Hyperspectral dark-field microscopy for pathogen detection based on spectral angle mapping

Early screening for food pathogens, specifically Escherichia coli O157:H7 in ready-to-eat food remains a central challenge, hyperspectral imaging (HSI) has the potential to acquire both image and spectral information of the target and is simple enough to serve as an effective auxiliary technology in food monitoring. Herein, a visual method was developed based on spectral angle mapping (SAM) and dark-field microscopy for rapid detection and quantification of Escherichia coli. First, immunomagnetic nanoparticle (MNP-probe) and gold nanoparticle (AuNP-probe) probes were prepared to form a sandwich complex for efficient concentration of the target bacteria. The complexes were then dropped directly on a glass slide for colony counting with hyperspectral dark field microscopy. The AuNP-probe bound to the complex was counted and SAM was used to evaluate the spectra and HSI image data to distinguish the target pixel from the background. The linear detection range of the method was estimated to be in the range between 6.1 × 10¹ and 6.1 × 10⁶ CFU/mL in the milk sample, and the detection limit (LOD) of this method was estimated to be ~61 CFU/mL. The average recovery of organisms in the milk sample was 106.0%, indicating that this method can be utilized to detect Escherichia coli in food samples and serve as an effective method to monitor pathogens in a complex sample.