Advanced multimodal methods in biomedicine : Raman spectroscopy and digital holographic microscopy

Moving towards label-free technologies is essential for many clinical and research applications. Raman spectroscopy is a powerful tool in the field of biomedicine for label-free cell characterisation and disease diagnosis, owing to its high chemical specificity. However, Raman scattering is a relatively weak process and can require long acquisition times, thus hampering its integration to clinical technologies. Multimodal analysis is currently pushing the boundaries in biomedicine, obtaining more information than would be possible using a single mode and overcoming any limitations specific to a single technique. Digital holographic microscopy (DHM) is a rapid and label-free quantitative phase imaging modality, providing complementary information to Raman spectroscopy, and is thus an ideal candidate for combination in a multimodal system. Firstly, this thesis explores the use of wavelength modulated Raman spectroscopy (WMRS), for the classification of immune cell subsets. Following this a multimodal approach, combining Raman spectroscopy and DHM, is demonstrated, where each technique is considered individually and in combination. The complementary modalities provide a wealth of information (both chemical and morphological) for cell characterisation, which is a step towards achieving a label-free technology for the identification of human immune cells. The suitability of WMRS to discriminate between closely related neuronal cell types is also explored. Furthermore optical spectroscopic techniques are useful for the analysis of food and beverages. The use of Raman and fluorescence spectroscopy to successfully discriminate between various whisky and extra-virgin olive oil brands is demonstrated, which may aid the detection of counterfeit or adulterated samples. The use of a compact Raman device is utilised, demonstrating the potential for in-field analysis. Finally, monodisperse and highly spherical nanoparticles are synthesised. A short study demonstrates the potential for these nanoparticles to benefit the techniques of surface enhanced Raman spectroscopy and optical trapping, by way of minimising variability

Optical spectroscopic analysis for the discrimination of Extra-Virgin Olive Oil

We thank the UK Engineering and Physical Sciences Research Council and the European Union FAMOS project (FP7 ICT, 317744) for funding.We demonstrate the ability to discriminate between five brands of commercially available extra-virgin olive oil (EVOO) using Raman spectroscopy or fluorescence spectroscopy. Data was taken on both a ‘bulk optics’ free space system and on a compact handheld device, each capable of taking both Raman and fluorescence data. With the compact Raman device we achieved an average sensitivity and specificity of 98.4% and 99.6% for discrimination, respectively. Our approach illustrates that both Raman and fluorescence spectroscopy can be used for portable discrimination of EVOOs. This technique may enable detection of EVOO that has undergone counterfeiting or adulteration. The main challenge with this technique is that oxidation of EVOO causes a shift in the Raman signal over time. It would therefore be necessary to retrain the database regularly.We demonstrate preliminary data to address this issue, which may enable successful discrimination over time. We show that by discarding the first principal component, which contains information on the variations due to oxidation, we can improve discrimination efficiency.PostprintPeer reviewe

Multimodal discrimination of immune cells using a combination of Raman spectroscopy and digital holographic microscopy

This work was supported by the UK Engineering and Physical Sciences Research Council under grant EP/J01771X/1, A European Union FAMOS project (FP7 ICT, 317744), and the ’BRAINS’ 600th anniversary appeal, and Dr. E. Killick. We would also like to thank The RS Macdonald Charitable Trust for funding support. KD acknowledges support of a Royal Society Leverhulme Trust Senior Fellowship. This work was also supported by the PreDiCT-TB consortium [IMI Joint undertaking grant agreement number 115337, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution (www.imi.europa.eu)]The ability to identify and characterise individual cells of the immune system under label-free conditions would be a significant advantage in biomedical and clinical studies where untouched and unmodified cells are required. We present a multi-modal system capable of simultaneously acquiring both single point Raman spectra and digital holographic images of single cells. We use this combined approach to identify and discriminate between immune cell populations CD4+ T cells, B cells and monocytes. We investigate several approaches to interpret the phase images including signal intensity histograms and texture analysis. Both modalities are independently able to discriminate between cell subsets and dual-modality may therefore be used a means for validation. We demonstrate here sensitivities achieved in the range of 86.8% to 100%, and specificities in the range of 85.4% to 100%. Additionally each modality provides information not available from the other providing both a molecular and a morphological signature of each cell.Publisher PDFPeer reviewe

