RESUMO
Cellular electron cryo-tomography (cryoET) produces high-resolution three-dimensional images of subcellular structures in a near-native frozen-hydrated state. These three-dimensional images are obtained by recording a series of two-dimensional tilt images on a transmission electron cryo-microscope that are subsequently back-projected to form a tomogram. Key to a successful experiment is however a high-quality sample. This chapter outlines a basic workflow for the preparation of cellular cryoET samples. It covers the preparation of infected cells on electron cryo-microscopy grids and the vitrification by plunge-freezing and clipping of grids into AutoGrid rims. It also provides a general overview of the workflow for thinning the vitrified cells by focused ion beam (FIB) milling. Although this book is dedicated to Rift Valley fever virus research, the present protocol may also be applied to any other research subject where high-resolution structural insight into intracellular processes is desired.
Assuntos
Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Tomografia com Microscopia Eletrônica/métodos , Microscopia Crioeletrônica/métodos , Animais , Imageamento Tridimensional/métodos , Vírus da Febre do Vale do Rift/ultraestrutura , Humanos , VitrificaçãoRESUMO
The synthesis and characterization of benz-1,4-dioxane crown ethers (CEs) and some of its homologues are described and analyzed. The effect of added C-atom within the CE ring (increasing the hydrophobicity of the CE ring by increasing the number of CH2-units) on the Li+ and Mg2+ complexation within a liquid-liquid extraction (LLE) is investigated and thermodynamically analyzed. The complex stability constant K, the change of entropy ΔS and enthalpy ΔH, and the Gibbs energy ΔG are determined. The enhanced hydrophobicity of the CE ring results in stronger complexation stability of the Mg2+ complex, while the Li+ complexes are less favored. This effect mainly occurs due to the increased entropy term with improved hydrophobicity of the CE. These results indicate a stronger extraction of Li+ in Mg2+-containing aqueous resources if more hydrophilic CEs are used.
