Three-dimensional imaging flow cytometry: current trends and future perspectives

Imaging flow cytometry (IFC) combines microscopic techniques and flow cytometry, enabling high-throughput, population-scale single-cell analysis with rich spatial detail and high molecular specificity. To date, most established IFC platforms are fundamentally built around 2D imaging, which is inherently limited in its ability to capture the complex 3D morphology, intracellular organization, and spatially distributed molecular features of single cells under flow. Driven by the growing demand for volumetric cellular information, IFC has progressively evolved from 2D imaging toward 3D imaging flow cytometry through a variety of optical and computational strategies. Existing 3D IFC approaches typically fall into scanning-based and non-scanning modalities, depending on whether volumetric information is assembled through sequential or parallel acquisition, with each paradigm offering complementary strengths in 3D imaging performance and cytometric throughput. Here, we systematically compare the underlying principles and performance characteristics of the most representative scanning-based and non-scanning 3D IFC technologies and discuss current challenges and future opportunities to advance volumetric cytometric analysis in biomedical and translational research.