Reagentless Identification of Single Bacterial Spores in Aqueous Solution by Confocal Laser Tweezers Raman Spectroscopy

We demonstrate that optical trapping combined with confocal Raman spectroscopy using a single laser source is a powerful tool for the rapid identification of micrometer-sized particles in an aqueous environment. Optical trapping immobilizes the particle while maintaining it in the center of the laser beam path and within the laser focus, thus maximizing the collection of its Raman signals. The single particle is completely isolated from other particles and substrate surfaces, therefore eliminating any unwanted background signals and ensuring that information is collected only from the selected, individual particle. In this work, an inverted confocal Raman microscope is combined with optical trapping to probe and analyze bacterial spores in solution. Rapid, reagentless detection and identification of bacterial spores with no false positives from a complex mixed sample containing polystyrene and silica beads in aqueous suspension is demonstrated. In addition, the technique is used to analyze the relative concentration of each type of particle in the mixture. Our results show the feasibility for incorporating this technique in combination with a flow cytometric-type scheme in which the intrinsic Raman signatures of the particles are used instead of or in addition to fluorescent labels to identify cells, bacteria, and particles in a wide range of applications.