Title: Raman microspectroscopy of living cells and biological tissues Author: Šárka Moudříková Department / Institute: Institute of Physics of Charles University Supervisor of the doctoral thesis: doc. RNDr. Peter Mojzeš, CSc., Institute of Physics of Charles University Abstract: Raman microscopy combines Raman spectroscopy with optical confocal microscopy and thus provides information on chemical composition of a sample with a µm3 resolution. In this thesis, Raman microscopy has been used to study microalgae-unicellular photosynthetic organisms that are greatly relevant for the Earth's environment as well as for biotechnological applications. Raman microscopy of photosynthetic organisms struggles with a highly intensive background of the spectra, which is formed by fluorescence of cellular photosynthetic apparatus. In this thesis, we have developed a fast and reliable photobleaching method that suppresses the unwanted background; this method has enabled us to study intracellular distribution of algal biomolecules such as proteins, starch, lipids and polyphosphate. We have investigated an evolution of these structures during a cell cycle of a model microalga Desmodesmus quadricauda. Next, we have developed a method for quantitative analysis of polyphosphate in a cellular culture of a microalga Chlorella...

Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

Ce travail de thèse est une approche originale de la compréhension des mécanismes physico-chimiques s'opérant à l'interface des restaurations céramo-céramiques par la microscopie confocale Raman. Il est scindé en trois parties. La première partie expose les caractéristiques d'une chape céramique à noyau zircone (Y-TZP) recouverte d'une céramique feldspathique. Les principaux facteurs responsables de la qualité de l'interface ont été recherchés par une revue de littérature. Le principe de la microscopie confocale Raman a été décrit. La seconde partie est la mise au point des méthodes d'analyse spectrale et d'imagerie Raman, en surface et en profondeur, sur matériaux isolés et à l'interface. La répartition des phases cristallines ou amorphes a été estimée de part et d'autre de cette dernière. La composition chimique élémentaire des composants et une ligne de balayage par traversée de l'interface ont été obtenues par spectroscopie dispersive en énergie (EDS). Le troisième chapitre explore les changements chimiques et morphologiques de l'interface sous différents procédés de fabrication : avec ou sans l'utilisation d'un Liner, avec ou sans utilisation d'une cuisson de régénération de l'Y-TZP. Les images Raman ont été traitées par la fonction d'analyse K-Means Cluster. Une cartographie de la distribution des éléments chimiques a été effectuée par EDS. / This thesis is an original approach to the understanding of the physical and chemical mechanisms operating at the interface of core-veneer all-ceramic restorations by confocal Raman microscopy. It is divided into three sections. The first part describes the characteristics of a zirconia core (Y-TZP) layered with feldspathic ceramic. The main factors affecting the interface have been investigated in the literature review. The principle of confocal Raman microscopy is described. The second part describes the development of spectral analysis and Raman imaging methods, on both the surface and at depth, on isolated materials and at the interface. The allocation of crystalline or amorphous phases was estimated around the interface. The elemental chemical analysis of the components and scanning line through the interface were obtained using energy dispersive X-ray spectroscopy (EDS). The third section explores the chemical and morphological changes of the interface under different manufacturing methods : with or without an optional liner material between the two components and with or without the use of a regeneration firing of the Y-TZP core. Raman images were processed by K-Means Cluster analysis function. The elemental distribution around the interface was estimated using EDS.

The effective diagnosis of leukemia subtypes requires the detection of multiple cell surface markers. Current methods of detection use mostly fluorophores, which are limited by their large spectral bandwidths, photobleaching, and incompatibility with histological stains used for morphological assessments. Antibody-conjugated Surface enhanced Raman scattering (SERS) nanoparticles is an alternative tool that overcomes these limitations. A current drawback of SERS is the lack of available tools to analyze the bioconjugation of antibodies to nanoparticles following EDC/sulfo-NHS cross-linking, which produces inconsistent results and determines the efficacy of SERS probe targeting. This study uses the flow cytometry approach to evaluate SERS particles by incorporating FITC and DyLight650 secondary antibodies. Flow cytometry was also used to assess targeting of particles to markers on LY10 cells and CLL cells and to detect SERS signals by inserting a 710 BP 10nm FWHM filter specific for MGITC.

The effective diagnosis of leukemia subtypes requires the detection of multiple cell surface markers. Current methods of detection use mostly fluorophores, which are limited by their large spectral bandwidths, photobleaching, and incompatibility with histological stains used for morphological assessments. Antibody-conjugated Surface enhanced Raman scattering (SERS) nanoparticles is an alternative tool that overcomes these limitations. A current drawback of SERS is the lack of available tools to analyze the bioconjugation of antibodies to nanoparticles following EDC/sulfo-NHS cross-linking, which produces inconsistent results and determines the efficacy of SERS probe targeting. This study uses the flow cytometry approach to evaluate SERS particles by incorporating FITC and DyLight650 secondary antibodies. Flow cytometry was also used to assess targeting of particles to markers on LY10 cells and CLL cells and to detect SERS signals by inserting a 710 BP 10nm FWHM filter specific for MGITC.

