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. ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, https://ror.org/029chgv08, Investigator Award in Science 220277/Z20/Z European Research Council, https://ror.org/0472cxd90, ERC-StG “NeuroVisEco” 677687, ERC AdG “Cones4Action” covered under the UK’s EPSRC guarantee scheme EP/Z533981/1 UK Research and Innovation, https://ror.org/001aqnf71, BBSRC, BB/R014817/1, BBSRC, BB/W013509/1 International Human Frontier Science Program Organization, https://ror.org/02ebx7v45, RGP001/2025 Leverhulme Trust, https://ror.org/012mzw131, PLP-2017-005, RPG-2021-026, RPG-2-23-042 Lister Institute of Preventive Medicine Gdańsk University of Technology, https://ror.org/006x4sc24, Nobelium DEC-4/1/2024/IDUB/I.1a/No, Platinum 1/1/2025/IDUB/I.1B/Pt

Observing the activity patterns of large neural populations throughout the brain is essential for understanding brain function. However, capturing neural interactions across widely distributed brain r...