Fluorescent nanodiamond phantoms for spatial resolution benchmarking in two- and three-photon microscopy

Reliable spatial-resolution benchmarking is essential for multiphoton microscopy, yet conventional fluorescent-bead samples are imperfect references for biological imaging. Their fluorescence and sample properties can vary with preparation, storage and excitation conditions, while the surrounding agarose does not reproduce the depth-dependent optical degradation encountered in tissue. Here, we evaluate fluorescent nanodiamond phantoms as a more robust alternative for two- and three-photon microscopy. Nanodiamonds provide well-defined point-spread-function measurements across different microscope configurations, can be repeatedly imaged over long periods using the same physical sample, and remain usable across a broad range of excitation powers. Crucially, their optically heterogeneous matrix produces progressive broadening of the measured PSF with imaging depth, capturing a defining feature of real biological imaging that conventional bead phantoms largely miss. Nanodiamond phantoms therefore provide a durable and experimentally forgiving reference for benchmarking not only nominal spatial resolution, but how that resolution is maintained as imaging conditions become more challenging.