Microbial Identification Through Multispectral Infrared Imaging of Colonies: A New Type of Morpho‐Spectral Fingerprinting

We describe a proof of concept for a new microbial identification technique using Direct Frequency Infrared (DFIR) multispectral imaging. This approach combines Quantum Cascade Laser (QCL) light sources with a microbolometer array in a lensless configuration to capture detailed multispectral images of microbial colonies. These optical fingerprints blend both morphological and spectral information, without the need for staining or colony picking. A proof-of-concept database was acquired, comprising 10 strains from 8 species across 4 distinct genera. In total, 2253 microbial colonies were imaged at 9 different mid-infrared wavelengths. Machine learning classification correctly identified up to 94.4% ± 1.6 of colonies fingerprints, efficiently discriminating even closely related strains. Reducing the number of wavelengths to 4 maintained high classification performance, demonstrating the method's robustness. The resulting system is faster and simpler than existing FTIR imaging systems, making it a promising tool for microbial identification.