Characterization and Visualization of Vesicles in the Endo-Lysosomal Pathway with Surface-Enhanced Raman Spectroscopy and Chemometrics.
ACS nano(2016)
Abstract
Surface-enhanced Raman spectroscopy (SERS) is an ultra-sensitive vibrational fingerprinting technique widely used in analytical and biosensing applications. For intracellular sensing, typically gold nanoparticles (AuNPs) are employed as transducers to enhance the otherwise weak Raman spectroscopy signals. Thus the signature patterns of the molecular nanoenvironment around intracellular unlabelled AuNPs can be monitored in a reporter-free manner by SERS. The challenge of selectively identifying molecular changes resulting from cellular processes in large and multidimensional data sets and the lack of simple tools for extracting this information has resulted in limited characterization of fundamental cellular processes by SERS. Here, this shortcoming in analysis of SERS data sets is tackled by developing a suitable methodology of reference-based PCA-LDA (principal component analysis-linear discriminant analysis). This method is validated and exemplarily used to extract spectral features characteristic of the endocytic compartment inside cells. The voluntary uptake through vesicular endocytosis is widely used for the internalisation of AuNPs into cells but the characterization of the individual stages of this pathway has not been carried out. Herein, we use reporter-free SERS to identify the stages of endocytosis of AuNPs in cells, visualize them and map the molecular changes via the adaptation and advantageous use of chemometric methods in combination with tailored sample preparation. Thus our study demonstrates the capabilities of reporter-free SERS for intracellular analysis and its ability to provide a way of characterizing intracellular composition. The developed analytical approach is generic and enables the application of reporter-free SERS to identify unknown components in different biological matrices and materials.
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Key words
surface-enhanced Raman spectroscopy,endocytosis,endosomes,lysosomes,linear discriminant analysis,chemometrics,cells
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