Christopher D. Syme

Molecular structures of viruses from Raman optical activity

A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity to chirality, ROA is an incisive probe of their aqueous solution structures at the molecular level. Protein ROA bands are especially prominent from which, as we have shown by comparison with the ROA spectra of proteins with known structures and by using a pattern recognition program, the folds of the major coat protein subunits may be deduced. Information about amino acid side-chain conformations, exemplified here by the determination of the sign and magnitude of the torsion angle chi(2,1) for tryptophan in fd, may also sometimes be obtained. By subtracting the ROA spectrum of the empty protein capsid (top component) of cowpea mosaic virus from those of the intact middle and bottom-upper components separated by means of a caesium chloride density gradient, the ROA spectrum of the viral RNA was obtained, which revealed that the RNA takes up an A-type single-stranded helical conformation and that the RNA conformations in the middle and bottom-upper components are very similar. This information is not available from the X-ray crystal structure of cowpea mosaic virus since no nucleic acid is visible.

Monitoring the Uptake and Redistribution of Metal Nanoparticles during Cell Culture Using Surface-Enhanced Raman Scattering Spectroscopy

We describe the uptake of silver nanoparticles by CHO (Chinese hamster ovary) cells and their subsequent fate as a result of cell division during culture, as monitored by surface-enhanced Raman scattering (SERS) spectroscopy. Mapping of populations of cells containing both labeled and native nanoparticles by SERS spectroscopy imaging provided a quantitative method by which the number of intracellular nanoparticles could be monitored. Initially, for a given amount of nanoparticles, the relationship between the number taken up into the cell and the time of incubation was explored. Subsequently, the redistribution of intracellular nanoparticles upon multiple rounds of cell division was investigated. Intracellular SERS signatures remained detectable in the cells for up to four generations, although the abundance and intensity of the signals declined rapidly as nanoparticles were shared with daughter cells. The intensity of the SERS signal was dependent both on stability of the label and their abundance (nanoparticle aggregation increases the extent of the SERS enhancement). The data show that while the labeled nanoparticles remain stable for prolonged periods, during cell division, the changes in signal could be attributed both to a decrease in abundance and distribution (and hence aggregation).

Solution structures of potato virus X and narcissus mosaic virus from Raman optical activity

Potato virus X (PVX) and narcissus mosaic virus (NMV) were studied using vibrational Raman optical activity (ROA) in order to obtain new information on the structures of their coat protein subunits. The ROA spectra of the two intact virions are very similar to each other and similar to that of tobacco mosaic virus (TMV) studied previously, being dominated by signals characteristic of proteins with helix bundle folds. In particular, PVX and NMV show strong positive ROA bands at approximately 1340 cm(-1) assigned to hydrated alpha-helix and perhaps originating in surface exposed helical residues, together with less strong positive ROA intensity in the range approximately 1297-1312 cm(-1) assigned to alpha-helix in a more hydrophobic environment and perhaps originating in residues at helix-helix interfaces. The positive approximately 1340 cm(-1) ROA band of TMV is less intense than those of PVX and NMV, suggesting that TMV contains less hydrated alpha-helix. Small differences in other spectral regions reflect differences in some loop, turn and side-chain compositions and conformations among the three viruses. A pattern recognition program based on principal component analysis of ROA spectra indicates that the coat protein subunit folds of PVX and NMV may be very similar to each other and similar to that of TMV. These results suggest that PVX and NMV may have coat protein subunit structures based on folds similar to the TMV helix bundle and hence that the helical architecture of the PVX and NMV particles may be similar to that of TMV but with different structural parameters.

Quantitative Characterization of Individual Microdroplets using Surface-Enhanced Resonance Raman Scattering Spectroscopy

Surface-enhanced resonance Raman scattering (SERRS) spectroscopy is a highly sensitive optical technique capable of detecting multiple analytes rapidly and simultaneously. There is significant interest in SERRS detection in micro- and nanotechnologies, as it can be used to detect extremely low analyte concentrations in small volumes of fluids, particularly in microfluidic systems. There is also rapidly growing interest in the field of microdroplets, which promises to offer the analyst many potential advantages over existing technologies for both design and control of microfluidic assays. While there have been rapid advances in both fields in recent years, the literature on SERRS-based detection of individual microdroplets remains lacking. In this paper, we demonstrate the ability to quantitatively detect multiple variable analyte concentrations from within individual microdroplets in real time using SERRS spectroscopy. We also demonstrate the use of a programmable pump control algorithm to generate concentration gradients across a chain of droplets.

