|
|
Use of optical and microfluidic techniques for analysis and separation of microbial cells
Večeríková, Paula ; Bernatová,, Silvie (referee) ; Samek,, Ota (advisor)
The presented diploma thesis primarily focuses on Raman spectroscopy and its connections to microfluidic techniques, along with potentially biotechnologically interesting products of bacterial strains Janthinobacterium lividum and Azotobacter vinelandii. The methods were chosen for their non-destructive nature, which allows for rapid cell analysis and comparison with gas chromatography. After analysis, cells can be further cultivated or a generation with excessive production of a selected metabolite can be prepared. Janthinobacterium lividum is a bacterial strain that produces the pigment violacein, which has significant biotechnological potential as a drug, antioxidant, fluorescent probe, or dye. The ability to distinguish between cells producing and not producing violacein can have wide applications in diagnosis and identification. In the experimental part, the spectra of three strains were measured, based on which Janthinobacterium lividum CCM 160 was selected for analysis using Raman tweezers, along with Cupriavidus necator H16. By evaluating with principal component analysis, the distinguishability of pigmented cultures from non-pigmented ones was determined, and a potential sorting marker of 1 140 cm-1 (corresponding to violacein) was selected for further measurements. The biotechnological potential of the Azotobacter vinelandii strain lies in its ability to fix nitrogen from the atmosphere, produce alginate, and polyhydroxybutyrate. The experimental part of the thesis focuses on identifying Raman spectra of five strains and the extracted alginates from them. Polyhydroxybutyrate (PHB) is a biodegradable polymer that replaces petrochemical plastics. For PHB production, Azotobacter vinelandii is an adept for commercial production, as it is one of the few that produces PHB directly from atmospheric nitrogen, which can lead to reduced production costs and thus higher representation of degradable plastics in the market. The highest response for PHB in Raman spectra was measured for Azotobacter vinelandii DSM 720 and the lowest for Azotobacter vinelandii DSM 85. Quantitative information for comparison was obtained from gas chromatography, which confirmed the measurements from Raman spectroscopy. By evaluating the measured Raman spectra of pure alginates, it is possible to assume that they are heteropolymeric fractions, as the spectra showed the expression of various monomers. Depending on the composition of subunits, the properties of alginate change, so it is necessary to know the composition for its adjustment for further use in medicine or the food industry. From the five Azotobacter vinelandii strains, producers with the highest and lowest PHB content were selected, which were subsequently analysed using Raman tweezers. By evaluating with principal component analysis, the distinguishability of these strains and their separability using the sorting parameter of 1 060 cm-1 was determined.
|
|
|
SMV-2022-44: Fabrication of optical tweezers for microrheology
Šerý, Mojmír
Fabrication of optical tweezers for microrheology. The result of the research is a laser microscope adapter, which allows the introduction of a beam of laser radiation through an optical fiber into the optical system of a microscope. The micro-object is captured in the focused laser beam near its focal point. The captured micro-object remains fixed in the field of view of the microscope and can be moved relative to its surroundings by movement of the microscope stage. The microscope objective is used to display the field of view of the micro-objects and also focuses the laser beam.
|
|
|
SMV-2022-45: Design of the Raman optical tweezers module
Šerý, Mojmír
Design of the Raman optical tweezers module. The result of the research is a laser microscope adapter, which allows the introduction of a beam of laser radiation through an optical fiber into the optical system of a microscope. The micro-object is captured in the focused laser beam near its focal point. The captured micro-object remains fixed in the field of view of the microscope and can be moved relative to its surroundings by movement of the microscope stage. The microscope objective is used to display the field of view of the micro-objects and also focuses the laser beam.
|
|
|
Stabilization of macroscopic particle in optical trap
Mlynář, Vojtěch ; Matěj, Zdeněk (referee) ; Brablc, Martin (advisor)
Cílem této práce je implementace a otestování algoritmů pro zpětnovazební stabilizaci částice v optické pasti. Tyto algoritmy jsou implementovány v programovatelném hradlovém poli Red Pitaya STEMlab 125-14 s využitím automatického generování kódu z modelů v programu Simulink. V práci je popsán fyzikální základ řízeného systému, jeho návaznost na teorii Kálmánova filtru, detaily implementovaných algoritmů v FPGA a procedury využité pro odhad parametrů. Implementované algoritmy byly úspěšně otestovány na reálné sestavě a výsledky jsou ukázány v závěru práce.
|
|
|
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.
