Nanodiamonds for spatial resolution benchmarking in two-photon microscopy

Summary Reliable and reproducible measurement of spatial resolution is essential for validating and comparing the performance of two-photon microscopy systems. We present fluorescent nanodiamonds as robust, photostable, reusable, and biocompatible probes for benchmarking spatial resolution. Owing to their nanoscale dimensions and stable fluorescence, nanodiamonds act as near-ideal point-like emitters, enabling accurate characterisation of the point spread function across varying imaging conditions. We demonstrate that nanodiamond-based phantoms serve as a reliable alternative to conventional fluorescent beads embedded in agarose, while at the same time offering advantages in stability and optical properties. Our results position nanodiamond phantoms as a next-generation calibration material that bridges ease of use and reproducibility, advancing quantitative imaging and cross-platform comparability in modern fluorescence microscopy.