Investigation of the stability of labelled nanoparticles for SE(R)RS measurements in cells

We explore the long-term stability of two different classes of labelled nanoparticles as intracellular SE(R)RS probes. Whilst chemisorbed labels gave stable responses inside cells for extended periods of time, signals from physisorbed labels could only be measured for short periods of time. These results help inform strategies for cellular imaging using vibrational spectroscopies.

Surface-enhanced coherent anti-Stokes Raman imaging of lipids

This work describes in detail a wide-field surface-enhanced coherent anti-Stokes Raman scattering (CARS) microscope, which enables enhanced detection of sample structures in close proximity (∼100  nm) of the substrate interface. Unlike conventional CARS microscopy, where the sample is illuminated with freely propagating light, the current implementation uses evanescent fields to drive Raman coherences across the entire object plane. By coupling the pump and Stokes excitation beams to the surface plasmon-polariton mode at the interface of a 30 nm thick gold film, we obtained strong CARS signals from cholesteryl oleate droplets adhered to the surface. The surface-enhanced CARS imaging system visualizes lipid structures with vibrational selectivity using illumination doses per unit area that are more than four orders of magnitude lower than in point-scanning CARS microscopy.

Intracellular multiplex detection and imaging of stable chemisorbed labels by SERS spectroscopy

SERS spectroscopy is currently gaining wider acceptance in biological research due to its ability to obtain signals from very low quantities of material, and to obtain information from within live cells. SERS spectroscopy yields very narrow bands (10-100 times narrower than typical fluorescence bands) and spectra suffer from minimal interference from aqueous media, making SERS spectroscopy ideal for multiplex detection of intracellular components. Typically for sensing, nanoparticles are labelled with suitable sensing molecules such as a dye or thiol. Nanoparticle labelling involves two different types of interaction between the label and the enhancing surface, chemisorption and physisorption. The former is considerably stronger and more stable than the latter and hence chemisorbed labels are more appropriate for intracellular nanosensor design. In this paper, we demonstrate the difference in stability of both types of Raman label inside live cells over periods of time. Chinese hamster ovary (CHO) cells were infused with a mixture of differently labelled stable nanosensors and were imaged using SERS microspectroscopy. We also demonstrate the applicability of SERS mapping for high-throughput multiplex detection using micropatterned cell arrays.

Frustration of crystallisation by a liquid-crystal phase.

Frustration of crystallisation by locally favoured structures is critically important in linking the phenomena of supercooling, glass formation, and liquid-liquid transitions. Here we show that the putative liquid-liquid transition in n-butanol is in fact caused by geometric frustration associated with an isotropic to rippled lamellar liquid-crystal transition. Liquid-crystal phases are generally regarded as being “in between” the liquid and the crystalline state. In contrast, the liquid-crystal phase in supercooled n-butanol is found to inhibit transformation to the crystal. The observed frustrated phase is a template for similar ordering in other liquids and likely to play an important role in supercooling and liquid-liquid transitions in many other molecular liquids.

Characterization of individual microdroplets by SERRS spectroscopy

Raman spectroscopy and its various derivatives continue to offer the analyst fast, powerful, non-invasive and nondestructive means by which to identify multiple analytes simultaneously and in real time. By virtue of the huge enhancements possible in Raman scattering, generated by both surface enhancement and the resonance Raman effect, or when coupled with other techniques such as confocal microscopy, Raman spectroscopy is becoming more and more applicable to the types of assay being conducted in lab-on-a-chip applications, such as flow cytometry, cell patterning and trapping, and microarrays, all of which often involve the detection of extremely low quantities of analyte. Surface enhanced Raman scattering (SERS, or when coupled with the resonance Raman phenomenon, SERRS) spectroscopy has proven to be of particular use as a robust optical detection method in microfluidic environments. In this paper, we demonstrate the use of SERRS multiplex detection to quantitatively characterize individual microdroplets in a continuous stream whose contents are gradually varied using a bespoke pump control algorithm.