Benchmarking classification abilities of novel optical photothermal IR spectroscopy at the single-cell level with bulk FTIR measurements.

Fourier-transform infrared (FTIR) spectroscopy is a simple, fast and inexpensive method with a history of use for bacterial analysis. However, due to the limitations placed on spatial resolution inherent to infrared wavelengths, analysis has generally been performed on bulk samples, leading to biological variance among individual cells to be buried in averaged spectra. This also increases the bacterial load necessary for analysis, which can be problematic in clinical settings where limiting incubation time is valuable. Optical photothermal-induced resonance (O-PTIR) spectroscopy is a novel method aiming to bypass this limitation using a secondary lower wavelength laser, allowing for infrared measurements of a single bacterium. Here, using , and strains as a model and FTIR as a benchmark, we examined O-PTIR's ability to discriminate single-cell samples at the intergenetic, interspecific and intraspecific levels. When combined with chemometric analysis, we showed that O-PTIR is capable of discriminating different between genera, species and strains within species to a degree comparable with FTIR. Furthermore, small variations in the amide bands associated with differences in the protein structure can still be seen in spite of smaller sample sizes. This demonstrates the potential of O-PTIR for single-cell bacterial analysis and classification.