Understanding the complexity of cellular biology often requires capturing and processing an enormous amount of data. In high-content drug screens, each cell is labeled with several different fluorescent markers and frequently thousands to millions of cells need to be analyzed in order to characterize biology’s intrinsic variability. In this work, we demonstrate a new microlens-based multispectral microscope designed to meet this throughput-intensive demand. We report multispectral image cubes of up to 1.30 gigapixels in the spatial domain, with up to 13 spectral samples per pixel, for a total image size of 16.8 billion spatial-spectral samples. To our knowledge, this is the largest multispectral microscopy dataset reported in the literature. Our system has highly reconfigurable spectral sampling and bandwidth settings and we have demonstrated spectral unmixing of up to 6 fluorescent channels.
Oxygen is transported throughout the body by hemoglobin in red blood cells. While the oxygen affinity of blood is well understood and is routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of red blood cell volume and hemoglobin concentration are taken millions of times per day by clinical hematology analyzers and are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume and hemoglobin concentration for individual red blood cells in high-throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.5%, which corresponds to the maximum slope of the oxygen-hemoglobin dissociation curve. In addition, single-cell oxygen affinity is positively correlated with hemoglobin concentration, but independent of osmolarity, which suggests variation in the hemoglobin to 2-3 DPG ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a new dimension to blood cell analysis and other measurements of single-cell variability.