UMF-stomata: An unsupervised multi-focus fusion framework for microscopic stomatal phenotyping.

Stomatal traits are key microscopic phenotypes for evaluating plant physiology, stress responses, and crop breeding potential. However, in vivo high-magnification microscopy often suffers from a shallow depth of field, causing noticeable defocus blur across different spatial locations and making it difficult to capture clear and complete stomatal structures in a single image. Multi-focus image fusion offers a practical solution, yet existing methods typically rely on supervised training, paired data, or hand-crafted rules, limiting their use in real agricultural microscopy scenarios. In this study, we propose an unsupervised multi-focus fusion framework for reconstructing fully focused stomatal microscopic images. The method integrates two-dimensional feature extraction with three-dimensional cross-focal-plane modeling to capture both spatial details and complementary information across focal planes. A max-response-guided spatial gating module is introduced to enhance focused regions while suppressing defocused responses. Additionally, dual sharpness priors based on perceptual features and wavelet high-frequency information enable pixel-wise pseudo-supervised learning without requiring all-in-focus ground-truth images. The model also predicts a probabilistic focal-plane volume for interpretable all-in-focus reconstruction. Experiments on a maize multi-focus image dataset demonstrate that the proposed method achieves superior or competitive performance across multiple fusion metrics, with entropy (EN), edge information preservation (Q AB∕F), Chen-Blum contrast metric (Q CB), and visual information fidelity for fusion (VIFF) reaching 7.43, 0.21, 0.41, and 1.01, respectively. Ablation studies confirm the effectiveness of the 3D modeling, spatial gating, and dual-prior sharpness supervision. More importantly, when the fused images serve as input to a YOLO-based stomatal instance segmentation model, the proposed method yields the best segmentation accuracy, with mAP50 and mAP50-95 reaching 0.9937 and 0.9121, respectively. Phenotypic measurements derived from the segmentation masks show high consistency with manual annotations, with the highest coefficient of determination R 2 = 0.97 achieved for stomatal count. These results indicate that the framework can act as an effective front-end module for automated microscopic stomatal phenotyping in agriculture.