Disorders of oxygen transport are commonly attributed to inadequate carrying capacity (anemia) but may also relate to inefficient gas exchange by red blood cells (RBCs), a process that is poorly characterized yet assumed to be rapid. Without direct measurements of gas exchange at the single-cell level, the barriers to O₂ transport and their relationship with hematological disorders remain ill defined. We developed a method to track the flow of O₂ in individual RBCs by combining ultrarapid solution switching (to manipulate gas tension) with single-cell O₂ saturation fluorescence microscopy. O₂ unloading from RBCs was considerably slower than previously estimated in acellular hemoglobin solutions, indicating the presence of diffusional barriers in intact cells. Rate-limiting diffusion across cytoplasm was demonstrated by osmotically induced changes to hemoglobin concentration (i.e., diffusive tortuosity) and cell size (i.e., diffusion pathlength) and by comparing wild-type cells with hemoglobin H (HbH) thalassemia (shorter pathlength and reduced tortuosity) and hereditary spherocytosis (HS; expanded pathlength). Analysis of the distribution of O₂ unloading rates in HS RBCs identified a subpopulation of spherocytes with greatly impaired gas exchange. Tortuosity imposed by hemoglobin was verified by demonstrating restricted diffusivity of CO₂, an acidic gas, from the dissipative spread of photolytically uncaged H⁺ ions across cytoplasm. Our findings indicate that cytoplasmic diffusion, determined by pathlength and tortuosity, is a major barrier to efficient gas handling by RBCs. Consequently, changes in RBC shape and hemoglobin concentration, which are common manifestations of hematological disorders, can have hitherto unrecognized and clinically significant implications on gas exchange.

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