Advanced cryo-tomography workflow developments - correlative microscopy, milling automation and cryo-lift-out.

Cryo-electron tomography (cryo-ET) is a groundbreaking technology for 3D visualisation and analysis of biomolecules in the context of cellular structures. It allows structural investigations of single proteins as well as their spatial arrangements within the cell. Cryo-tomograms provide a snapshot of the complex, heterogeneous and transient subcellular environment. Due to the excellent structure preservation in amorphous ice, it is possible to study interactions and spatial relationships of proteins in their native state without interference caused by chemical fixatives or contrasting agents. With the introduction of focused ion beam (FIB) technology, the preparation of cellular samples for electron tomography has become much easier and faster. The latest generation of integrated FIB and scanning electron microscopy (SEM) instruments (dual beam microscopes), specifically designed for cryo-applications, provides advances in automation, imaging and the preparation of high-pressure frozen bulk samples using cryo-lift-out technology. In addition, correlative cryo-fluorescence microscopy provides cellular targeting information through integrated software and hardware interfaces. The rapid advances, based on the combination of correlative cryo-microscopy, cryo-FIB and cryo-ET, have already led to a wealth of new insights into cellular processes and provided new 3D image data of the cell. Here we introduce our recent developments within the cryo-tomography workflow, and we discuss the challenges that lie ahead. LAY DESCRIPTION: This article describes our recent developments for the cryo-electron tomography (cryo-ET) workflow. Cryo-ET offers superior structural preservation and provides 3D snapshots of the interior of vitrified cells at molecular resolution. Before a cellular sample can be imaged by cryo-ET, it must be made accessible for transmission electron microscopy. This is achieved by preparing a 200-300 nm thin cryo-lamella from the cellular sample using a cryo-focused ion beam (cryo-FIB) microscope. Cryo-correlative light and electron microscopy (cryo-CLEM) is used within the workflow to guide the cryo-lamella preparation to the cellular areas of interest. We cover a basic introduction of the cryo-ET workflow and show new developments for cryo-CLEM, which facilitate the connection between the cryo-light microscope and the cryo-FIB. Next, we present our progress in cryo-FIB software automation to streamline cryo-lamella preparation. In the final section we demonstrate how the cryo-FIB can be used for 3D imaging and how bulk-frozen cellular samples (obtained by high-pressure freezing) can be processed using the newly developed cryo-lift-out technology.

Risk element sorption/desorption characteristics of dry olive residue: a technique for the potential immobilization of risk elements in contaminated soils.

Olive oil production is one of the most relevant agroindustrial activities in the Mediterranean region and generates a huge amount of both solid and semi-solid wastes, the uncontrolled disposal of which might lead to serious environmental problems. Due to its organic matter and mineral nutrient content, the waste material can be applied to agricultural soil as a fertilizer. However, due to its high organic matter content, dry olive residue (DOR), commonly called "alperujo," has the potential to immobilize risk elements in contaminated soils. The main objective of this study was to assess the possible effect of DOR on sorption of risk elements such as cadmium (Cd), lead (Pb), and zinc (Zn) in the soil. A set of batch sorption experiments were carried out to assess the ability of DOR to adsorb Cd, Pb, and Zn where the effect of the preceding biotransformation of DOR by four species of fungi: Penicillium chrysogenum, Coriolopsis floccosa, Bjerkhandera adusta, and Chondrostereum purpureum was compared. The Freundlich and Langmuir sorption isotherms were calculated to assess the sorption characteristics of both transformed and non-transformed DOR. The results showed good potential sorption capacity of DOR, especially for Pb and to a lesser extent for Cd and Zn. Better sorption characteristics were reported for the biotransformed DOR samples, which are expected to show higher humification of the organic matter. However, the desorption experiments showed weakness and instability of the DOR-bound elements, especially in the case of Zn. Thus, future research should aim to verify the DOR sorption pattern in contaminated soil as well as the potential stabilization of the DOR element bounds where the increase of the pH levels of the DOR samples needs to be taken into account.

A method of imaging ultrathin foils with very low energy electrons.

We demonstrate the possibility to examine the free-standing foils of thicknesses in units of nm in the scanning low energy electron microscope, using both reflected and transmitted electrons. Very high contrast has been obtained in dependence on the thickness and structure of the foil. A contribution of secondary electrons to the forward scattered electron signal is discussed and a way of suppressing it is presented. Examples of reflected, total transmitted and dark field transmitted electron signal for two graphene-like samples are shown. Dependence of the transmitted signal on the electron energy is observed.