Předložená dizertační práce se zabývá kombinací optických mikromanipulací s mikrospektroskopickými metodami. Využili jsme laserovou pinzetu pro transport a třídění živých mikroorganismů, například jednobuněčných řas, či kvasinek. Ramanovskou spektroskopií jsme analyzovali chemické složení jednotlivých buněk a tyto informace jsme využili k automatické selekci buněk s vybranými vlastnostmi. Zkombinovali jsme pulsní amplitudově modulovanou fluorescenční mikrospektroskopii, optické mikromanipulace a jiné techniky ke zmapování stresové odpovědi opticky zachycených buněk při různých časech působení, vlnových délkách a intenzitách chytacího laseru. Vyrobili jsme různé typy mikrofluidních čipů a zkonstruovali jsme Ramanovu pinzetu pro třídění mikro-objektů, především živých buněk, v mikrofluidním prostředí.

Miniaturization of devices to study chemical interactions and processes in liquid samples has led to the emergence of microfluidics and construction of lab-on-a-chip systems. Present work was devoted to implementation, development and testing of microfluidic systems with detection by confocal Raman microscopy and surface enhanced Raman scattering under the conditions of training department. Several options of performing standard macroscopic measurements in microscopic scales were explored. A method for measuring thermal stability of biopolymers in microsystems with contactless detection of temperature has been designed and tested. Furthermore, possibilites for studying the SERS effect within microfluidic channels were explored. It was demonstrated that the microfluidic chips provide promising opportunity to study hydrodynamics of liquids at microscopic level and chemical reactions and kinetics.

The aim of this thesis was using surface-enhanced Raman scattering (SERS) microspectroscopy for the study of biomolecules and biological systems. The main probe molecule was cationic porphyrin H2TMPyP, however, other porphyrins, tryptophan and two lipids were also used. The sensitivity and reproducibility of several solid SERS substrates: (i) Au and Ag nanoparticles (NPs) immobilized via a bifunctional linker, (ii) AgNPs immobilized by drying, (iii) highly ordered Au and Ag film-over-nanosphere (FON) and (iv) Ag-coated insect wings were compared. On most of the solid substrates, the lowest detected H2TMPyP concentration was ~10-8 M. The highest sensitivity was provided by the dried drops of AgNPs/analyte mixture, where concentrations 1×10-10 M TMPyP, 1×10-5 M tryptophan, 2×10-7 M DSPC and 3×10-7 M DMTAP were detected. Nevertheless, the spectral reproducibility was decreased due to porphyrin metallation and perturbation of the lipid spectra in comparison to their Raman spectra from solution. The highest reproducibility was achieved by AuFON and Ag-coated insect wings. Finally, the AgNPs modified by PEG polymers were tested for intracellular application using HeLa cancer cells. Metallation of H2TMPyP served to probe the accessibility of PEG- AgNPs surface. The results proved that the accessibility...

Raman microspectroscopy enables one to acquire spectra of Raman scattering with a spatial resolution in the order of a few μm3 and thus to study the natural composition of biological objects such as tissues, single cells and cellular organelles in a non-invasive way. In this work, we used Raman microspectroscopy to investigate vacuoles of the opportunistic human yeast pathogen Candida albicans. Large sets of Raman spectra of vacuoles were collected based on different cultivation protocols. The sets of the spectra were evaluated using the multivariate statistical method of singular value decomposition. Based on the spectral analysis, we characterized the chemical composition of the vacuoles. We found out that the vacuoles of cells cultured differently or in different media vary particularly in the concentration of polyphosphate, represented in the spectra by the peak near 1155 cm-1 . Interestingly, the wavenumber position of the polyphosphate peak may also be shifted by several cm-1 . We studied these shifts in vitro with sodium hexametaphosphate as a model of vacuolar polyphosphate. Based on these experiments, we suggest that the peak position is significantly influenced by the concentration of divalent cations.

