Identification of single bacterial cells in aqueous solution using conflocal laser tweezers Raman spectroscopy

We report on a rapid method for reagentless identification and discrimination of single bacterial cells in aqueous solutions using a combination of laser tweezers and confocal Raman spectroscopy (LTRS). The optical trapping enables capturing of individual bacteria in aqueous solution in the focus of the laser beam and levitating the captured cell well off the cover plate, thus maximizing the excitation and collection of Raman scattering from the cell and minimizing the unwanted background from the cover plate and environment. Raman spectral patterns excited by a near-infrared laser beam provide intrinsic molecular information for reageatless analysis of the optically isolated bacterium. In our experiments, six species of bacteria were used to demonstrate the capability of the confocal LTRS in the identification and discrimination between the diverse bacterial species at various growth conditions. We show that synchronized bacterial cells can be well-discriminated among the six species using principal component analyses (PCA). Unsynchronized bacterial cells that are cultured at stationary phases can also be well-discriminated by the PCA, as well as by a hierarchical cluster analysis (HCA) of their Raman spectra. We also show that unsynchronized bacteria selected from random growth phases can be classified with the help of a generalized discriminant analysis (GDA). These findings demonstrate that the LTRS may find valuable applications in rapid sensing of microbial cells in diverse aqueous media.