Optical manipulation and spectroscopy of single functional globin-containing cells in microfluidic systems

Doctoral thesis, 2006

Recent advances in microbiology and biomedicine require development of tools to monitor the response of single cells to environmental stimuli under controlled physiological conditions. In this thesis, I describe how this can be achieved by combining various spectroscopic techniques with optical tweezers and microfluidic systems. Apart from absorption spectroscopy the technique of resonance Raman microscopy has been used to examine the oxygenation cycle of optically trapped cells containing heme-proteins, i.e. hemoglobin in red blood cells, neuroglobin overexpressed in Escherichia coli bacteria, and the nerve globin of the polychaete annelid Aphrodite aculeate in the ganglia of the nerve chain. Resonance Raman spectroscopy is an excellent tool to investigate the structure-function relationship of heme proteins. The porphyrin group in the heme absorbs visible light and a resonance effect leads to a selective enhancement of the porphyrin vibrations, hence, the method gives information about the oxygenation as well as the spin state of the heme iron. An important issue in the field of biochemistry, biomedicine and drug discovery is the mimicking of in vivo conditions in an in vitro environment. There is a demand to monitor chemical reactions in real time with good control over the diffusion of substances. Microfluidic technology can fulfil many of these requirements. Within this work, I developed a microfluidic system equipped with a pump that is connected to a switch enabling the flushing of buffers purged with O2 or N2 to create aerobic or anaerobic conditions in a flow cell. By doing so, I was able to monitor the oxygenation cycle of heme proteins reversibly over an extended period of time, confirming that the set-up meets the demand of physiological conditions.