HyperBird: A Hyperspectral Microscopic Imaging Robot for High-Throughput Plant Phenotyping

Abstract

The automation and standardization of hyperspectral imaging, particularly at high spatial resolution, are essential for advancing plant phenomics and supporting diverse plant science research. We developed HyperBird, a hyperspectral microscopic imaging robot, to automate the acquisition of up to 351 leaf-disc samples in a single tray within approximately 2.4 h and thereby support scalable plant experiments. System calibration established a spatial resolution of 24.3 μ m (full width at half maximum; FWHM), a spectral resolution of 1.78 nm (FWHM), and a depth of field of 4.53 mm, producing hyperspectral data cubes of 2195 × 2000 × 950 pixels across the 400–1000 nm spectral range. System-level and biological-sample consistency assessments confirmed stable spectral measurements during extended scanning sessions and across independently prepared trays. Thermal evaluation confirmed that the 150 W illumination design introduced minimal sample heating during scanning. HyperBird was first evaluated using grape downy and powdery mildews, where no consistent measurable effect of repeated imaging on pathosystem development was detected under the tested experimental conditions. Subsequently, HyperBird was used to characterize spatiotemporal spectral progression in grapevine leaves inoculated with Plasmopara viticola . An automated regional spectral tracing pipeline was developed to isolate spectra from retrospectively traced regions and compare them with whole-leaf averaged spectra across 0–9 days post-inoculation (DPI). Categorical mixed-effects modeling showed that these spatially resolved regional spectra exhibited significant disease-associated spectral change by 5 DPI, with a sharp transition at 6 DPI, whereas whole-leaf spectra showed delayed and weaker disease-aligned changes beginning at 7 DPI. These results demonstrate that HyperBird enables high-throughput, spatially resolved hyperspectral imaging for quantifying localized plant disease progression and provides a scalable platform for studying spectral biology across plant phenotyping applications.

Plant phenotyping relevance

HyperBirdの高スループット・ハイパースペクトル画像取得ロボットを開発し、校正、再現性、加熱影響、病害進展の定量性能を検証しており、植物フェノタイピング手法が中心である。

Code and data availability

The paper's Data Availability statement points to a public GitHub repository containing the authors' code and processed data supporting the phenotyping analyses; raw hyperspectral image data are request-only.