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

Endoscopic sensing of alveolar pH

Previously unobtainable measurements of alveolar pH were obtained using an endoscope-deployable optrode. The pH sensing was achieved using functionalized gold nanoshell sensors and surface enhanced Raman spectroscopy (SERS). The optrode consisted of an asymmetric dual-core optical fiber designed for spatially separating the optical pump delivery and signal collection, in order to circumvent the unwanted Raman signal generated within the fiber. Using this approach, we demonstrate a ~100-fold increase in SERS signal-to-fiber background ratio, and demonstrate multiple site pH sensing with a measurement accuracy of ± 0.07 pH units in the respiratory acini of an ex vivo ovine lung model. We also demonstrate that alveolar pH changes in response to ventilation

Universal surface-enhanced Raman tags : individual nanorods for measurements from the visible to the infrared (514 – 1064 nm)

Surface-enhanced Raman scattering (SERS) is a promising imaging modality for use in a variety of multiplexed tracking and sensing applications in biological environments. However, the uniform production of SERS nanoparticle tags with high yield and brightness still remains a significant challenge. Here, we describe an approach based on the controlled co-adsorption of multiple dye species onto gold nanorods to create tags that can be detected across a much wider range of excitation wavelengths (514 – 1064 nm) compared to conventional approaches that typically focus on a single wavelength. This was achieved without the added complexity of nanoparticle aggregation or growing surrounding metallic shells to further enhance the surface-enhanced resonance Raman scattering (SERRS) signal. Correlated Raman and scanning electron microscopy mapping measurements of individual tags were used to clearly demonstrate that strong and reproducible SERRS signals at high particle yields (>92 %) were readily achievable. The polyelectrolyte-wrapped nanorod-dye conjugates were also found to be highly stable as well as non-cytotoxic. To demonstrate the use of these universal tags for the multimodal optical imaging of biological specimens, confocal Raman and fluorescence maps of stained immune cells following nanoparticle uptake were acquired at several excitation wavelengths and compared with dark-field images. The ability to colocalize and track individual optically encoded nanoparticles across a wide range of wavelengths simultaneously will enable the use of SERS alongside other imaging techniques for the real-time monitoring of cell-nanoparticle interactions

Raman spectroscopy: techniques and applications in the life sciences

Raman spectroscopy is an increasingly popular technique in many areas including biology and medicine. It is based on Raman scattering, a phenomenon in which incident photons lose or gain energy via interactions with vibrating molecules in a sample. These energy shifts can be used to obtain information regarding molecular composition of the sample with very high accuracy. Applications of Raman spectroscopy in the life sciences have included quantification of biomolecules, hyperspectral molecular imaging of cells and tissue, medical diagnosis, and others. This review briefly presents the physical origin of Raman scattering explaining the key classical and quantum mechanical concepts. Variations of the Raman effect will also be considered, including resonance, coherent, and enhanced Raman scattering. We discuss the molecular origins of prominent bands often found in the Raman spectra of biological samples. Finally, we examine several variations of Raman spectroscopy techniques in practice, looking at their applications, strengths, and challenges. This review is intended to be a starting resource for scientists new to Raman spectroscopy, providing theoretical background and practical examples as the foundation for further study and exploration