Elektron kryo-mikroskopické techniky v biologickém výzkumu a nanotechnologiích

Příprava biologických vzorků pro transmisní elektronovou mikroskopii není triviální úkol. Vzorky musí odolat vakuu přítomném v mikroskopu, a proto je často nutné uplatnit nefyziologické postupy při jejich zpracování. Tyto postupy obvykle zahrnují fixaci na bázi aldehydů, nahrazení vody alkoholem (t.j. dehydrataci/substituci), a zalití do pryskyřice, která vytváří podporu pro následnou přípravu tenkých řezů, které pak mohou být vloženy do mikroskopu. V posledním desetiletí získala dominantní postavení v oblasti výzkumu buněčné biologie metoda kryo-fixace (vitrifikace) za pomoci ultrarychlého vysokotlakého zmrazování a následná kryo-substituce a zalití vzorků do pryskyřice při nízkých teplotách. Tímto způsobem byli úspěšně vitrifikovány různé biologické vzorky s tloušťkou až několik stovek mikrometrů do stavu, který byl srovnatelný s jejich in vivo strukturou. Kryo-fixace izolovaných biologických objektů (s omezenou tloušťkou do několika mikrometrů) je možná i v tenké vrstvě vitrifikované vody za pomoci imerzní kryo-fixace při normálním tlaku. V kombinaci s kryo-elektronovou mikroskopií se tato metoda stala nejefektivnejším a základním principem pro tvorbu elektron kryo-mikroskopických obrázků plně hydratovaných vzorků s velmi vysokým rozlišením na úrovni několika desetin nanometrů. Obě tyto metody...Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...Ústav buněčné biologie a patologie 1. LF UKInstitute of Cell Biology and Pathology First Faculty of Medicine Charles UniversityFirst Faculty of Medicine1. lékařská fakult

Characterising the elastic and viscoelastic interaction between the cell and its matrix in 3D: because it takes two to salsa dance

The extracellular matrix (ECM) is a three-dimensional, acellular component of all organs and tissues. The ECM has elastic and viscoelastic properties, quantified through the elastic modulus (i.e. stiffness) and stress relaxation, respectively, that guide cell fate. Stiffness and stress relaxation drive cellular plasticity in homeostasis and disease. Therefore, to represent the mechanics of the ECM in vitro it is necessary to employ models that recapitulate these properties. Among said models are hydrogels: polymeric networks whose mass mainly consists of water. Importantly, hydrogel viscoelasticity remains an understudied property, notably in cell-loaded materials. Hence, this thesis investigated the elastic and viscoelastic properties of diverse cell-free and cell-loaded hydrogels. The hydrogels evaluated in this thesis include organ-derived ECM, gelatine methacryloyl, agarose, human-derived platelet-poor plasma, alginate and pluronic. These hydrogels have tissue engineering and regenerative medicine (TERM) potential. Particular emphasis was placed on investigating and mathematically modelling the elastic and viscoelastic fate of cell-loaded hydrogels. Our data show that increasing polymer concentration tailored hydrogel elasticity and viscoelasticity. Hydrogel architecture, composition and the bonds forming the polymer network dictated hydrogel elasticity and viscoelasticity. Also, cells altered hydrogel stiffness and stress relaxation in a polymer type, hydrogel concentration and time-dependent manner. A generalised Maxwell model of viscoelasticity further revealed cell-induced changes in hydrogel time-dependent mechanics. Overall, this thesis furthers our understanding of cell-matrix biology in vitro. The data presented here also have implications for the TERM field and areas of hydrogel-based research for cellular applications

The drying of foods using supercritical carbon dioxide

Food drying techniques such as air and freeze drying are not ideal: high temperatures used during air drying result in degradation of nutrients and sensorial properties, while freeze drying is expensive and therefore only applicable to high value foods. As an alternative to such drying techniques, drying with supercritical carbon dioxide was investigated here. Initially, carrot was dried using this technique. Addition of a co-solvent (ethanol) to the supercritical fluid was used as a method to increase the water solubility in the supercritical fluid and therefore aid drying. Analysis of the dried and rehydrated product structure, rehydration properties and mechanical properties was carried out which gave an indication of product quality. Drying of agar, containing varying concentrations of sugar was carried out on a laboratory and pilot plant scale. Gel structure and gel properties were studied. Addition of sugar to agar gel pieces improved structural retention considerably during drying. Fourier transform infrared analysis was used to investigate interactions that may be responsible for structural differences seen during supercritical drying. Changes in experimental parameters such as flow rate and depressurisation rate did not appear to have a significant effect on the dried gel structure. The supercritical drying technique investigated allowed food products to be dried and unique structures to be created with different rehydration and textural properties to the equivalent food products dried by air or freeze drying

Novel detectors and algorithms for electron nano-crystallography

In the past decade, advances in structure determination with electron microscopy of organic, beam sensitive, materials have been significant. The newly developed techniques, triggered by new microscope systems and new cameras, made it possible to acquire 3D structural information from these samples to a resolution which was impossible to achieve before. Knowledge is required to improve structure solution and every aspect of the process involved, from treatment of radiation sensitive materials, sample preparation, TEM imaging and diffraction systems all the way to how data must be interpreted. In this thesis I explained multiple new techniques and methods developed by us, using both new microscopes as well as a new type of detector: Timepix. I describe how these tools can help to overcome (what were) the most important problems and bottlenecks in detection of very low dose electron diffraction.UBL - phd migration 201