Les propriétés photoniques des tissus durs dentaires nous ont permis d’étudier l’email et la dentine a un niveau moléculaire (in vitro) en utilisant des techniques de microscopie optique non linéaires. La microscopie confocale Raman est technique d’imagine de haute résolution permettant d’analyse d’échantillon sans préparation spécifique ni marquage. Cette méthode nous a permis de reconstituer une cartographie de la réticulation du collagène et de la cristallinité au niveau de la jonction émail-dentine et cela avec une résolution spatiale non atteinte jusque-là. Cette analyse chimique de la jonction émail-dentine a permis de redéfinir la largeur de cette zone de transition. Cette largeur est nettement supérieure à celles proposées par les études précédentes. Par ailleurs, l’étude portant sur les changements de fluorescence intrinsèque entre les tissues dentaires sains et cariés suggèrent l’implication de la protoporphyrin IX et de la pentosidine dans l’expression de la fluorescence rouge des tissus cariés. La microscopie multiphotonique quant à elle nous a permis de détecter la lésion carieuse et de suivre son développement en utilisant la génération de seconde harmonique (SHG) et la fluorescence par excitation à deux photons (2PEF). Nos études ont démontré la validité du ratio SHG/2PEF comme paramètre fiable pour la détection de la lésion carieuse. Les études proposées par cette thèse montrent le potentiel des propriétés photoniques de l’émail et de la dentine en utilisant les microscopies Raman et multiphotoniques dans l’étude de ces tissus au niveau moléculaire. Cela offre de nouvelles perspectives en recherche et en applications cliniques. / Photonic properties of dental hard tissues allowed us to proceed to in vitro analysis of enamel and dentin on a molecular level. Confocal Raman microscopy has been used to produce a mapping of collagen cross-link and crystallinity of human dentin–enamel junction (DEJ) with a spatial resolution not achieved up to now. The method is a non-invasive, label-free and a high spatial resolution imaging technique. This chemical analysis of DEJ led us to redefine a wider width of this transition zone and advance our understanding of dental histology. A study on the intrinsic fluorescence changes of sound and carious tissues using conventional fluorescence microscopy suggests the involvement of protoporphyrin IX and pentosidine in the fluorescence red-shift observed in carious tissues. Multiphoton microscopy allowed to detect nonlinear optical signal changes during caries process using second harmonic generation (SHG) and two-photon excitation fluorescence (2PEF). Our studies led us to propose the ratio SHG/2PEF as valuable parameter to monitor caries lesion. Collectively, advances described in this thesis show the potential of photonic properties of enamel and dentin using Raman and multiphoton microcopies for molecular investigations on sound as much as on carious tissues. It opens new perspective in dental research and clinical applications.

Many freshwater, terrestrial or marine microalgae contain various microcrystalline inclusions that they use in their life cycle. However, the identification of the molecular composition of these inclusions via many physical or chemical methods is often very difficult and susceptible to many measurement errors. Therefore, the chemical composition of these microcrystals in many microalgae has not been determined at all or may be incorrect. One of the high precision methods capable of determining the composition of microcrystalline bodies within microalgae is Raman confocal microscopy. This very promising method of optical vibrational spectroscopy allows rapid and non-destructive molecular analysis of objects. Raman microscopy does not require chemical extraction, modification or other color marking or staining of the sample. Therefore, it can directly measure living cells at various stages of their natural development. The chemical composition of the sample is then characterized by its corresponding Raman vibrational spectrum. The aim of this diploma thesis is to determine the presence of microcrystals in different species of microalgae, study the conditions of their occurrence and identify their chemical composition via Raman microscopy.

Unicellular algae (microalgae) are able to produce a number of substances such as starches, oils, proteins, carotenoids, polyphosphates, or crystalline purines directly from inorganic sources by photosynthesis. Different species of microalgae can be used for the economic production of various biomolecules. Due to their autotrophic nature, microalgae are also unique as they can synthesize complex isotopically labeled biomolecules from simple isotopically labeled inorganic substances. Analysis of the chemical composition of microalgae by means of chemical-analytical methods is relatively complex, time-consuming, and laborious. Confocal Raman microscopy represents one of the optical methods by which the chemical composition of microalgae can be determined in situ, i.e. directly within intact cells. This technique combining confocal optical microscopy with Raman spectroscopy enables fast and non- destructive analysis of the chemical composition of substances in the investigated objects, including the effect of isotopic labeling. The chemical composition of the investigated objects is reflected by their Raman spectra, in the case of Raman mapping of microscopic objects by their chemical maps. In this work, a specific case of isotopic labeling was studied, namely the effect of heavy water (D2O) on the deuteration...

