Laser Tweezers Raman Spectroscopy (LTRS) to Detect Effects of Chlorine Dioxide on Individual Nosema bombycis Spores

The microsporidium Nosema bombycis (Nb) causes pebrine, a fatal disease in sericulture. Nb is effectively killed by chlorine dioxide (ClO₂); however, the precise killing mechanism remains unclear. We used laser tweezers Raman spectroscopy (LTRS) to monitor the action of ClO₂ on individual Nb spores in real time. Raman peaks of ClO₂ appeared in Nb spores, corresponding to decreased peaks of trehalose that gradually disappeared. A peak (1658 cm^-1) corresponding to the protein α-helix significantly weakened while that (1668 cm^-1) corresponding to irregular protein structures was enhanced; their intensities were negatively correlated in a certain time range and dependent on ClO₂ concentration. The intensities of peaks at 782 cm^-1 (nucleic acids) and 1004 cm^-1 (phenylalanine of protein) did not change evidently even under extremely high ClO₂ concentrations. Thus, ClO₂ rapidly permeates the Nb spore wall, changing the protein secondary structure to lose biological function and destroy permeability, causing trehalose to leak out. These effects are ClO₂ concentration-dependent, but no other obvious changes to biomacromolecules were detected. Single-cell analysis using LTRS is an effective method to monitor the action of chemical sporicides on microbes in real time, providing insight into the heterogeneity of cell stress resistance.