|
|
|
Behaviour of Objects in Structured Light Fields and Low Pressures
Flajšmanová, Jana ; Čižmár, Tomáš (referee) ; Marago, Onofrio (referee) ; Zemánek, Pavel (advisor)
Studium chování opticky zachycených částic nám umožňuje porozumět základním fyzikálním jevům plynoucím z interakce světla a hmoty. Předkládaná práce podává vysvětlení zesílení tažné síly působící na opticky svázané částice ve strukturovaném světelném poli, tzv. tažném svazku. Ukazujeme, že pohyb dvou opticky svázaných objektů v tažném svazku je silně závislý na jejich vzájemné vzdálenosti a prostorové orientaci, což rozšiřuje možnosti manipulace hmoty pomocí světla. Následně se práce zaměřuje na levitaci opticky zachycených částic ve vakuu. Představujeme novou metodologii na charakterizaci vlastností slabě nelinearního Duffingova oscilátoru reprezentovaného opticky levitující částicí. Metoda je založena na průměrování trajektorií s určitou počáteční pozicí ve fázovém prostoru sestávajícím z polohy a rychlosti částice a poskytuje informaci o parametrech oscilátoru přímo ze zaznamenaného pohybu. Náš inovativní postup je srovnán s běžně užívanou metodou založenou na analýze spektrální hustoty polohy částice a za využití numerických simulací ukazujeme její použitelnost i v nízkých tlacích, kde nelinearita hraje významnou roli.
|
|
|
Transitional motion analysis of the optically trapped particle in under-damped regime
Sűsserová, Martina ; Damková, Jana (referee) ; Harabiš, Vratislav (advisor)
The bachelor thesis deals with the analysis of the movement of optically trapped nanoparticles at low pressures and familiarizes with methods commonly used in these conditions. The practical part of this work was done in Matlab, where the undesirable signal interference was filtered out from the original data and their influence on the phase portraits of the acquired transients was analyzed. Furthermore, the damped oscillator parameters are obtained from the records of average trajectories and the distribution of position data during the transient compared with distribution of position data at stationary state by histograms.
|
|
|
Transitional motion analysis of the optically trapped particle in under-damped regime
Sűsserová, Martina ; Damková, Jana (referee) ; Harabiš, Vratislav (advisor)
This bachelor thesis deals with the analysis of the transition phenomenon, which occurs when the nanoparticles enter the laser beam. Thereafter the particle is anchored around the point of focus of this beam. The main objectives of the thesis were to illustrate the dependence of the trajectory positions in relation to its instantaneous values and to obtain the parameters of the attenuated harmonic oscillator whose character is characteristic for the particle inserting the beam. Another objective of the thesis was to apply frequency filters to signal data and to observe their effects on phase diagrams and also to visualize the behavior of the particle in the vicinity of the focus in the stationary state.
|
|
|
Measurement of extinction spectra of optically trapped plasmon nano-particles
Flajšmanová, Jana ; Jonáš,, Alexander (referee) ; Brzobohatý, Oto (advisor)
This thesis deals with the dark-field imaging and the optical spectroscopy of optically trapped plasmonic nanoparticles. The optical trapping and the characterization of a single particle or multiple nanoparticles as well are demonstrated. The number of the optically trapped particles can be estimated from the dark-field scattering intensity. Experiments show the presence of the interparticle coupling among trapped metallic nanoparticles which has not been observed in case of dielectric particles. The scattering spectra of the plasmonic nanoparticles were compared with theoretical models based on the Mie theory and the Discrete dipole approximation.
|
|
|
Controlling and Evaluation of Laser Micromanipulation Experiments
Kaňka, Jan ; Jákl, Petr (referee) ; Provazník, Ivo (advisor)
This work is focused on the development of a user friendly software interface using the LabViewTM environment that simplifies running of various experiments using laser micromanipulations and laser microspectroscopy of living microorganisms. Both techniques have been developing very fast for the last decade and belong to the growing group of contact-less and nondestructive techniques for manipulation and diagnostics of individual living microorganisms, cells, or viruses. Within this project we mastered the driving of peripheries, calibration of CCD scene, real-time image processing of the CCD scene, automatic selection of the cell for further laser processing, acquisition and processing of the Raman spectrum from living microorganisms. The final goal of our activity is fully automatic laser-based sorter of living cells depending on their chemical compositions. This work has been elaborated at the Institute of Scientific Instruments of the ASCR, v.v.i. under the supervision of prof. Pavel Zemanek.
|
guest ::