Optical trapping of ultrasmooth gold nanoparticles in liquid and air

This work is supported by the UK Engineering and Physical Sciences Research Council for funding through Grant (Nos. EP/P030017/1, EP/J01171X/1, EP/K016342/1, and EP/M506631/1) and the Leverhulme Trust (No. RPG-2015-042).Optical manipulation of gold nanoparticles has emerged as an exciting avenue for studies in nanothermometry, cell poration, optical binding, and optomechanics. However, conventional gold nanoparticles usually depart from a spherical shape, making such studies less controlled and leading to potential artifacts in trapping behavior. We synthesize ultrasmooth gold nanoparticles, which offer improved circularity and monodispersity. In this article, we demonstrate the advantages of such nanoparticles through a series of optical trapping experiments in both liquid and air. Compared to their conventional counterparts, ultrasmooth gold nanoparticles exhibit up to a two-fold and ten-fold reduction in standard deviation for trap stiffness measurements in liquid and air, respectively. They will enable more controlled studies of plasmon mediated light-matter interactions.Publisher PDFPeer reviewe

The use of Wavelength Modulated Raman Spectroscopy in label-free identification of T lymphocyte subsets, Natural Killer cells and Dendritic cells

This work was funded by a Cancer Research United Kingdom, Engineering and Physical Sciences Research Council (grant EP/J01771X/1), Medical Research Council and Department of Health England Imaging Programme (MC, MM KD), and by A European Union FAMOS project (FP7 ICT, 317744) to KD.Determining the identity of cells of the immune system usually involves destructive fixation and chemical staining, or labeling with fluorescently labeled antibodies recognising specific cell surface markers. Completely label-free identification would be a significant advantage in conditions where untouched cells are a priority. We demonstrate here the use of Wavelength Modulated Raman Spectroscopy, to achieve label-free identification of purified, unfixed and untouched populations of major immune cell subsets isolated from healthy human donors. Using this technique we have been able to distinguish between CD4+ T lymphocytes, CD8+ T lymphocytes and CD56+ Natural Killer cells at specificities of up to 96%. Additionally, we have been able to distinguish between CD303+ plasmacytoid and CD1c+ myeloid dendritic cell subsets, the key initiator and regulatory cells of many immune responses. This demonstrates the ability to identify unperturbed cells of the immune system, and opens novel opportunities to analyse immunological systems and to develop fully label-free diagnostic technologies.Publisher PDFPeer reviewe

Cluster plots showing the first three principal components for each cell subset isolated from three individuals, with their corresponding first three loadings shown on the right.

<p>(a) CD4⁺, CD8⁺ T cells and CD56⁺ NK cells. (b) CD4⁺ and CD8⁺ T cells. (c) CD4⁺ T cells and CD56⁺ NK cells. (d) CD8⁺ T cells and CD56⁺ NK cells. (3D rotating views of these plots are available to view in the supplementary information).</p

Flow cytometric and functional characterisation of purified cell subsets.

<p>(a) CD4 staining of isolated CD4⁺ T cells. (b) IL-2 ELISA of CD4⁺ T cells stimulated with or without anti-CD3/CD28 beads. (c) CD8 staining or isolated CD8⁺ T cells. (d) IFNγ ELISPOT assay of PBMC and purified CD8⁺ T cells incubated with and without EBV derived peptide AVFDRKSDAK. (e) CD56 staining of isolated NK cells. (f) NK cell degranulation assay—CD107a staining of NK cells incubated without (left panel) or with (right panel) MHC class I deficient. 221 cells at a 10:1 effector to target ratio. (g) CD303 staining of isolated pDC. (h) CD1c staining of isolated mDC. The x-axis in each flow cytometry plot indicates fluorescent intensity. The left hand peak in each flow cytometry plot indicates control staining with an irrelevant antibody. Representative white-light microscopy images of each of the purified cell populations used in Raman spectroscopy experiments are also shown.</p

Cluster plot showing the first three principal components for the analysis of pDC and mDC subsets.

<p>The corresponding first three loadings are shown on the right.</p

Confusion matrix for CD4⁺, CD8⁺ and CD56⁺ cell subsets.

<p>The majority of numbers occur on the diagonal indicating good discrimination between the three cells subsets.</p><p>Confusion matrix for CD4⁺, CD8⁺ and CD56⁺ cell subsets.</p