Label-free Spectroscopic Analysis of Optically Trapped Red Blood Cells

Sickle cell disease (SCD) is one of the most common severe monogenic diseases worldwide. Globally there are around 275,000 babies born with SCD each year. In the United States, SCD affects approximately 90,000 to 100,000 people. However, there is no widely available cure for this disease. Recently, several new therapeutic approaches are conceived to be potential universal cures for SCD by mechanism. First, the use of induced pluripotent stem cells as a source of hematopoietic progenitors for gene therapy has successfully corrected sickle-cell disease in mice, which provides rationales for ongoing research into alternative curative strategies. The second approach under development is the targeted therapy to reverse the fetal-to-adult switch which happens immediately after birth through molecular mechanisms. Nevertheless, the efficacy of those therapeutic approaches needs to be proven by an assay which can perform functional analysis of the living RBCs at the single cell level. Such an assay doesn't exist. To address the unmet biomedical need of a suitable single cell tool for functional RBC analysis, the major goal of this dissertation is to investigate the mechanical-chemical relationships within single RBCs using laser tweezers Raman spectroscopy (LTRS) and develop inexpensive assays for high-throughput functional analysis of RBCs at the single cell level based on the insight revealed by such a study.;Micro-Raman spectroscopy has been proven to be a powerful analytical technique for single cell analysis. LTRS, which couples the micro-Raman spectroscopy with optical tweezers, provides a flexible way of immobilizing or perturbing the biological cells in the laser focus during the slow Raman spectral acquisition. First, using LTRS we discovered the mechanically induced oxy-deoxy transition of single RBCs trapped in a single beam optical tweezers. Second, we discovered that the oxygenation states of normal adult, sickle and fetal red blood cells evolve differently under the perturbation of increased imposed optical forces by adjusting the laser powers in the LTRS setup. This discovery provides the rationale to apply LTRS for functional analysis of the oxygen carrying capacity of the RBCs corrected by various therapeutic approaches at the single cell level.;To understand the heterogeneous nature of cell populations and cellular response to external stimuli, it is vital to perform measurements on a large number of single cells to obtain a robust statistical insight. However, large-scale single cell analysis using LTRS is impeded by the low analytical throughput of current LTRS technology. To address these limitations, several technology development efforts were made towards this direction. First, 1D multi-focal laser tweezers Raman spectroscopy (M-LTRS) technique for parallel Raman spectral acquisition of multiple individual biological cells has been demonstrated to improve the analytical throughput by ∼ 10 times. Second, the concept of 2D multifocal laser tweezers Raman spectroscopy (M-LTRS) technique has been demonstrated using polystyrene beads. Third, to specifically boost the analytical throughput for the functional analysis of RBC disorders at the single cell level, laser tweezers absorption spectroscopy (LTAS) is proposed and demonstrated as an alternative technique for characterizing the oxygenation state of RBCs that is inexpensive and has an analytical throughput potentially ∼ 1000 times greater than that of LTRS. Lastly, a novel automation scheme for LTRS/LTAS using scattered light to trigger the spectral acquisition has been proposed and implemented to enable the continuous sampling of a large number of cells.