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A unique optical instrument is described which uses the firefly bioluminescent reaction to measure adenosine triphosphate (ATP) levels in single red blood cells. The method allows chemical content level to be associated with individual cell features. The optical instrument consists of a phase contrast microscope to view cells, a pulsed argon-ion laser to rupture the cell membrane, and a photon counting system to measure the bioluminescent yield. The technique has been calibrated against a standard ATP measurement using bulk analysis methods. The ATP loss mechanism for blood cells in a controlled depletion experiment was also investigated.

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The densest 0.1%--1% of circulating red cells were separated from fresh blood, and the ATP content of a representative sample of such cells was determined. The dense (“old”) cells had decreased amounts of ATP relative to unfractionated cells. However, the dense cells were also smaller, and the concentration of ATP in these cells was the same as in controls. Therefore, it seems unlikely that loss of cellular ATP is a causative factor in removal of senescent red cells from the circulation.