ABSTRACT Single-cell analysis is a transformative capability in modern biological and medical research that enables capturing the heterogeneity between cells within an entire cell population. Single-cell analysis can inspect biological, chemical, and physicochemical parameters at the single-cell level. While there are numerous analytical approaches to single-cell analysis, magnetic resonance and especially electron spin resonance (ESR), is yet to contribute to this field, mostly because of a sensitivity issue and the lack of appropriate bio-stable intracellular spin probes. This proposal aims to develop a highly sensitive ESR-based flow cytometry technology (instrumentation and molecular spin-probes) that enables single-cell analysis of chemical and physicochemical parameters. The current project focuses on the simultaneous measurement of pO2 and microviscosity at the single-cell level to develop and demonstrate the concept. However, the technology can be applied to assess other parameters in the future, such as microviscosity, pH, oxygen concentration, redox status, and glutathione. We will achieve our goal by the following three specific aims. In SA1, we will synthesize and characterize intracellular biostable trityl spin probes sensitive to the oxygen concentration and microviscosity. In SA2, we will develop a compact, highly sensitive ESR surface resonator embedded in a cell microfluidic device to measure a continuous flow of single cells. Finally, in SA3, we will validate our technology with the single-cell measurement for a population of a few hundred cells of a cancer cell line. The completion of this R21 project will expand the current capability in single-cell analysis, allowing assessing multiple parameters that cannot be fully characterized by the currently available techniques with significant new applications downstream.