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Intracellular pH (pH i ) homeostasis is intertwined with a myriad of normal cellular behaviors as well as pathological processes. As such, small molecule probes for the measurement of pH i are invaluable tools for chemical biology, facilitating the study of the role of pH in cellular function and disease. The field of small molecule pH i sensors has traditionally been dominated with probes based on fluorescent scaffolds. In this study, a series of low molecular weight (<260) oligoyne compounds have been developed which exhibit pH sensitive alkyne stretching frequencies (ν alkyne ) in Raman spectroscopy. The modular design of the compounds enabled tuneability of their pK a (H) through simple structural modification, such that continuous pH sensitivity is achieved over the range 2-10. Alkyne stretching bands reside in the 'cell-silent' region of the Raman spectrum (1800-2600 cm -1 ) and are readily detectable in a cellular environment with subcellular spatial resolution. This enabled the application of a pH sensitive oligoyne compound to the ratiometric sensing of pH i in prostate cancer (PC3) cells in response to drug treatment. We propose that probes based on Alkyne Tag Raman Imaging offer an entirely new platform for the sensing of pH i , complementary to fluorescence microscopy.

The ability to probe through barriers and tissue non-invasively is an urgent unmet need in both the security and biomedical imaging fields. Surface enhanced Raman spectroscopy (SERS) has been shown to yield superior enhancement in signal over conventional Raman techniques. Furthermore, by utilising a resonant Raman reporter to produce surface enhanced resonance Raman spectroscopy (SERRS), even greater enhancement in chemical signal can be generated. Here we show the benefit of using red-shifted chalcogenpyrylium based Raman reporters for probing through large thicknesses of plastic and tissue barriers using a conventional Raman instrument. In addition, the benefit of using a resonant Raman reporter for superior levels of through barrier detection is demonstrated, and we aim to show the advantage of using resonant nanotags in combination with conventional Raman spectroscopy to probe through plastic and tissue barriers. Raman signals were collected from SERRS active nanotags through plastic thicknesses of up to 20 mm, as well as the detection of the same SERRS nanotags through up to 10 mm of tissue sections using a handheld conventional Raman spectrometer. The ability to detect SERRS-active nanotags taken up into ex vivo tumour models known as multicellular tumour spheroids (MTS), through depths of 5 mm of tissue is also shown. The advantages of applying multivariate analysis for through barrier detection when discriminating analytes with similar spectral features as the barrier is also clearly demonstrated. To the best of our knowledge, this is the first report of the assessment of the maximum level of through barrier detection using a conventional handheld Raman instrument for SERS applications as well as demonstration of the power of resonant nanotags for probing through barriers using conventional Raman spectroscopy.

Resonant chalcogenpyrylium nanotags demonstrate an exceptional surface enhanced Raman scattering (SERS) performance for use in SORS applications. Using surface enhanced spatially offset Raman spectroscopy (SESORS), nanotags modified with a chalcogenpyrylium dye were observed at concentrations as low as 1 pM through 5 mm of tissue. Calculated limits of detection suggest that these SERS nanotags can be detected at 104 fM using surface enhanced spatially offset resonance Raman scattering (SESORRS) demonstrating their potential for in vivo applications.

De novo lipid synthesis is upregulated in cancer cells and inhibiting these pathways has displayed anti-tumour activity. Here we use Raman spectroscopy, focusing solely on high wavenumber spectra, to detect changes in lipid composition in single cells in response to drugs targeting de novo lipid synthesis. Unexpectedly, the beta-blocker propranolol showed selectively towards cancerous PC3 compared to non-cancerous PNT2 prostate cells, demonstrating the potential of this approach to identify new anti-cancer drug leads. A unique and simple ratiometric approach for intracellular lipid investigation is reported using statistical analysis to create phenotypic 'barcodes', a globally applicable strategy for Raman drug-cell studies. High wavenumber spectral analysis is compatible with low cost glass substrates, easily translatable into the cytological work stream. The analytical strength of this technique could have a significant impact on cancer treatment through vastly improved understanding of cancer cell metabolism, and thus guide drug design and enhance personalised medicine strategies.

Through utilizing the depth penetration capabilities of SESORS, multiplexed imaging and classification of three singleplex nanotags and a triplex of nanotags within breast cancer tumour models is reported for the first time through depths of 10 mm using a handheld SORS instrument.

