Resolving fish haemoglobin: Simultaneous pH and spectrophotometric recordings in blood microvolumes advance oxygen binding measurements

Oxygen transport measurements have been widely used to explain evolutionary adaptation and performance in fishes. Simultaneous measurements of blood pH and oxygen saturation are essential to understand oxygen transport as it occurs in vivo, but has been challenging to monitor with existing methods, particularly in minute blood volumes. We equipped a gas diffusion chamber with a broad-range fibre-optic spectrophotometer and a micro-pH optode and successfully recorded changes of pigment oxygenation and pH in only 15 µl of whole fish blood at 0°C. Highly resolved spectra ranging from 280 – 900 nm captured the complex absorbance features of Antarctic fish haemoglobin in response to changing oxygen and carbon dioxide partial pressures. After consideration of photobleaching and intrinsic fluorescence, pH optodes recorded pH shifts of 0.03 pH units. With this modified diffusion chamber experimental biologists are able to accurately characterize oxygen binding with minimal sample consumption and manipulation under diverse experimental settings.