Importance of substrate and photo-induced effects in Raman spectroscopy of single functional erythrocytes

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Abstract
Hemoglobin (Hb) in single erythrocytes (red blood cells), adsorbed on polylysine-coated glass surfaces, was studied using resonance Raman spectroscopy and global Raman imaging. The erythrocytes were found to be sensitive to both surface adsorption and laser illumination. Substrate-dependent changes of the cell membrane shape were observed immediately after cell adsorption, while a photo-induced increase of fluorescence was observed for visible excitation (λ=514.5 nm). Concurrent changes in Raman spectra revealed a conversion of oxy-Hb (2+) to the inactive met-Hb state (3+). These effects severely complicate the interpretation of Raman images. However, at a low accumulated photon dose, the preparation method enabled the recording of Raman spectra during the oxygenation cycle of a single erythrocyte in buffer, which illustrates the feasibility of Raman investigations of functional cells in in-vivo environments. © 2003 Society of Photo-Optical Instrumentation Engineers.