Modular dual-channel 3D microscopy for resolving fast nanoscale dynamics in soft matter

Fast volumetric microscopy is essential for studying dynamic heterogeneous soft matter, but combining 3D imaging with independent detection channels remains challenging, often compromising acquisition speed or resolution. Prism-based multiplane microscopes provide instantaneous 3D imaging by splitting emission into multiple focal planes, yet existing implementations use only one input face of the prism. Here, we introduce M³Scope, a dual-entry prism multiplane microscope that exploits the previously unused second prism input face to create an independent second detection channel. This architecture enables simultaneous 16-plane acquisition across two channels while preserving the temporal (>100 fps) and volumetric advantages of multiplane imaging and without sacrificing signal-to-noise ratio. By exchanging the optical element in a magnetic cube, the same architecture supports dual-color fluorescence, polarization-resolved scattering, and correlative brightfield-fluorescence imaging. We demonstrate the approach in soft-matter microrheology: dual-color 3D tracking reveals probe-size-dependent dynamics during polyacrylamide gelation; polarization-resolved multiplane imaging enables simultaneous translational and rotational tracking of gold bipyramids in high-viscosity media; and brightfield-fluorescence imaging links pNIPAM structural evolution to local tracer mobility. These results establish dual-entry prism multiplane microscopy as a simple route to simultaneous, multimode 3D measurements of nanoscale dynamics in soft matter and biological systems.