Label-free molecular imaging and discrimination of stem cells by Raman micro-spectroscopy

This thesis is focused on the development of Raman micro-spectroscopy for label free imaging and discrimination of stem cells. The thesis is divided into six chapters. Chapter 1 gives an overview of the existing techniques used for molecular analysis of cells, with emphasis on methods that allow non-invasive label-free imaging. A literature review of the main relevant applications of Raman micro-spectroscopy for imaging cells was also included. Chapter 2 discusses the basic theoretical principles of Raman scattering and design of Raman micro-spectrometers. The practical aspects related to the design of an optimised Raman micro-spectrometer are presented in Chapter 3 along with experimental characterisation of its performance. The chapter concludes with examples of Raman spectral maps of endothelial cells. Chapter 4 and Chapter 5 present experimental results obtained by Raman micro-spectroscopy for molecular analysis of live neural and mesenchymal stem cells. In these investigations Raman spectroscopy was used to identify, image and quantify spectral markers for label-free discrimination between glial cells and their neural progeny. The potential of Raman micro-spectroscopy to measure timecourse molecular changes of individual bone nodules was demonstrated in Chapter 5. Future work and final conclusion are discussed in Chapter six of this thesis

Value vs. Growth: un nou paradigma?

Treballs Finals del Grau de d'Administració i Direcció d'Empreses, Facultat d'Economia i Empresa, Universitat de Barcelona, Curs: 2022-2023, Tutor: Bàrbara Llacay PintatUn dels debats que més controvèrsia ha generat al llarg del temps, dins del món de les inversions de renda variable és la distinció entre la inversió en valor coneguda com value i la inversió de creixement growth. Aquestes dues estratègies d'inversió són l'objecte d'estudi d'aquest treball de fi de grau, on es pretén donar una resposta al dilema de quina estratègia aporta rendiments superiors i a què es deu aquesta diferència entre les rendibilitats obtingudes. Partint d'estudis previs i d'una gran quantitat de literatura acadèmica publicada anteriorment, es revisen els resultats assolits pels investigadors en el passat i les seves conclusions. Mitjançant una metodologia d'estudi avalada pels experts en el tema, s'obtenen resultats que ens permeten veure l'evolució de les diferents estratègies en els últims catorze anys, on ens trobem una situació que ha canviat respecte a tota l'experiència prèvia

Label-free molecular imaging and discrimination of stem cells by Raman micro-spectroscopy

This thesis is focused on the development of Raman micro-spectroscopy for label free imaging and discrimination of stem cells. The thesis is divided into six chapters. Chapter 1 gives an overview of the existing techniques used for molecular analysis of cells, with emphasis on methods that allow non-invasive label-free imaging. A literature review of the main relevant applications of Raman micro-spectroscopy for imaging cells was also included. Chapter 2 discusses the basic theoretical principles of Raman scattering and design of Raman micro-spectrometers. The practical aspects related to the design of an optimised Raman micro-spectrometer are presented in Chapter 3 along with experimental characterisation of its performance. The chapter concludes with examples of Raman spectral maps of endothelial cells. Chapter 4 and Chapter 5 present experimental results obtained by Raman micro-spectroscopy for molecular analysis of live neural and mesenchymal stem cells. In these investigations Raman spectroscopy was used to identify, image and quantify spectral markers for label-free discrimination between glial cells and their neural progeny. The potential of Raman micro-spectroscopy to measure timecourse molecular changes of individual bone nodules was demonstrated in Chapter 5. Future work and final conclusion are discussed in Chapter six of this thesis

Monitoring the mineralisation of bone nodules in vitro by space- and time-resolved Raman micro-spectroscopy.

Raman microscopy was used as a label-free method to study the mineralisation of bone nodules formed by mesenchymal stem cells cultured in osteogenic medium in vitro. Monitoring individual bone nodules over 28 days revealed temporal and spatial changes in the crystalline phase of the hydroxyapatite components of the nodules

Applications of Raman micro-spectroscopy to stem cell technology: label-free molecular discrimination and monitoring cell differentiation.

Stem cell therapy is widely acknowledged as a key medical technology of the 21st century which may provide treatments for many currently incurable diseases. These cells have an enormous potential for cell replacement therapies to cure diseases such as Parkinson's disease, diabetes and cardiovascular disorders, as well as in tissue engineering as a reliable cell source for providing grafts to replace and repair diseased tissues. Nevertheless, the progress in this field has been difficult in part because of lack of techniques that can measure non-invasively the molecular properties of cells. Such repeated measurements can be used to evaluate the culture conditions during differentiation, cell quality and phenotype heterogeneity of stem cell progeny. Raman spectroscopy is an optical technique based on inelastic scattering of laser photons by molecular vibrations of cellular molecules and can be used to provide chemical fingerprints of cells or organelles without fixation, lysis or use of labels and other contrast enhancing chemicals. Because differentiated cells are specialized to perform specific functions, these cells produce specific biochemicals that can be detected by Raman micro-spectroscopy. This mini-review paper describes applications of Raman micro-scpectroscopy to measure moleculare properties of stem cells during differentiation in-vitro. The paper focuses on time- and spatially-resolved Raman spectral measurements that allow repeated investigation of live stem cells in-vitro