In order to improve patient survival and reduce the amount of unnecessary and traumatic biopsies, non-invasive detection of cancerous tumours is of imperative and urgent need. Multicellular tumour spheroids (MTS) can be used as an ex vivo cancer tumour model, to model in vivo nanoparticle (NP) uptake by the enhanced permeability and retention (EPR) effect. Surface enhanced spatially offset Raman spectroscopy (SESORS) combines both surface enhanced Raman spectroscopy (SERS) and spatially offset Raman spectroscopy (SORS) to yield enhanced Raman signals at much greater sub-surface levels. By utilizing a reporter that has an electronic transition in resonance with the laser frequency, surface enhanced resonance Raman scattering (SERRS) yields even greater enhancement in Raman signal. Using a handheld SORS spectrometer with back scattering optics, we demonstrate the detection of live breast cancer 3D MTS containing SERRS active NPs through 15 mm of porcine tissue. False color 2D heat intensity maps were used to determine tumour model location. In addition, we demonstrate the tracking of SERRS-active NPs through porcine tissue to depths of up to 25 mm. This unprecedented performance is due to the use of red-shifted chalcogenpyrylium-based Raman reporters to demonstrate the novel technique of surface enhanced spatially offset resonance Raman spectroscopy (SESORRS) for the first time. Our results demonstrate a significant step forward in the ability to detect vibrational fingerprints from a tumour model at depth through tissue. Such an approach offers significant promise for the translation of NPs into clinical applications for non-invasive disease diagnostics based on this new chemical principle of measurement.

Conventional two dimensional (2D) monolayer cell culture has been considered the 'gold standard' technique for in vitro cellular experiments. However, the need for a model that better mimics the three dimensional (3D) architecture of tissue in vivo has led to the development of Multicellular Tumour Spheroids (MTS) as a 3D tissue culture model. To some extent MTS mimic the environment of in vivo tumours where, for example, oxygen and nutrient gradients develop, protein expression changes and cells form a spherical structure with regions of proliferation, senescence and necrosis. This review focuses on the development of techniques for chemical analysis of MTS as a tool for understanding in vivo tumours and a platform for more effective drug and therapy discovery. While traditional monolayer techniques can be translated to 3D models, these often fail to provide the desired spatial resolution and z-penetration for live cell imaging. More recently developed techniques for overcoming these problems will be discussed with particular reference to advances in instrument technology for achieving the increased spatial resolution and imaging depth required.

Intracellular redox potential is a highly regulated cellular characteristic and is critically involved in maintaining cellular health and function. The dysregulation of redox potential can result in the initiation and progression of numerous diseases. Redox potential is determined by the balance of oxidants and reductants in the cell and also by pH. For this reason a technique for quantitative measurement of intracellular redox potential and pH is highly desirable. In this paper we demonstrate how surface enhanced Raman scattering (SERS) nanosensors can be used for multiplexed measurement of both pH and redox potential in live single cells.

The intracellular pH plays an important role in various cellular processes. In this work, we describe a method for monitoring of the intracellular pH in endothelial cells by using surface enhanced Raman spectroscopy (SERS) and 4-mercaptobenzoic acid (MBA) anchored to gold nanoparticles as pH-sensitive probes. Using the Raman microimaging technique, we analysed changes in intracellular pH induced by buffers with acid or alkaline pH, as well as in endothelial inflammation induced by tumour necrosis factor-α (TNFα). The targeted nanosensor enabled spatial pH measurements revealing distinct changes of the intracellular pH in endosomal compartments of the endothelium. Altogether, SERS-based analysis of intracellular pH proves to be a promising technique for a better understanding of intracellular pH regulation in various subcellular compartments.

Solid-state (13)C magic-angle spinning (MAS) NMR spectroscopy is used to investigate the structure of the Cu(II)-based metal-organic frameworks (MOFs), HKUST-1 and STAM-1, and the structural changes occurring within these MOFs upon activation (dehydration). NMR spectroscopy is an attractive technique for the investigation of these materials, owing to its high sensitivity to local structure, without any requirement for longer-range order. However, interactions between nuclei and unpaired electrons in paramagnetic systems (e.g., Cu(II)-based MOFs) pose a considerable challenge, not only for spectral acquisition, but also in the assignment and interpretation of the spectral resonances. Here, we exploit the rapid T(1) relaxation of these materials to obtain (13)C NMR spectra using a spin-echo pulse sequence at natural abundance levels, and employ frequency-stepped acquisition to ensure uniform excitation of resonances over a wide frequency range. We then utilise selective (13)C isotopic labelling of the organic linker molecules to enable an unambiguous assignment of NMR spectra of both MOFs for the first time. We show that the monomethylated linker can be recovered from STAM-1 intact, demonstrating not only the interesting use of this MOF as a protecting group, but also the ability (for both STAM-1 and HKUST-1) to recover isotopically-enriched linkers, thereby reducing significantly the overall cost of the approach.