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 (