2D and 3D optical imaging of SERS nanotags intracellularly

Adoption of a multi-marker nanotag approach will led to better disease characterisation whilst simultaneously enabling targeting of multiple disease markers or organelles. The employed nanotag method controllably aggregated nanoparticles with 1,6-hexamethylene diamine (1,6-HMD), before polymer coating with polyvinylpyrrolidone (PVP) and labelling with small molecule reporters; 4-mercaptopyridine (MPY), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 4-nitrobenzenethiol (NBT) and 2-naphthalenethiol (2-NPT). Within a multiple component suspension reporters were identified by their unique peak and when present within single cells or populations they were additionally identified using component direct classical least squares (DCLS). Within a single cell three of the four components (MPY, DTNB and NBT) were positively identified. 2D SERS imaging can monitor nanotag uptake but it provides no conclusive evidence of cellular inclusion. The simultaneous determination of cellular uptake and nanotag identification was however achieved using combined 3D Raman and SERS imaging. Three of the four components were detected within a single cell and by combining 2D sections from the 3D images it was possible to determine their intracellular location. Determination of intracellular localisation was achieved using principal component analysis (PCA) since it resulted in the resolution of a subcellular compartment. However, the ultimate success of the system will only be realised when active targeting is demonstrated. Nanotags were functionalised with peptide sequences specific for the endoplasmic reticulum (ER) and trans-Golgi network (TGN). Both nanotag systems were found to locate within lipid rich regions of the cell but they could not be positively confirmed as the ER or TGN. To identify these structures and confirm localisation, further chemometric methods must be investigated including hierarchical cluster analysis (HCA). In conclusion, the SERS nanotags were suitable imaging agents for 2 and 3D cell interrogation. 3D imaging simultaneously permitted organelle resolution and the intracellular localisation of the SERS nanotags. Targeting systems were developed and in future work their localisation within organelles will be confirmed by the application of advanced chemometric methods.Adoption of a multi-marker nanotag approach will led to better disease characterisation whilst simultaneously enabling targeting of multiple disease markers or organelles. The employed nanotag method controllably aggregated nanoparticles with 1,6-hexamethylene diamine (1,6-HMD), before polymer coating with polyvinylpyrrolidone (PVP) and labelling with small molecule reporters; 4-mercaptopyridine (MPY), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 4-nitrobenzenethiol (NBT) and 2-naphthalenethiol (2-NPT). Within a multiple component suspension reporters were identified by their unique peak and when present within single cells or populations they were additionally identified using component direct classical least squares (DCLS). Within a single cell three of the four components (MPY, DTNB and NBT) were positively identified. 2D SERS imaging can monitor nanotag uptake but it provides no conclusive evidence of cellular inclusion. The simultaneous determination of cellular uptake and nanotag identification was however achieved using combined 3D Raman and SERS imaging. Three of the four components were detected within a single cell and by combining 2D sections from the 3D images it was possible to determine their intracellular location. Determination of intracellular localisation was achieved using principal component analysis (PCA) since it resulted in the resolution of a subcellular compartment. However, the ultimate success of the system will only be realised when active targeting is demonstrated. Nanotags were functionalised with peptide sequences specific for the endoplasmic reticulum (ER) and trans-Golgi network (TGN). Both nanotag systems were found to locate within lipid rich regions of the cell but they could not be positively confirmed as the ER or TGN. To identify these structures and confirm localisation, further chemometric methods must be investigated including hierarchical cluster analysis (HCA). In conclusion, the SERS nanotags were suitable imaging agents for 2 and 3D cell interrogation. 3D imaging simultaneously permitted organelle resolution and the intracellular localisation of the SERS nanotags. Targeting systems were developed and in future work their localisation within organelles will be confirmed by the application of advanced chemometric methods

Surface enhanced Raman spectroscopy (SERS) : potential applications for disease detection and treatment

The implementation of Raman and surface enhanced Raman spectroscopy (SERS) for the detection of disease has increased in recent years. The reasons for their increased implementation have often been attributed to their well-known advantages, including the production of narrow spectral bands, which are characteristic of the molecular components present, their non-destructive method of analysis and the sensitivity and specificity which they can confer. This review analyses a range of diseases which can be detected by Raman or SERS, particularly those in vitro, ex vivo and in vivo. The sophistication of the investigated systems varied widely but the suitability of Raman and SERS for medical diagnostics and future implementation in a clinical environment is clearly demonstrated

3D optical imaging of multiple SERS nanotags in cells

Recent studies have clearly demonstrated that Raman and surface enhanced Raman scattering (SERS) spectroscopies are information rich, non-destructive techniques for the monitoring of subtle intracellular changes. However, despite the demonstrated and sophisticated applications of these techniques in cell studies there still remains a lack of accompanying 3D images. Herein we demonstrate for the first time combined 3D Raman and SERS imaging for the simultaneous confirmation of the cellular inclusion and multiple component detection of SERS nanotags. We also report on the 3D elucidation of the cell nuclei by multivariate analysis methods. Imaging in 3D will be critical to understanding architectural changes between diseased and healthy cells, and tissues. It will also provide non-destructive definitive proof of cellular uptake whilst simultaneously confirming targeting of SERS nanotags to their intended destinations

Confocal SERS Mapping of Glycan Expression for the Identification of Cancerous Cells

Lectin-functionalized silver nanoparticles have been successfully designed for use as molecular imaging agents to investigate carbohydrate–lectin interactions at the surface of mammalian cells, using surface-enhanced Raman scattering (SERS). Carbohydrate-lectin interactions are key to many cellular processes and are responsible for controlling an array of cellular interactions. In this study, lectin-functionalized silver nanoparticles were used to detect the expression of carbohydrate species at the cellular interface. The carbohydrate–lectin interactions were demonstrated using three different lectin species for three distinct cell types. Due to the known difference between the expressions of glycans in cancerous versus noncancerous cells of the same origin, this approach has been expanded to study both cancerous and noncancerous prostate cells. This has been achieved via confocal SERS mapping of the expression of the key glycan, sialic acid, on the surface of each of these cell types. In achieving such discrimination, a novel method has been created by which glycan expression can be reproducibly monitored. Comparative studies were performed using both fluorescence and SERS. SERS provided an increased discrimination over fluorescence when analyzing cell subsets to discriminate between cancerous and noncancerous cells. The success of this method means that it could be used to complement the current gold standard histopathological techniques

Dataset for Ultra-low background Raman sensing using a negative-curvature fibre and no distal optics

Collected data for the results presented in "Ultra-low background Raman sensing using a negative-curvature fibre and no distal optics" demonstrating the use of a single hollow core negative curvature fibre for Raman and surface enhanced Raman spectroscopy sensing. This new optical fibre shows a 1000x background reduction compared to conventional fibres while maintaining the same collection efficiency. The dataset includes Raman and SERS spectra from different samples and attenuation data for a range of wavelengths for the negative curvature fibre