Label-free functional imaging of single red blood cells (RBCs) in vivo holds the key to uncovering the fundamental mechanism of oxygen metabolism in cells. To this end, we developed single-RBC photoacoustic flowoxigraphy (FOG), which can image oxygen delivery from single flowing RBCs in vivo with millisecond-scale temporal resolution and micrometer-scale spatial resolution. Using intrinsic optical absorption contrast from oxyhemoglobin (HbO 2 ) and deoxyhemoglobin (HbR), FOG allows label-free imaging. Multiple single-RBC functional parameters, including total hemoglobin concentration (C Hb ), oxygen saturation (sO 2 ), sO 2 gradient (∇sO 2 ), flow speed (v f ), and oxygen release rate (rO 2 ), have been quantified simultaneously in real time. Working in reflection instead of transmission mode, the system allows minimally invasive imaging at more anatomical sites. We showed the capability to measure relationships among sO 2 , ∇sO 2 , v f , and rO 2 in a living mouse brain. We also demonstrated that single-RBC oxygen delivery was modulated by changing either the inhalation gas or blood glucose. Furthermore, we showed that the coupling between neural activity and oxygen delivery could be imaged at the single-RBC level in the brain. The single-RBC functional imaging capability of FOG enables numerous biomedical studies and clinical applications. Although individual parameters such as oxygen saturation (sO 2 ) (1, 2), partial oxygen pressure (pO 2 ) (3, 4), or blood flow speed (v f ) (4-7) may partially indicate tissue oxygenation, none of them can provide a complete description of oxygen transport. To quantify tissue oxygenation in vivo, three primary imaging modalities have been applied: positron emission tomography (PET) (8), functional magnetic resonance imaging (fMRI) (9, 10), and diffuse optical tomography (DOT) (11). Although these methods may provide deep functional imaging, they all suffer from millimeter-scale spatial resolutions. Recently, photoacoustic (PA) microscopy was proposed to measure the oxygen metabolic rate at feeding and draining blood vessels (12). However, the assessment is limited to a relatively large region; moreover, the feeding and draining blood vesselsespecially those surrounding a tumor-may be numerous and difficult to identify. Because micrometer-sized RBCs are the basic elements for delivering most of the oxygen to cells and tissues, the need for direct imaging of oxygen release from flowing single RBCs in vivo is imperative. A molecular imaging method based on the measurement of ground-state recovery time has been investigated for hemoglobin sensing (13), but in vivo measurement of oxygen saturation has not been implemented. Dual-wavelength spectrophotometry has been applied to measure oxygen release for decades (14), but transmission-mode imaging limits its application to very thin tissue. Many biomedical problems-for example, tumor or neuroscience studies-require minimally invasive imaging at various anatomical sites. A new method for in vivo imaging of single-RBC oxygen release...