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Design of optomechanical module for chemical mapping using Laser-Induced Breakdown Spectroscopy
Švábíková, Anna ; Samek, Ota (referee) ; Pořízka, Pavel (advisor)
Tato diplomová práce se zabývá návrhem optomechanického modulu pro chemické mapování metodou spektroskopie laserem buzeného plazmatu (LIBS). Cílem je vyvinout modul, který bude umožňovat analýzu spektrálních čar zinku v ultrafialové (UV) oblasti. V práci jsou popsány teoretické základy metody LIBS a následně je provedena rešerše zaměřená na problematiku dálkové LIBS analýzy. V diplomové práci jsou prezentovány možné optické návrhy fokusační a sběrné optiky, z nichž jsou vybrané následně otestovány. Výsledkem práce je konstrukční návrh modulu.
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Multi-element analysis of soft tissues in paraffin by laser induced breakdown spectroscopy
Kopřivová, Hana ; Samek, Ota (referee) ; Pořízka, Pavel (advisor)
This thesis deals with the analysis of paraffin-embedded soft tissues using laser-induced breakdown spectroscopy (LIBS) and the complementary technique of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Specifically, the work focuses on optimizing soft tissue LIBS measurement parameters for femtosecond and nanosecond pulsed laser ablation at different wavelengths on mouse kidneys as a soft tissue model matrix. In addition, elemental analyses were performed on several samples of the most commonly occurring human skin cancers (malignant melanoma, basal cell carcinoma, and squamous cell carcinoma) using the above-mentioned analytical methods. Maps of the spatial distributions of biogenic elements were obtained. Aim of this work is to show a potential of new analytical methods that are not intended to replace the classical histological examination completely. However, these techniques could be used for preliminary screening of tumor tissue or purely as a complementary technique for histologists to diagnose cancers. Both methods could offer a completely new perspective on soft tissue cancer diagnosis in the future.
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Analysis of secondary metabolites from Streptomyces by Raman spectroscopy in vivo
Mikulová, Adéla ; Petříčková, K. ; Bobek, J. ; Chroňáková, Alica ; Samek, Ota ; Pilát, Zdeněk
The work deals with the study of Raman spectra of secondary metabolites of bacteria of the genus Streptomyces, especially polyene antifungal agents. Purified isolates and commercial standards of these compounds were used to obtain reference spectra from which spectral markers characterizing specific details of the chemical structure were derived. Verification of the observed markers was performed using in silico Raman spectra predictions. Subsequently, these findings were used for the spectroscopic characterization of the composition of secondary metabolites in vivo, directly in bacteria growing on a solid medium. The developed methodologies will help to optimize the conditions for the biotechnological production of antibiotics.
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SERS-TAGS: Selective immobilization and detection of bacteria using specific antibodies and surface-enhanced Raman scattering
Benešová, Markéta ; Bernatová, Silvie ; Samek, Ota ; Pokorná, Zuzana ; Mika, Filip ; Kizovský, Martin ; Pilát, Zdeněk
Raman spectroscopy is a non-destructive instrumental analytical technique based on Raman scattering, which is the inelastic scattering of photons that occurs when interacting with electrons in chemical bonds. With the help of Raman spectroscopy, chemical compounds, their mixtures and biological samples, including living organisms, can be analyzed very quickly, non-contactly and non-destructively. The performed measurements can be compared with spectra databases. A problem with Raman spectroscopy is that samples sometimes give a weak signal that is often overlaid by intense fluorescence. SERS (surface-enhanced Raman spectroscopy) is used to amplify Raman scattering.
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Development and optimization of laser assisted spark discharge optical emission spectroscopy
Kleštinec, Róbert ; Samek, Ota (referee) ; Pořízka, Pavel (advisor)
This master's thesis addresses the improvement of the detection capability and sensitivity of the LIBS method through the utilization of a secondary energy source in the form of spark discharge. The use of spark discharge for plasma excitation significantly extends its lifetime and provides an opportunity to enhance the analytical properties of the existing LIBS system. In the experimental part, both conductive and non-conductive samples were analyzed. In cement paste samples, chlorine was successfully detected without the need for additional rare gas atmosphere.
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Multi-element analysis of soft tissues in paraffin by laser induced breakdown spectroscopy
Kopřivová, Hana ; Samek, Ota (referee) ; Pořízka, Pavel (advisor)
This thesis deals with the analysis of paraffin-embedded soft tissues using laser-induced breakdown spectroscopy (LIBS) and the complementary technique of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Specifically, the work focuses on optimizing soft tissue LIBS measurement parameters for femtosecond and nanosecond pulsed laser ablation at different wavelengths on mouse kidneys as a soft tissue model matrix. In addition, elemental analyses were performed on several samples of the most commonly occurring human skin cancers (malignant melanoma, basal cell carcinoma, and squamous cell carcinoma) using the above-mentioned analytical methods. Maps of the spatial distributions of biogenic elements were obtained. Aim of this work is to show a potential of new analytical methods that are not intended to replace the classical histological examination completely. However, these techniques could be used for preliminary screening of tumor tissue or purely as a complementary technique for histologists to diagnose cancers. Both methods could offer a completely new perspective on soft tissue cancer diagnosis in the future.
