Intrinsically co-registered extended-depth-of-focus OCM and two-photon microscopy for label-free volumetric analysis...

Beam engineering effectively overcomes the resolution-depth of focus trade-off in high-NA optical coherence microscopy (OCM). Conventional OCM is limited by this constraint, hindering stable volumetric acquisition. To overcome this, we present an intrinsically registered imaging platform based on Bessel beam-enabled extended-depth-of-focus OCM for quantitative, label-free volumetric phenotyping of three-dimensional cell models. Annular illumination extends the focal range to 193 µm, enabling stable scattering contrast in dense spheroids. By exploiting structural scattering signatures associated with cellular disintegration, we demonstrate volumetric viability mapping with 94.57 ± 2.69% aggregate viability concordance relative to fluorescence references. The platform differentiates drug-specific responses and captures pathological transitions in spheroids and organoids, establishing a quantitative paradigm for volumetric biological phenotyping.