Ultraquantitative Raman Spectral Cytometry to Measure the Cargo Capacity of Individual Macrophages

Abstract

This dissertation demonstrates the usefulness of Raman microscopy as a multipurpose, 'high content' cytometric analysis tool for characterizing the phenotype of diverse macrophage populations in terms of their molecular contents, in absolute quantitative terms. Experimentally, the results indicate how a Raman microscope can be used to map the spatial distribution of drug cargo contents of macrophages at a single cell level; to ascertain the charged state of drug molecules and their location within single cells; to determine different phospholipidosis phenotypes in terms of lipid and protein contents or the associated lipid/protein ratios within individual cells; and, to determine the existence of different macrophage subpopulations in terms of lipid, protein, DNA or (drug) cargo contents and their spatial distribution within the cell. Furthermore, the thesis argues for Raman cytometry as having distinct advantages in relation to flow cytometry or other semi-quantitative indirect cytometric analysis, especially by allowing for an absolute approach to the characterization of cellular phenotypes using limited number of cells. In terms of its broader applicability, Raman microscopy can demonstrably be used to obtain insights from macrophages obtained from bronchoalveolar lavages; for comparative analysis of the biomolecular composition of different cell types at the level of the individual cells (e.g. macropahges vs. fibroblasts); and for assessing drug distribution in complex tissue samples. Direct quantitation of total cellular contents reveals how drug exposure and accumulation interplays with the accumulation of phospholipids in alveolar “foam cell” macrophages. Since airway and alveolar macrophages are readily accessible in humans, this methodology could potentially be used to assess amiodarone exposure in the lungs, in a minimally invasive manner. Lastly, in terms of its relevance to pharmaceutical sciences, the Raman technique was also used to obtain insights into microscopic drug transport pathways and the function of controlled-release drug delivery devices, in a manner that could be used for optimizing a specific drug formulation. We envision this microanalysis calibration platform as the foundation for many future biomedical applications, ranging from diagnostic assays to pathological analysis to advanced pharmaco/toxicokinetic research studies. The findings from this study represent a significant advancement in the cytometry field and open the doors of quantitative scientific perception to the entirety of the intracellular biomolecular matrix without artificial chemical tags, providing an approach by which scientists and clinicians may holistically explore the unadulterated biochemical realm within single cells: the building blocks of life.