Analysis of Red Blood Cells under Oxygen-deprived Environments

Raman spectroscopy and microscopy have proven to be powerful label-free analytical techniques for determining the oxygenation level of red blood cells (RBCs).  The known conformational differences between normal, sickle and fetal red blood cells (RBCs) indicate that studying them in an initial deoxygenated state using Raman spectroscopy and microscopy at the single cell level may reveal additional insight into the fundamental molecular conformations of different types of hemoglobin. To carry out these experiments in our lab requires additional instrumentation to maintain the cells in an oxygen-deprived environment during Raman analysis.  We resolve this by fabricating a chamber that will isolate the cell samples from the outside atmosphere while continuously purging the inside atmosphere with pure nitrogen gas, which is monitored using a flow meter.  A slight positive pressure inside (<1 psi) prevents oxygen from leaking inside, guaranteeing that the inside atmosphere will maintain an oxygen free environment throughout the duration of the experiment.  We propose to carry out experiments to identify RBC Raman spectra that reflect deoxygenated conditions in a pure nitrogen atmosphere and monitor the deoxygenation process of RBCs in real time when the nitrogen is initially filled in. In addition, we propose to investigate and identify Raman markers that can distinguish between normal, sickle and fetal RBCs under the oxygen-deprived environment.