Cytoplasmic RNA in undifferentiated neural stem cells: a potential label-free Raman spectral marker for assessing the undifferentiated status

Raman microspectroscopy (rms) was used to identify, image, and quantify potential molecular markers for label-free monitoring the differentiation status of live neural stem cells (NSCs) in vitro. Label-free noninvasive techniques for characterization of NCSs in vitro are needed as they can be developed for real-time monitoring of live cells. Principal component analysis (PCA) and linear discriminant analysis (LDA) models based on Raman spectra of undifferentiated NSCs and NSC-derived glial cells enabled discrimination of NSCs with 89.4% sensitivity and 96.4% specificity. The differences between Raman spectra of NSCs and glial cells indicated that the discrimination of the NSCs was based on higher concentration of nucleic acids in NSCs. Spectral images corresponding to Raman bands assigned to nucleic acids for individual NSCs and glial cells were compared with fluorescence staining of cell nuclei and cytoplasm to show that the origin of the spectral differences were related to cytoplasmic RNA. On the basis of calibration models, the concentration of the RNA was quantified and mapped in individual cells at a resolution of ~700 nm. The spectral maps revealed cytoplasmic regions with concentrations of RNA as high as 4 mg/mL for NSCs while the RNA concentration in the cytoplasm of the glial cells was below the detection limit of our instrument (~1 mg/mL). In the light of recent reports describing the importance of the RNAs in stem cell populations, we propose that the observed high concentration of cytoplasmic RNAs in NSCs compared to glial cells is related to the repressed translation of mRNAs, higher concentrations of large noncoding RNAs in the cytoplasm as well as their lower cytoplasm volume. While this study demonstrates the potential of using rms for label-free assessment of live NSCs in vitro, further studies are required to establish the exact origin of the increased contribution of the cytoplasmic RNA

Analysis of interaction between the apicomplexan protozoan Toxoplasma gondii and host cells using label-free Raman Spectroscopy

Label-free imaging using Raman micro-spectroscopy (RMS) was used to characterize the spatio-temporal molecular changes of T. gondii tachyzoites and their host cell microenvironment. Raman spectral maps were recorded from isolated T. gondii tachyzoites and T. gondii-infected human retinal cells at 6 hr, 24 hr and 48 hr post-infection. Principal component analysis (PCA) of the Raman spectra of paraformaldehyde-fixed infected cells indicated a significant increase in the amount of lipids and proteins in the T. gondii tachyzoites as the infection progresses within host cells. These results were confirmed by experiments carried out on live T. gondii-infected cells and were correlated with an increase in the concentration of proteins and lipids required for the replication of this intracellular pathogen. These findings demonstrate the potential of RMS to characterize time- and spatially-dependent molecular interactions between intracellular pathogens and the host cells. Such information may be useful for discovery of pharmacological targets or screening compounds with potential neuro-protective activity for eminent effects of changes in brain infection control practices

Real-time investigation of skin blood flow changes induced by topical capsaicin

Capsaicin induces a localized inflammatory process known as neurogenic inflammation upon its topical administration on the skin, due to the release of various neuropeptides from the cutaneous sensory nerve endings. In this study, we investigated real-time skin blood flow changes that occur in neurogenic inflammation induced by topical capsaicin by means of in vivo reflectance confocal microscopy. 27 healthy subjects (15 women and 12 men, mean age ± Standard Deviation: 22.62±4.47) were administered topical capsaicin solution (Capsaicin group) or immersion oil (Control group) on the dorsal side of their non-dominant hand. At different time intervals during administration (0, 10, 25, and 40 minutes), cutaneous blood flow was evaluated using reflectance confocal microscopy and compared between the two groups. Blood flow values were higher during topical capsaicin, with significant increase after 25 (P=0.0160, Dunn’s multiple comparisons test) and 40 minutes (P=0.0132, Dunn’s multiple comparisons test) after its administration when compared with the initial 0 min value. Furthermore, the differences in the blood flow changes between the two groups were significant at 25 min (P=0.0182, Dunn’s multiple comparisons test) and 40 min (P=0.0296, Dunn’s multiple comparisons test) after capsaicin administration. Reflectance confocal microscopy allows in vivo, real-time evaluation of cutaneous blood flow changes within the capsaicin-induced inflammation, and this method might serve as a research model to test neurovascular reactivity. </p

Proceedings of Abstracts, School of Physics, Engineering and Computer Science Research Conference 2022

© 2022 The Author(s). This is an open-access work distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. For further details please see https://creativecommons.org/licenses/by/4.0/. Plenary by Prof. Timothy Foat, ‘Indoor dispersion at Dstl and its recent application to COVID-19 transmission’ is © Crown copyright (2022), Dstl. This material is licensed under the terms of the Open Government Licence except where otherwise stated. To view this licence, visit http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3 or write to the Information Policy Team, The National Archives, Kew, London TW9 4DU, or email: [email protected] present proceedings record the abstracts submitted and accepted for presentation at SPECS 2022, the second edition of the School of Physics, Engineering and Computer Science Research Conference that took place online, the 12th April 2022