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Optical Micromanipulation Techniques Combined with Microspectroscopic Methods
Pilát, Zdeněk ; Prášil,, Ondřej (referee) ; Mojzeš, Peter (referee) ; Zemánek, Pavel (advisor)
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.
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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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Study of isotopically labeled substances in living cells by means of Raman microscopy
Bura, Radek ; Mojzeš, Peter (advisor) ; Pilát, Zdeněk (referee)
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...
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Experimental fluorescence device for dielectrophoretic sorting of droplets in microfluidic chips
Ježek, Jan ; Pilát, Zdeněk ; Šmatlo, Filip ; Zemánek, Pavel
At present, many chemical and biological disciplines use different forms of spectroscopy for their observations. One of the most common methods is fluorescence spectroscopy. During the last seven years, microfluidic techniques began developing rapidly, using channels in which two immiscible liquids create droplets with diameters from units to tens to hundreds of micrometers. These droplets serve as liquid microcontainers containing the analysed sample and the necessary reagents. Using special microfluidic techniques, it is possible to fuse droplets with different contents (controlled triggering of chemical reactions), to change the concentration of reactants in the droplet with high speed (concentration gradients), or sort the droplets by content (creation of new cell strains).\n\n
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Optical Micromanipulation Techniques Combined with Microspectroscopic Methods
Pilát, Zdeněk ; Prášil,, Ondřej (referee) ; Mojzeš, Peter (referee) ; Zemánek, Pavel (advisor)
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.
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Compact device for fluorescence measurement by optical fiber in microfluidic chip
Ježek, Jan ; Pilát, Zdeněk ; Zemánek, Pavel
In our paper we present a device that combines fluorescence spectroscopy with fiber optics. The device allows high speed detection (in the order of kHz) of the fluorescence signal, which is coming from the sample by an inserted optical fiber, e.g. from a micro-droplet flow in a microfluidic chip, from the liquid flowing in the transparent capillary, etc. The device uses a laser diode at a wavelength suitable for excitation of fluorescence, excitation and emission filters, optics for focusing the laser radiation into the optical fiber, and a highly sensitive fast photodiode for detection of fluorescence.
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