Rapid Serovar Classification Using AI-Enabled Hyperspectral Microscopy with Enhanced Data Preprocessing and Multimodal Fusion.

serovar identification typically requires multiple enrichment steps using selective media, consuming considerable time and resources. This study presents a rapid, culture-independent method leveraging artificial intelligence (AI) to classify serovars from rich hyperspectral microscopy data. Five serovars (Enteritidis, Infantis, Kentucky, Johannesburg, 4,[5],12:i:-) were analyzed from samples prepared using only sterilized de-ionized water. Hyperspectral data cubes were collected to generate single-cell spectra and RGB composite images representing the full microscopy field. Data analysis involved two parallel branches followed by multimodal fusion. The spectral branch compared manual feature selection with data-driven feature extraction via principal component analysis (PCA), followed by classification using conventional machine learning models (i.e., -nearest neighbors, support vector machine, random forest, and multilayer perceptron). The image branch employed a convolutional neural network (CNN) to extract spatial features directly from images without predefined morphological descriptors. Using PCA-derived spectral features, the highest performing machine learning model achieved 81.1% accuracy, outperforming manual feature selection. CNN-based classification using image features alone yielded lower accuracy (57.3%) in this serovar-level discrimination. In contrast, a multimodal fusion model combining spectral and image features improved accuracy to 82.4% on the unseen test set while reducing overfitting on the train set. This study demonstrates that AI-enabled hyperspectral microscopy with multimodal fusion can streamline serovar identification workflows.