Fig. 4. Example of a calibration experiment of Oxyphor R2 in the haemolymph of a Hb-poor Daphnia magna. A circular drop of the dye-loaded haemolymph sample was equilibrated at seven different ambient oxygen tensions (P_O2amb=0, 1.02, 2.05, 3.07, 4.09, 5.12, 6.14 kPa; 0–30% air saturation). The phosphorescence lifetime () of the sample was imaged and is depicted in pseudo-colour presentation (top row). Pixel positions outside the sample image were masked out by setting pixel intensity to black colour. The relationship between the reciprocal of the mean of each image and P_O2amb (open circles) was then analyzed by linear regression analysis using the Stern–Volmer equation (^–1=₀^–1 + k_qP_O2amb), which yielded the estimates of the lifetime at zero-oxygen concentration (₀) and of the quenching constant (k_q). ₀ and k_q were used to transform phosphorescence lifetime images (top row) into oxygen partial pressure (P_O2) images (bottom row). The numbers below the gallery indicate the mean value of each P_O2 image.