Label-free identification of spore-forming bacteria using ultra-broadband multiplex CARS (coherent anti-Stokes Raman scattering) microspectroscopy

Spore-forming bacteria accumulate dipicolinic acid (DPA) to form spores to survive in extreme environments. Vibrational spectroscopy is widely used to detect DPA to elucidate the existence of the bacteria, while a vegetative cell, another form of spore-forming bacteria, has not studied deeply. Here we applied coherent anti-Stokes Raman scattering (CARS) microscopy to spectroscopically identify both spores and vegetative cells without staining or molecular tagging. The spores were identified by the strong CARS signals due to dipicolinic acid (DPA). Furthermore, we observed bright spots in the vegetative cells in the CARS image at 1735 cm-1. The vegetative cells contained molecular species with C=O bonds because of this vibrational mode being associated with the carbonyl group. One of the candidate molecular species is diketopimelic acid, a DPA precursor. The results indicate that the observed vegetative cell is in the sporulation process. CARS spectra can be used to monitor the maturation or formation of spores.