Importance of substrate and photo-induced effects in Raman spectroscopy of single functional erythrocytes.
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 (lambda=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.
Ramser
Kerstin
K
Chalmers University of Technology, Department of Applied Physics, S-412 96 Göteborg, Sweden. k.ramser@fy.chalmers.se
Bjerneld
Erik J
EJ
Fant
Camilla
C
Käll
Mikael
M
D018929:Cell Culture Techniques / Q000379:methods*; D018914:Cells, Immobilized; D004912:Erythrocytes / Q000737:chemistry* / Q000378:metabolism*; D005240:Feasibility Studies; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010101:Oxygen Consumption / Q000502:physiology; D011107:Polylysine / Q000737:chemistry; D013059:Spectrum Analysis, Raman / Q000379:methods*
D006454:Hemoglobins; D011107:Polylysine; D010100:Oxygen