How flat is your sample? An opportunistic survey of 3D tilt in public fluorescence microscopy data

Sample planarity is rarely monitored in fluorescence microscopy quality control, yet focal plane deviations across the field of view are a potential source of measurement error. Here I describe FlatStat, a tool that estimates sample tilt automatically from any 3D fluorescence stack, without prior knowledge of sample content, by fitting a plane to the Z-map of maximum intensity. Applied to an Argolight calibration slide and biological samples on a laser-scanning confocal system, FlatStat yielded reproducible slope and direction measurements attributable to the instrument rather than the sample. To establish community reference values, FlatStat was extended to Python and applied opportunistically to 1204 image stacks from 22 projects in the Image Data Resource, yielding 4670 tilt measurements. Slopes spanned several orders of magnitude across projects; inter-channel coherence confirmed that measured tilt reflects physical stage and mounting geometry rather than channel-specific biological topography. Unfortunately, instrument and sample preparation metadata were largely absent from the corpus, limiting causal inference. Finally, controlled tilt experiments on fluorescent beads showed that chromatic shift increased modestly with tilt (∼57 nm over the full range tested), while lateral and axial resolutions were essentially unaffected.