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bioRxiv · 10.64898/2026.01.12.699010

Nanodiamonds for spatial resolution benchmarking in two-photon microscopy

Janiak, F. K.·Forsthofer, M.·Czubek, J.·Szczerska, M.·Baden, T.
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Abstract

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.

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Janiak, F. K., Forsthofer, M., Czubek, J., Szczerska, M., Baden, T.. 2026-01-13. Nanodiamonds for spatial resolution benchmarking in two-photon microscopy. https://doi.org/10.64898/2026.01.12.699010

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