Raman microspectroscopy and imaging provides insights into heme aggregation and denaturation within human erythrocytes

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Abstract
The oxygenation process of a human erythrocyte is monitored using a Raman microimaging technique. Raman images of the 1638 cm−1 band are recorded in the oxygenated and deoxygenated state using only 120 s of laser exposure and ∼1 mW of defocused laser power. The images show hemoglobin oxygenating and deoxygenating within the cell. Prolonged laser imaging exposure (−1 are identified as markers for heme aggregation. Raman images recorded of cells after prolonged laser exposure appear to show heme aggregation commencing in the middle and moving toward the periphery of the cell. UV-visible spectra of erythrocytes show the Soret band to be broader and red shifted (∼3 nm) at temperatures between 45 and 55° indicative of excitonic interactions. It is postulated that the enhancement of the aggregation marker bands observed at 632.8-nm excitation results primarily from excitonic interactions between the aggregated hemes in response to protein denaturation. The results have important medical implications in detecting and monitoring heme aggregation associated with hemopathies such as sickle cell disease. © 2005 Society of Photo-Optical Instrumentation Engineers.