Raman imaging for the investigation of Mycobacterium smegmatis in a mother machine

The mother machine (MM) is a microfluidic device designed to trap and measure single bacteria for long-term experiments, enabling the study of bacterial growth and cell cycle dynamics. In this work, the concept of a MM is adapted to fit the requirements for investigating Mycobacterium smegmatis, a bacterium used extensively as a model system in tuberculosis research. A MM chip was fabricated using soft lithography, and a protocol for sample preparation and filling of the side channels for mycobacteria was established. Trapped bacteria were measured using Raman spectroscopy to introduce a new analysis approach inside the MM. The subsequent data evaluation demonstrates the potential for obtaining spatially resolved chemical information on a single cell within a side channel of the MM through Raman imaging. Furthermore, the combination of Raman imaging and stable isotope labelling, as applied in this study, demonstrates the viability of bacteria inside a side channel and opens up possibilities to study cell cycles in long-term experiments.