Since the 1900’s it has been known that microorganisms are capable of generating electrical power through extracellular electron transfer by converting the energy found organic compounds (Potter, 1911). Microbial fuel cells (MFCs) has garnered more attention recently, and have shown promise in several applications, including wastewater treatment (Yakar et al., 2018), bioremediation (Rosenbaum & Franks, 2014), biosensors (ElMekawy et al., 2018) desalination (Zhang et al., 2018) and as an alternative renewable energy source in remote areas (Castro et al., 2014). In MFCs catalytic reactions of microorganisms oxidize an electron donor through extracellular electron transfer to the anode, under anaerobic conditions, with the cathode exposed to an electron acceptor, facilitating an electrical current (Zhuwei, Haoran & Tingyue, 2007; Lovley, 2006). For energy production in remote areas a low cost and easily accessible feed stock is required for the MFCs. Sweet sorghum is a drought tolerant feedstock with high biomass and sugar yields, good water-use efficiency, established production systems and the potential for genetic improvements. Because of these advantages sweet sorghum stalks were proposed as an attractive feedstock (Rooney et al., 2010; Matsakas & Christakopoulos, 2013). Dried sweet sorghum stalks were, therefore, tested as a raw material for power generation in a MFC, with anaerobic sludge from a biogas plant as inoculum (Sjöblom et al., 2017a). Using sorghum stalks the maximum voltage obtained was 546±10 mV, the maximum power and current density of 131±8 mW/m2 and 543±29 mA/m2 respectively and the coulombic efficiency was 2.2±0.5%. The Ohmic resistances were dominant, at an internal resistance of 182±17 Ω, calculated from polarization data. Furthermore, hydrolysis of the dried sorghum stalks did not improve the performance of the MFC but slightly increased the total energy per gram of substrate. During the MFC operation, the sugars were quickly fermented to formate, acetate, butyrate, lactate and propionate with acetate and butyrate being the key acids during electricity generation. Efficient electron transfer between the microorganisms and the electrodes is an essential aspect of bio-electrochemical systems such as microbial fuel cells. In order to design more efficient reactors and to modify microorganisms, for enhanced electricity production, understanding the mechanisms and dynamics of the electron transport chain is important. It has been found that outer membrane C-type cytochromes (OMCs) (including omcS and omcZ discussed in this study) play a key role in the electron transport chain of Geobacter sulfurreducens, a well-known, biofilm forming, electro-active microorganism  (Millo et al., 2011; Lovley, 2008). It was found that Raman microscopy is capable of providing biochemical information, i.e., the redox state of c-type cytochromes (cyt-C) without damaging the microbial biofilm, allowing for in-situ observation. Raman microscopy was used to observe the oxidation state of OMCs in a suspended culture, as well as in a biofilm of an MFC. First, the oxidation state of the OMCs of suspended cultures from three G. sulfurreducens strains (PCA, KN400 and ΔpilA) was analyzed. It was found that the oxidation state can also be used as an indicator of the metabolic state of the cells, and it was confirmed that PilA, a structural pilin protein essential for long range electron transfer, is not required for external electron transfer. Furthermore, we designed a continuous, anaerobic MFC enabling in-situ Raman measurements of G. sulfurreducens biofilms during electricity generation, while poised using a potentiostat, in order to monitor and characterize the biofilm. Two strains were used, a wild strain, PCA, and a mutant, ΔOmcS. The cytochrome redox state, observed through the Raman spectra, could be altered by applying different poise voltages to the electrodes. This change was indirectly proportional to the modulation of current transferred from the cytochromes to the electrode. This change in Raman peak area was reproducible and reversible, indicating that the system could be used, in-situ, to analyze the oxidation state of proteins responsible for the electron transfer process and the kinetics thereof.

