From projection to true surface: Curvature-corrected SEM morphometrics of diatom frustules under hydration variability.

Diatom frustules exhibit highly organised silica architectures with submicron features, which are widely studied. Nowadays, scanning electron microscopy (SEM) is commonly used for studying their morphology. However, SEM provides a two-dimensional projection that distorts the true size of three-dimensional features. In addition, samples for SEM observations are usually prepared by removing organic parts and drying the remaining parts made of bio-silica (so-called frustule). Here, we combine high-resolution SEM and liquid-cell SEM imaging with confocal microscopy to construct a curvature-aware correction model that accounts for the surface area distortion in SEM imaging caused by the in-plane projection of three-dimensional frustule geometry, demonstrated using Diploneis didyma frustules as a case study. The paper describes how automated image analysis enabled the detection of individual pores, the localisation of their centroids, and the calculation of curvature-corrected surface areas. True diameters of the frustules are provided, and drying-induced shrinkage of pores is obtained. Species length-based normalisation of morphometric data reduced variability and allow to infer systematic dependence of pore sizes on position on frustules. The results establish a reliable framework for quantitative characterisation of diatom frustules and can be applied across disciplines requiring accurate morphometrics of biogenic silica structures. LAY DESCRIPTION: Curvature-corrected SEM morphometrics, paired with hydrated/dehydrated imaging, offer a reliable quantitative framework for diatom frustule pore-size assessment. Drying shrinks pores by ∼10%, so dry-sample measurements should be corrected when targeting living/wet morphology. Curvature correction compensates SEM projection distortion, with uncorrected pores underestimated by ∼34% on average, enabling realistic edge-to-centre comparisons. The protocol is broadly transferable beyond Diploneis didyma with minor adjustments and supports applications in taxonomy, biomonitoring, and bioinspired design.