Microphotometric method for measuring the oxygenation and deoxygenation rate in a single red blood cell

A new reaction apparatus combining a microscope and a photometric device was developed for kinetic studies of a single red cell. A monolayer of red cells was placed in a closed reaction cuvette set on a microscope stage, a light beam of 5 to 10 mum in diameter was directed into one of the red cells, and the light transmission change in the cell was analyzed. The light beam with a wavelength range shorter than 460 nm was made by placing a narrow iris diaphragm in the light path. The space in the cuvette prevented the red cells from drying thereby providing favorable physiological conditions during measurements. The cuvette was filled with reagent gas mixtures of O2, CO2, and N2 which came in contact with the red cells. Transmission change due to the reaction was detected separately at two wavelengths of 418 and 402 nm by means of two photomultipliers mounted on the microscope. The linearity was tested by comparison between SO2 measured with a Van-Slyke apparatus and the microphotometer. Both SO2 measurements agreed well with each other, but the latter was about 3% greater than the former at around 50% SO2. Using this apparatus the oxygenation and deoxygenation velocities were measured over an entire O2-saturation range. The velocity factors showed good agreement with those obtained by using conventional flow methods.