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The aim of this diploma thesis is the application of vibrational spectroscopy in pharmaceutical analysis in studying solid pharmaceutical forms. The surface of tablet samples containing the active substance from the group of statins has been studied especially by the methods infrared microscopy. Spectral maps of samples were collected thanks to the techniques of specular reflection, attenuated total reflection (ATR) and "inverse" ATR after determining optimal conditions for measurements. In order to evaluate these measured maps, one-dimensional analysis and principal component analysis were used. As the same samples of tablets were also measured by Raman microscopy, the comparison has been provided. The measured distribution maps enable both a determination of substances in the sample and conclusion concerned a method of tablets' preparation. The method in this case is a granulation, which has been found out from a comparison of maps of generic and original medicament. The specular reflection method was selected to be the most appropriate technique for obtaining the maps of the surface of a tablet, via confrontation of particular methods consequently with consideration of their advantages and disadvantages in the measurement and data processing.

Title: Optimization of surface-enhanced Raman scattering spectroscopy for study of biologically important biomolecules and their interactions Author: Natália Šmídová Department: Institute of Physics of Charles University Supervisor of the doctoral thesis: Doc. RNDr. Marek Procházka, PhD. Abstract: The main goal of this thesis was to optimize surface-enhanced Raman scattering (SERS) spectroscopy for study of biologically important biomolecules. For that purpose we focused on substrates based on gold colloidal nanoparticles immobilized to silanized glass plates. Stable, uniform and highly reproducible SERS-active substrates have been prepared by using aminopropyltrimethoxysilane and citrate- reduced gold nanoparticles thermally stabilized after their immobilization. Model biomolecules 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin (TMPyP) and 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TSPP) were studied on these substrates by using a classical Raman spectrometer in macro-mode and a confocal Raman microspectrometer. Conditions for SERS spectroscopy of porphyrins were optimized with respect to sensitivity and reproducibility. SERS microspectroscopy showed several advantages over SERS measurements in macromode: possibility of surface spectral mapping, easier manipulation with samples, shorter...

Mikroplastik (1–1000 µm) kommt praktisch überall in der Umwelt vor, aber immer noch ist die Iden-tifizierung und Quantifizierung eine anspruchsvolle und zeitintensive Aufgabe. Erste analytisch Metho¬den beginnen sich zu etablieren, jedoch sind die benötigten Instrumente komplex und der Probendurchsatz für Routineuntersuchungen in den meisten Fällen noch zu gering. Diese Arbeit widmet sich zunächst dem Potenzial der Nahinfrarot (NIR)-Spektroskopie diese Lücke zu schließen. Exemplarisch wird ein günstiges Verfahren mit großem Probendurchsatz zur Bestimmung von Mikro¬plastik-Gesamtgehalten der verbreiteten Verpackungskunststoffe Polyethylen (PE), Polystyrol (PS) und Polypropylen (PP) in Böden und Kompost entwickelt. Neben der Untersuchung von Mikroplastik-Gesamtgehalten einer Probe ist auch die Charakterisierung individueller Partikel von großer Bedeutung. Die bildgebende Fourier-Transform-Infrarot (FTIR)-Mikrospektroskopie ist hierfür sehr gut geeignet. Allerdings ist es eine Herausforderung Mikroplastik in den aus mehreren Million Spektren bestehenden hyperspektralen Bildern zu identifizieren. Eine schnelle und zuverlässige Mikroplastikerkennung wird hier durch eine explorative Analyse und automatisierte Klassifizierung der Spektren erreicht. Zusammenfassend zeigt diese Arbeit, dass die optische Spektroskopie im mittleren und nahen Infrarot über ihre bisherige Anwendung hinaus ein großes Potenzial besitzen, die Mikroplastik-Analytik kostengünstiger, einfacher und schneller zu gestalten. / Microplastics (1-1000 µm) are ubiquitous in the environment, but their identification and quantification is still a challenging and time-consuming task. The first established methods require complex instruments and the sample throughput is still too low for routine analysis in most cases. This work first addresses the potential of near-infrared (NIR) spectroscopy to fill this gap. A low-cost method with large sample throughput is developed for the determination of total microplastic contents of the common packaging plastics polyethylene (PE), polystyrene (PS) and polypropylene (PP) in soils and compost. In addition to the investigation of total microplastic levels in a sample, the characterization of individual particles is also of great importance. Fourier transform infrared (FTIR) imaging microspectroscopy is well suited for this purpose. However, it is challenging to identify microplastics in hyperspectral images consisting of several million spectra. Fast and reliable microplastic detection is achieved by exploratory analysis and automated classification of the spectra. In summary, this work shows that mid- and near-infrared optical spectroscopy have great potential beyond their current application to make microplastics analysis cheaper, easier, and faster.

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