Calibration and measurement of single-cell elastic modulus by time-of-flight and fluorescence signal analysis in a serial microfluidic cytometer

The mechanical properties of cells, particularly elastic modulus, are important indicators of cellular state, function, and disease. However, conventional mechanophenotyping methods such as atomic force microscopy (AFM) are low-throughput and lack the ability to integrate biochemical characterization. We hypothesize that we can leverage data collected from a novel multi-region microfluidic optical cytometer to determine particle elastic modulus at high throughput. To test this, we adapted a serial microfluidic fluorescence cytometer to perform simultaneous mechanophenotyping and fluorescence measurements using time-of-flight (TOF) and fluorescence spectral time-series analysis (STA). Calibration with polyacrylamide microparticles of known size (8.9 µm to 23 µm diameter) and stiffness (0.1 kPa to 9.1 kPa) demonstrated strong correlations between TOF, particle diameter, and elastic modulus, achieving per-particle TOF CVs below 8 % and resolving elastic modulus differences within 20 % agreement of AFM data. By integrating size estimation from STA into a physically informed regression model, we deconvolved the effects of size and deformation on TOF to yield elastic modulus measurements. We validated this method using live MG-63 osteosarcoma cells, obtaining size and elastic modulus estimates (median 0.9 kPa) consistent with atomic force microscopy. Importantly, the device maintained > 90 % cell viability and enabled single-cell measurements with built-in error quantification. An alternative to both low-throughput, gold-standard methods and newer microfluidic approaches that are harsher on cells and lack error metrics, this platform offers a high-throughput, label-compatible approach to single-cell mechanophenotyping.