Light-addressable measurements of cellular oxygen consumption rates in microwell arrays based on phase-based phosphorescence lifetime detection

A digital light modulation system that utilizes a modified commercial digital micromirror device (DMD) projector, which is equipped with a UV LED as a light modulation source, has been developed to spatially direct excited light toward a microwell array device to detect the oxygen consumption rate (OCR) of single cells via phase-based phosphorescence lifetime detection. The microwell array device is composed of a combination of two components: an array of glass microwells containing Pt(II) octaethylporphine (PtOEP) as the oxygen-sensitive luminescent layer and a microfluidic module with pneumatically actuated glass lids set above the microwells to controllably seal the microwells of interest. By controlling the illumination pattern on the DMD, the modulated excitation light can be spatially projected to only excite the sealed microwell for cellular OCR measurements. The OCR of BHK-21 fibroblast cells cultivated on the PtOEP layer within a sealed microwell has been successfully measured at 104 ± 2.96 amol s^-1 cell^-1. Repeatable and consistent measurements indicate that the oxygen measurements did not adversely affect the physiological state of the measured cells. The OCR of the cells exhibited a good linear relationship with the diameter of the microwells, ranging from 400 to 1000 μm and containing approximately 480 to 1200 cells within a microwell. In addition, the OCR variation of single cells in situ infected by Dengue virus with a different multiplicity of infection (M.O.I) was also successfully measured in real-time. This proposed platform provides the potential for a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery.