**Featured Application: Phenotype classification of zebrafish larvae from brightfield microscopic images.**

**Abstract:** Classification of different zebrafish larvae phenotypes is useful for studying the environmental influence on embryo development. However, the scarcity of well-annotated training images and fuzzy inter-phenotype differences hamper the application of machine-learning methods in phenotype classification. This study develops a deep-learning approach to address these challenging problems. A convolutional network model with compressed separable convolution kernels is adopted to address the overfitting issue caused by insufficient training data. A two-tier classification pipeline is designed to improve the classification accuracy based on fuzzy phenotype features. Our method achieved an averaged accuracy of 91% for all the phenotypes and maximum accuracy of 100% for some phenotypes (e.g., dead and chorion). We also compared our method with the state-of-the-art methods based on the same dataset. Our method obtained dramatic accuracy improvement up to 22% against the existing method. This study offers an effective deep-learning solution for classifying difficult zebrafish larvae phenotypes based on very limited training data.

**Keywords:** zebrafish larva; microscopy image analysis; deep neural network
