(a) In awake mice, imaging with a cool water immersion objective decreases capillary RBC velocity (left, n = 6 capillaries, three mice), flow (middle, n = 8 capillaries, four mice) and Po2 Mean (right, n = 8 capillaries, four mice). The effect is reversible upon heating the objective. Upper insets illustrate typical velocity, flow and Po2 measurements. (b) Inset, Schematic of EATs: Po2 at the RBC border (Po2 RBC), Po2 at distance from a RBC (Po2 InterRBC) which gives an estimate of pericapillary Po2, average Po2 in the capillary (Po2 Mean). 1-D scatter plots show that lowering temperature reversibly decreases Po2 RBC, ‘tissue’ Po2 and RBC saturation. Note that all Po2 values are calculated using calibrations curves acquired at the corresponding temperatures. (c) In mice anesthetized with ketamine/medetomidine and breathing air supplemented with oxygen (30%), resting Po2 is high but still increases upon heating the objective, brain temperature reaching ~35.7 C° and Po2 ~70 mmHg (velocity, n = 18 capillaries, eight mice; blood flow and Po2, n = 9 capillaries, three mice). See Materials and methods for the statistical tests. *, **, ***, **** for p<0.05, 0.01, 0.001 and 0.0001 respectively, NS: non significant.