Confocal Raman microscopy offers a particular pathway for monitoring chemical fingerprints of intracellular components like the cell membrane, organelles, and nucleus. Nevertheless, conventional Raman acquisitions of fixed or randomly dispersed cells on a substrate, such as a microscope slide, might be severely limited for biomedical investigations. Backscattered Raman signal is likely to be overshadowed by contributions from substrate fluorescence. Also, biological assays for drug discovery, infection, and tissue engineering may require monitoring live cells individually over a time interval spanning from a few hours to a few days. To meet the needs of cell assay monitoring, we propose an acoustofluidic device that forms a levitating cell aggregation (one layer) in a cylindrical chamber of 10 {mu}L volume that operates at 1 MHz frequency. The integrated system comprises a lab-on-a-chip device and a confocal Raman microscope. In this setup, a cell can be selectively Raman-investigated with micrometre accuracy, without any substrate interference. Based on a set of carefully designed experiments, we demonstrate that polystyrene microparticles are assembled and held standstill, enabling a full Raman spectrum to be taken of a single particle in less than a minute. The signal-to-background ratio is improved by a thousandfold when compared with conventional Raman acquisition. The Raman-acoustofluidic system is showcased for obtaining the spectrum of a single cell among an enriched population of macrophages of mice. The obtained results confirm the methods robustness for applications in single-cell analysis.
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