Esta Tese de doutorado teve por objetivo aumentar a compreensão dos aspectos sinérgicos na corrosão de bens culturais feitos com chumbo (Pb) e avaliar a agressividade ambiental sobre bens culturais em igrejas e museus brasileiros para a proposição de estratégias de conservação. Neste contexto, foram investigados, em microambientes simulados, os efeitos sinérgicos entre formaldeído e ácido acético, ácido fórmico ou dióxido de carbono na corrosão de Pb; foi feita a síntese e caracterização de diferentes formiatos gerados nos processos de corrosão de Pb por formaldeído ou ácido fórmico; foi avaliada a agressividade ambiental sobre bens culturais da Igreja de São Francisco (São Paulo, ambiente urbano industrial), da Igreja dos Freis Carmelitas (Santos, ambiente urbano industrial costeiro), do Museu de Arte Contemporânea da USP (MAC, São Paulo, reserva técnica da nova sede) e do Museu de Arqueologia e Etnologia da USP (MAE, São Paulo, reserva técnica), através de cupons metálicos (chumbo e cobre) expostos nos ambientes interno e externo dessas instituições e através do uso de monitores miniaturizados; foi feita a otimização destes monitores e, finalmente, foi considerado o desenvolvimento de um nariz eletrônico (EN) baseado em microbalanças de quartzo (QCMs) para o uso em conservação preventiva. Nestas investigações microscopia Raman foi a principal técnica empregada, sendo que as demais foram FTIR-ATR, µ-FTIR-ATR, µ-FTIR, nano-FTIR (SINS), s-SNOM, AFM, SEM, SEM-EDX, XRD e SR-XRD. Os resultados apontaram para a necessidade de individualização dos objetos artísticos tanto para a proposição de estratégias de conservação quanto para o entendimento de sua reatividade, mostraram a agressividade ambiental de todas as instituições investigadas, indicaram a potencialidade de ENs em conservação preventiva e demonstraram a utilidade de técnicas de alta resolução lateral, como nano-FTIR, em investigações de corrosão metálica. Mais especificamente, as investigações de sinergia entre poluentes aumentaram o entendimento da reatividade de Pb e permitiram a proposição de esquemas mais completos de sua reatividade, levando a discussões acerca da conservação desses bens. A agressividade química ambiental das igrejas e museus a bens culturais foi inferida pela caracterização de cupons metálicos expostos em seus interiores e exteriores, enquanto que a agressividade relacionada a fatores físicos, como temperatura e umidade relativa, foi inferida através do uso de monitores miniaturizados expostos somente nas igrejas. Com esse monitoramento, estratégias de conservação foram propostas. Para a devida caracterização dos cupons metálicos, expostos nas igrejas e museus ou nos microambientes simulados, a síntese e a caracterização de diferentes formiatos de Pb foi essencial. Por fim, visando melhorar o desempenho dos monitores miniaturizados e ampliar sua aplicabilidade (com seu futuro uso como EN), um novo dispositivo foi construído e um estágio na Universidade de Aveiro, em Portugal, foi realizado para o desenvolvimento de um EN baseado em QCMs. / The objective of this doctoral thesis was to increase the understanding of synergistic aspects in the corrosion of cultural objects made with lead (Pb) and to assess the environmental aggressiveness on cultural objects of Brazilian churches and museums in order to propose conservation strategies. In this context, in simulated micro-environments, the synergistic effects between formaldehyde and acetic acid, formic acid or carbon dioxide in Pb corrosion were investigated; the syntheses and characterization of different formates generated by formaldehyde or formic acid in Pb corrosion processes was conducted; the environmental aggressiveness on cultural objects of the church of São Francisco (São Paulo, urban industrial environment), of the church of the Carmelite Friars (Santos, urban industrial littoral environment), of the Museum of Contemporary Art of the USP (MAC, São Paulo, new site) and of the Museum of Archaeology and Ethnology of the USP (MAE, São Paulo) was assessed exposing metal coupons (lead and copper) in their internal and external environments and using miniaturized monitors; these monitors were optimized and finally, the development of an \'electronic nose\' (EN) based on quartz crystal microbalances (QCMs) was considered for preventive conservation. In these investigations the principal technique employed was Raman microscopy, and FTIR-ATR, µ-FTIR-ATR, µ-FTIR, nano-FTIR (SINS), s-SNOM, AFM, SEM, SEM-EDX, XRD and SR-XRD were also used. The results pointed out to the necessity of individualization of the artistic objects when investigating their reactivity or when proposing conservation strategies, showed the environmental aggressiveness of all the investigated institutions, indicated the potential of ENs in preventive conservation and demonstrated the utility of techniques of high lateral resolution such as nano-FTIR for the investigation of metal corrosion. More specifically, the investigations of synergistic effects between gaseous pollutants increased the understanding of Pb reactivity and allowed to propose more completed schemes of its reactivity, which allowed discussions on the conservation of these objects. The aggressiveness related to the environmental chemical composition of the churches and museums was inferred through the characterization of metal coupons exposed in their indoor and outdoor environments, whilst the aggressiveness related to physical factors such as temperature and relative humidity was inferred through the use of miniaturized monitors exposed only in the churches. With this monitoring, conservation strategies were proposed. For the well characterization of the metal coupons, exposed in the churches and museums or in simulated microenvironments, the syntheses and characterization of different Pb formates were essential. Finally, aiming at still improving the performance of the miniaturized monitors and increasing their applicability (in their future use as EN), a new device was constructed, and an internship was considered in the University of Aveiro, Portugal, for the development of an EN based on QCMs.