Multiplexed live-cell profiling with Raman probes

Abstract

Single-cell multiparameter measurement has been increasingly recognized as a key technology toward systematic understandings of complex molecular and cellular functions in biological systems. Despite extensive efforts in analytical techniques, it is still generally challenging for existing methods to decipher a large number of phenotypes in a single living cell. Herein we devise a multiplexed Raman probe panel with sharp and mutually resolvable Raman peaks to simultaneously quantify cell surface proteins, endocytosis activities, and metabolic dynamics of an individual live cell. When coupling it to whole-cell spontaneous Raman micro-spectroscopy, we demonstrate the utility of this technique in 14-plexed live-cell profiling and phenotyping under various drug perturbations. In particular, single-cell multiparameter measurement enables powerful clustering, correlation, and network analysis with biological insights. This profiling platform is compatible with live-cell cytometry, of low instrument complexity and capable of highly multiplexed measurement in a robust and straightforward manner, thereby contributing a valuable tool for both basic single-cell biology and translation applications such as high-content cell sorting and drug discovery.

Tetraplex Immunophenotyping of Cell Surface Proteomes via Synthesized Plasmonic Nanotags and Portable Raman Spectroscopy

“…Multiplex detection is essential for the comprehensive immunophenotyping of cancer cell surface proteomes, and SERS is a technique with unique multiplexing advantages. ,,− To demonstrate multiplexing capability, we lastly demonstrated tetraplex immunophenotyping of breast cancer cell surface proteomes using the portable SERS immunophenotyping assay. In the tetraplex detection, cells were labeled with a cocktail of SERS nanotags for simultaneous signal readouts.…”

Application of Advanced Non-Linear Spectral Decomposition and Regression Methods for Spectroscopic Analysis of Targeted and Non-Targeted Irradiation Effects in an In-Vitro Model

“…t-SNE decomposition has previously been used to cluster and visualise Raman spectra from single neural pluripotent stem cells at various stages of differentiation and cell lines of various lineages exposed to various different chemotherapeutic agents [ 57 , 58 ]. As an approach to the decomposition of large multi-variate datasets t-SNE preserves local data structures at the expense of global structure, in comparison with PCA which attempts to preserve global data structure [ 56 ].…”

Miniaturized Chemical Tags for Optical Imaging

“…[92] By capitalizing on the high spectral resolution of Raman spectroscopy, multiplexed detection of 14 biomarkers for live cell profiling was reported. [93] In this study, the conjugation of Rdots to antibodies and aptamers was used to analyze cell surface protein abundance, endocytosis activity and metabolic dynamics. Through integration of fluorescence microscopy into a similar SRS platform, Ozeki and co-workers demonstrated faster visualization of multiplexed samples using an image-based cytometry approach.…”