High-throughput multimodal optofluidic biophysical imaging cytometry

Traditional biophysical cytometry has been limited by its low-dimensional phenotyping characteristics, often relying on only one or a few cellular biophysical phenotypes as readouts. This has perpetuated the perception that biophysical cytometry lacks the power to determine cellular heterogeneity. Here, we introduce a multimodal biophysical cytometry platform, termed quantitative phase morpho-rheological (QP-MORE) cytometry, which simultaneously captures a collection of high-resolution biophysical and mechanical phenotypes of single cells at ultrahigh throughput (>10 000 cells per s). Combined with a microfluidic constriction channel design, QP-MORE integrates ultrafast single-cell quantitative phase imaging (QPI) and high-throughput deformability cytometry to resolve subcellular structures and whole-cell rheology in a single pass. QP-MORE's optofluidic design enables label-free, multi-contrast imaging of cells flowing at ∼1 m s⁻¹, achieving subcellular resolution unmatched by existing deformability-based platforms. To validate its precision, we developed a robust calibration protocol ensuring high accuracy in morpho-rheological measurements. We also deployed QP-MORE to dissect drug-induced biophysical heterogeneity in HL60 leukemia and MDA-MB-231 breast cancer cells treated with latrunculin B (actin depolymerizer) and cytochalasin D (actin capping agent). QP-MORE not only revealed drug-specific subcellular biophysical signatures, but also achieved 99% accuracy in classifying drug mechanisms, surpassing deformability cytometry (78–94%). This underscores the potential of QP-MORE in expanding the capability of biophysical cytometry, especially in advancing our understanding of cellular heterogeneity and drug interactions.