title: Characterization and Visualization of Vesicles in the Endo-Lysosomal Pathway with Surface-Enhanced Raman Spectroscopy and Chemometrics.
abstract: Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive 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 unlabeled 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 internalization 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 and visualize the stages of endocytosis of AuNPs in cells 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.
authors.affiliation: Sector for Biological and Soft Systems, Cavendish Laboratory, Department of Physics, University of Cambridge , 19 JJ Thomson Avenue, Cambridge CB3 0HE, United Kingdom.|Institute of Life Sciences and Department of Chemistry, University of Southampton , Highfield Campus, SO17 1BJ Southampton, United Kingdom.|John van Geest Centre for Brain Repair, University of Cambridge , Forvie Site, Robinson Way, Cambridge CB2 0PY, United Kingdom.
authors.forename: Anna
authors.initials: A
authors.lastname: Huefner
authors.affiliation: John van Geest Centre for Brain Repair, University of Cambridge , Forvie Site, Robinson Way, Cambridge CB2 0PY, United Kingdom.
authors.forename: Wei-Li
authors.initials: WL
authors.lastname: Kuan
authors.affiliation: Cambridge Advanced Imaging Centre, Department of Physiology, Development and Neuroscience, Anatomy Building, Cambridge University , Downing Street, Cambridge CB2 3DY, U.K.
authors.forename: Karin H
authors.initials: KH
authors.lastname: Müller
authors.affiliation: Cambridge Advanced Imaging Centre, Department of Physiology, Development and Neuroscience, Anatomy Building, Cambridge University , Downing Street, Cambridge CB2 3DY, U.K.
authors.forename: Jeremy N
authors.initials: JN
authors.lastname: Skepper
authors.affiliation: John van Geest Centre for Brain Repair, University of Cambridge , Forvie Site, Robinson Way, Cambridge CB2 0PY, United Kingdom.
authors.forename: Roger A
authors.initials: RA
authors.lastname: Barker
authors.affiliation: Institute of Life Sciences and Department of Chemistry, University of Southampton , Highfield Campus, SO17 1BJ Southampton, United Kingdom.
authors.forename: Sumeet
authors.initials: S
authors.lastname: Mahajan
mesh_terms: D001692:Biological Transport / Q000502:physiology; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry; D016002:Discriminant Analysis; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure*; D006046:Gold / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure*; D053768:Metal Nanoparticles / Q000737:chemistry*; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure*; D025341:Principal Component Analysis; D012313:RNA / Q000737:chemistry; D013059:Spectrum Analysis, Raman / Q000379:methods*
chemical_list: D012313:RNA; D006046:Gold; D004247:DNA
keywords: cells; chemometrics; endocytosis; endosomes; linear discriminant analysis; lysosomes; surface-enhanced Raman spectroscopy