. It uses the transient absorption of endogenous hemoglobin molecules to image RBCs, and replies on excited- state dynamics difference between oxyhemoglobin and deoxyhemoglobin to determine sO2. Compared to the existing two-photon oxygen probe, our new technique potentially offers 3-4 orders of magnitude improvement in sO2 imaging speed. More importantly, it can be readily integrated with other high-throughput microvessel and neuron measurements. In Aim 1, we will develop and validate a high sensitivity transient absorption microscope for in vivo sO2 imaging of the brain cortex. A new dual-wavelength laser will be used to maximize sO2 sensitivity and imaging depth. Accuracy of sO2 imaging will be determined by comparison with an improved phosphorescent oxygen probe Oxyphor2. In Aim 2, we will create a high-throughput volumetric imaging microscope by combining TAM imaging with Bessel beam excitation and a novel interlaced scanning method. The new microscope will be optimized for simultaneous volumetric imaging of capillary sO2, vessel diameter, blood flow, flux, and neuronal activity. This breakthrough capability will enable real-time assessment of oxygen delivery through each capillary in the microvessel network at an unprecedented spatial and temporal resolution. We anticipate broad applications of this technology in studying neurovascular coupling, microvascular diseases, aging, and neurodegeneration.