Extremely thin layer plastification for focused-ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells.

Since the recent boost in the usage of electron microscopy in life-science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused-ion beam scanning electron microscopy (FIB-SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold-labelled breast cancer SKBR3 cells was to visualise gold-labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 µm2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back-scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB-SEM. Cross-sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.

Cryogenic EBSD on ice: preserving a stable surface in a low pressure SEM.

Naturally deformed ice contains subgrains with characteristic geometries that have recently been identified in etched surfaces using high-resolution light microscopy (LM). The probable slip systems responsible for these subgrain boundary types can be determined using electron backscattered diffraction (EBSD), providing the etch features imaged with reflected LM can be retained during EBSD data acquisition in a scanning electron microscope (SEM). Retention of the etch features requires that the ice surface is stable. Depending on the pressure and temperature, sublimation of ice can occur. The equilibrium temperature for a low pressure SEM operating at 1 × 10(-6) hPa is about -112°C and operating at higher temperatures causes sublimation. Although charging of uncoated ice samples is reduced by sublimation, important information contained in the etch features are removed as the surface sublimes. We developed a method for collecting EBSD data on stable ice surfaces in a low pressure SEM. We found that operating at temperatures of <-112°C reduced sublimation so that the original etch surface features were retained. Charging, which occurred at low pressures (<1.5 × 10(-6) to 2.8 × 10(-5) hPa) was reduced by defocusing the beam. At very low pressures (<1.5 × 10(-6) hPa) the spatial resolution with a defocused beam at 10 kV was about 3 µm in the x-direction at -150°C and 0.5 µm at -120°C, because at higher temperature charging was less and only a small defocus was needed to compensate it. Angular resolution was better than 0.7° after orientation averaging. Excellent agreement was obtained between LM etch features and EBSD mapped microstructures. First results are shown, which indicate subgrain boundary types comprised of basal (tilt and twist) and nonbasal dislocations (tilt boundaries).

Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low-kV BSE imaging.

Tomography in a focused ion beam (FIB) scanning electron microscope (SEM) is a powerful method for the characterization of three-dimensional micro- and nanostructures. Although this technique can be routinely applied to conducting materials, FIB-SEM tomography of many insulators, including biological, geological and ceramic samples, is often more difficult because of charging effects that disturb the serial sectioning using the ion beam or the imaging using the electron beam. Here, we show that automatic tomography of biological and geological samples can be achieved by serial sectioning with a focused ion beam and block-face imaging using low-kV backscattered electrons. In addition, a new ion milling geometry is used that reduces the effects of intensity gradients that are inherent in conventional geometry used for FIB-SEM tomography.

Focused ion beam-scanning electron microscope: exploring large volumes of atherosclerotic tissue.

Atherogenesis is a pathological condition in which changes in the ultrastructure and in the localization of proteins occur within the vasculature during all stages of the disease. To gain insight in those changes, high-resolution imaging is necessary. Some of these changes will only be present in a small number of cells, positioned in a 'sea' of non-affected cells. To localize this relatively small number of cells, there is a need to first navigate through a large area of the sample and subsequently zoom in onto the area of interest. This approach enables the study of specific cells within their in vivo environment and enables the study of (possible) interactions of these cells with their surrounding cells/environment. The study of a sample in a correlative way using light and electron microscopy is a promising approach to achieve this; however, it is very laborious and additional ultrastructural techniques might be very valuable to find the places of interest. In this report we show that the focused ion beam-scanning electron microscope is a powerful tool to study biological specimens in a correlative way. With this microscope one can scan for the area of interest at low magnification, in this case the atherosclerotic plaque, and subsequently zoom in, for further analysis on an ultrastructural level, rendering valuable and detailed two- and three-dimensional information of, in this case, the endothelial cells and the vessel wall. Moreover, in combination with pre-embedment labelling of surface exposed antigens, the method allows insight into the 3D distribution of these markers.

In-situ Observation of Hierarchical Self-Assembly Driven by Bicontinuous Gelation in Mixed Nanodisc Dispersions.

The search for new functional soft materials with precise and reconfigurable structures at the nano and meso-scale is a major challenge as well as objective of the current science. Patchy colloids of different shapes and functionalities are considered important new building blocks of a bottom-up approach towards rational design of new soft materials largely governed by anisotropic interactions. Herein, we investigate the self-assembly, growth of hierarchical microstructures and aging dynamics of 2D nano-platelets of two different aspect ratios (Laponite ~25 and Montmorillonite ~250) which form gels with different porosity that is achieved by tuning their mixing ratios. Qualitative in situ real-space studies are carried out, including fluorescent confocal microscopy imaging of the bicontinuous gelation process or final states, which provides dynamic visualization of the self-organization. The bicontinuous gels exhibit a foam-like morphology having pores of a few micrometers in size that can be tuned by varying the mixing ratio of nanoplatelets. It is shown that this new class of clay gels has unique and tunable physical properties that will find potential applications in the development of low cost lithium ion batteries, nanocomposites and nuclear waste management.