Microspectroscopy of red blood cells

Spectroscopic techniques have been widely employed to analyze properties of macromolecules and dynamics of intracellular events on bulk preparations of cells. The development of computer controlled microspectrophotometers has made possible the study of the same events in single cells, often providing significant and unexpected results. This paper briefly reviews experimental works carried out in our laboratories on single red blood cells. Microspectrophotometric techniques were applied which make use of the fact that ligand binding to intracellular haemoglobin is associated with optical changes. Information on the relative abundance of different haemoglobin components inside single erythrocytes of trout blood was obtained from spectra of air equilibrated samples, taking advantage of the extreme pH sensitivity of one of the four haemoglobin components. The kinetics of oxygen and carbon monoxide binding to haemoglobin has been followed and demonstrated to correspond to a zero order process, with a rate much slower than that characteristic for haemoglobin in solution. These results demonstrate that the process is diffusion limited; computer simulations suggest that ligand uptake is limited by the time required for the diffusion from the extracellular space of enough ligand molecules for total saturation of intraerythrocytic haemoglobin. Finally, oxygen dissociation curves in single red blood cells can be obtained by means of particular flow cell, with promising results for the study of physiological and pathological processes (namely red cell sickling in drepanocytosis).