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Raman microspectroscopy of living cells and biological tissues
Moudříková, Šárka ; Mojzeš, Peter (advisor) ; Matthäus, Christian (referee) ; Samek, Ota (referee)
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...
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Evolutionary engineering of cyanobacteria with respect to PHA accumulation
Vašířová, Kristýna ; Slaninová, Eva (referee) ; Samek, Ota (advisor)
The aim of this diploma thesis was to subject selected cyanobacterial strains to adaptive evolution and subsequently investigate the properties of the resulting adapted strains, especially their changes related to polyhydroxyalkanoates (PHA) accumulation. The theoretical part of the work describes in more detail the issue of cyanobacteria, PHA and their interconnection in the field of evolutionary engineering. Cyanobacterial strains Synechocystis sp 6803 and. Synechocystis salina CCALA 192 were used for evolutionary experiments. Selection pressures of hydrogen peroxide and copper. were applied to selected representatives. The resulting cultures and their ability to accumulate PHA were subsequently assessed by gas chromatography. Both of these selection pressures proved to be unsuitable, as strong growth inhibition was observed after their application to cultures, which did not allow the application of evolutionary engineering methods. In the second half of the experimental part, the provided adapted strains to 6% NaCl were monitored. Adaptation has been shown to have a positive effect on microorganisms, as they have a higher biomass content, better stress resistance and a slight increase in PHA accumulation.
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Raman spectroscopy of prokaryotic cells
Večeríková, Paula ; Bernatová,, Silvie (referee) ; Samek, Ota (advisor)
The presented bachelor‘s thesis deals with the Raman spectroscopy of bacterial cells. This method proves to be very suitable for monitoring the culture conditions of these bacterial strains because it is essentially non-destructive and fast compared to gas chromatography. Due to it‘s non-destructive nature, it is possible to further cultivate the measured cells. This analytical method can also be used to sort and select individual cells with increased ability to produce PHB. This would enable the possibility to select the generation of so-called PHB superproducers. Polyhydroxybutyrate (PHB) belongs to the group of polyhydroxyalkanoates (PHA), which serve as a substitute for plastics made in the petrochemical industry, whose consumption is increasing, especially during a pandemic. Current biodegradable substitutes are based on starch, which, in turn, depletes nutritional material for humans and animals. This bachelor thesis can serve as a basis outline for reducing the cost of PHB production, because Raman spectroscopy can be used in cell culture as a sensor of PHB content in response to a bacterial strain. In the experimental part of the work, the Raman spectra of selected bacteria were measured, where the result showed the greatest response to PHB in Chelatococcus shambunathi and the lowest in the thermophilic isolate BZ (Paenibacillus sp.). Quantitative information on the content of PHA in bacteria was obtained by gas chromatography, where the highest content was the bacteria- Chelatococcus shambunathi and the lowest Termobacillus composti. The subject of the second part was the separation of a mixture of PHA-producing and non-PHA-producing bacterial cultures. The result clearly proves that the mixture is separable based on the intensity of the selected sorting parameter.
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Analytical methods for qualitative and quantitative determination of PHA in cyanobacteria
Černayová, Diana ; Samek, Ota (referee) ; Sedláček, Petr (advisor)
The diploma thesis is confused to verify the applicability of selected physicochemical and spectroscopic methods for characterization of cyanobacteria, with special emphasis on possibilities of qualitative and quantitative analysis of polyhydroxyalkanoates (specifically polyhydroxybutyrate (PHB)) accumulated in cyanobacterial cells. The sample basis of the work was formed by cultures of cyanobacterial strains of Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. The cultures were were cultivated in several ways to cover the widest possible range of physiological conditions and PHB contents, in particular using an autotrophic way of cultivation on shakers and multicultural culture method in a basic culture medium,and in media enriched with 2% salt (NaCl ) as well as mixotrophic culture media with different types of the carbon substrate. After few weeks of cultivation, cyanobacterial cultures were obtained and complexly analyzed by following techniques- cell suspensions were analyzed by flow cytometry and UV-VIS spectrometry (transmission and diffusion transmission mode), dry cell biomass was characterised by gas chromatography to obtain a exact amount of PHB, and then FT-IR spectrometry and thermogravimetric analysis. The work aimed to assess whether any of these methods can be a quick and affordable alternative to the determination of PHB content to the most commonly used method of gas chromatography, but also to assess what additional information about the physiological state of cyanobacterial cells can provide test methods. The highest correlation on PHB content was determined for the parameters determined by infrared spectroscopy, in which specific peaks from the characteristic wavelengths for polyhydroxybutyrate were important. Weak correlations on PHB content were achieved in thermogravimetric analysis and cytometry, using the hydrophobic fluorescent probe BODIPY 439/503, which bound to lipophilic parts of cells. In addition to the determination of PHB, it was possible to determine pigments present in cyanobacteria (such as chlorophyll, phycocyanin and carotenoids) by flow cytometry and UV-VIS diffusion transmission spectrometry. In the end, results from all used techniques were compared by PCA analysis to determine the similarity of all analyzed samples.
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