RAMAN MICROSPECTROSCOPY OF HUMAN BLOOD CELLS

Abstract

In this thesis, we introduce the study on different types of human blood cells using laser Raman microspectroscopy. Human neutrophilic granulocytes, eosinophilic granulocytes, helper T lymphocytes, cytotoxic T lymphocytes and monocytes donated by healthy people were studied by Raman microspectroscopy. Raman spectra were recorded from different parts of these cells, including the cytoplasm and nucleus region. Differences were found between the Raman spectra recorded from the cytoplasm and nucleus region. In the spectra recorded from the nucleus region, many peaks can be assigned to the molecular vibration or DNA. And in the spectra recorded from the cytoplasm region, almost all the peaks can be assigned to the molecular vibration of proteins. For laser excitation at 488nm, we observed resonance enhancement in the Raman spectra recorded from the cytoplasm region of neutrophilic and eosinophilic granulocytes. The resonance enhancement is due to myeloperoxidase (MPO) in neutrophilic granulocytes and eosinophil peroxidase (EPO) in eosinophilic granulocytes. For laser excitation at 782nm, we also recorded good Raman spectra from neutrophilic and eosinophilic granulocytes, which represent the first non-resonant Raman spectra recorded from neutrophilic and eosinophilic granulocytes. We also studied human neutrophilic and eosinophilic granulocytes donated by patients with infection, using Raman microspectroscopy. In the Raman spectra recorded from the cytoplasm region of neutrophilic granulocytes, the peaks due to MPO are much stronger than that in the spectra recorded from healthy cells, which means that the MPO content increased in the neutrophilic granulocytes of patients with infection. In the Raman spectra recorded from the cytoplasm region of eosinophilic granulocytes, the peaks due to EPO decreased greatly in relative intensity compared to that in the spectra recorded from healthy cells, which means that the EPO content decreased in the eosinophilic granulocytes donated by patients with infection. These results imply that it is possible to monitor the in vivo micro-biological process in living human cells using laser Raman microspectroscopy. We have tried four types of substrates, including Ti coated silicon wafer, aluminum foil wrapped on glass slide, aluminum thin film coated on glass slide and silver thin film coated on glass slide. Our experiment indicate that only cells prepared on Ti coated wafer and aluminum foil wrapped on glass slide can give good Raman spectra.