Chris T.W.M. Schneijdenberg

Integrated fluorescence and transmission electron microscopy

Correlative microscopy is a powerful technique that combines the strengths of fluorescence microscopy and electron microscopy. The first enables rapid searching for regions of interest in large fields of view while the latter exhibits superior resolution over a narrow field of view. Routine use of correlative microscopy is seriously hampered by the cumbersome and elaborate experimental procedures. This is partly due to the use of two separate microscopes for fluorescence and electron microscopy. Here, an integrated approach to correlative microscopy is presented based on a laser scanning fluorescence microscope integrated in a transmission electron microscope. Using this approach the search for features in the specimen is greatly simplified and the time to carry out the experiment is strongly reduced. The potential of the integrated approach is demonstrated at room temperature on specimens of rat intestine cells labeled with AlexaFluor488 conjugated to wheat germ agglutinin and on rat liver peroxisomes immunolabeled with anti-catalase antibodies and secondary AlexaFluor488 antibodies and 10nm protein A-gold.

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.

The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy

There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing. For transfer, handling and subsequent milling, the tubes are placed in a novel connective device (ferrule) that protects the sample from devitrification and contamination and passes through all operation steps. A piezo driven sample positioning stage (cryo-nano-bench, CNB) with three degrees of freedom was additionally developed to enable accurate milling of frozen-hydrated lamellas. With the CNB, high-pressure frozen samples can be milled to produce either thin lamellas (<100 nm), for direct imaging by high-resolution cryo-TEM or thicker lamellas (300-500 nm) for cryo-electron tomography. The sample remains vitreous throughout the process by using the presented tools and methods. The results are an important step towards investigating larger cells and even tissue in there natural state which in the end will enable us to gain better insights into cellular processes.

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.

FACTORS CONTROLLING THE RHYTHMIC CONTRACTION OF COLLAGEN GELS BY NEONATAL HEART CELLS

SummaryA floating collagen matrix culture of neonatal rat heart myocardial cells shows rhythmic contractions which are dependent on localization of cells, cell density, and collagen concentration. The rhythmic contractions of the collagen matrix can be registered by a device scanning the optical density at the edge of the gel and have been observed over a temperature range from 9° to 40° C. The results of the present study underline the usefulness of myocardial cell populated collagen matrixes for studies on coherent contractions of heart cell cultures.

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).

In-situ integrity control of frozen-hydrated, vitreous lamellas prepared by the cryo-focused ion beam-scanning electron microscope

Recently a number of new approaches have been presented with the intention to produce electron beam transparent cryo-sections (lamellas in FIB-SEM terminology) from hydrated vitreously frozen cryo samples with a Focused Ion Beam (FIB) system, suitable for cryo-Transmission Electron Microscopy (cryo-TEM). As the workflow is still challenging and time consuming, it is important to be able to determine the integrity and suitability (cells vs. no cells; vitreous vs. crystalline) of the lamellas. Here we present an in situ method that tests both conditions by using the cryo-Scanning Electron Microscope (cryo-SEM) in transmission mode (TSEM; Transmission Scanning Electron Microscope) once the FIB-made lamella is ready. Cryo-TSEM imaging of unstained cells yields strong contrast, enabling direct imaging of material present in the lamellas. In addition, orientation contrast is shown to be suitable for distinguishing crystalline lamellas from vitreous lamellas. Tilting the stage a few degrees results in changes of contrast between ice grains as a function of the tilt angle, whereas the contrast of areas with vitreous ice remains unchanged as a function of the tilt angle. This orientation contrast has subsequently been validated by cryo-Electron BackScattered Diffraction (EBSD) in transmission mode. Integration of the presented method is discussed and the role it can play in future developments for a new and innovative all-in-one cryo-FIB-SEM life sciences instrument.

Is acidosis the clue to the loss of structure and functioning of the sarcolemma

The only way for a tissue or organ to survive ischemia is by reperfusion or restoration of the blood flow. However, if the ischemic period is too long reperfusion leads to a Ca2+ overload of the myocardial cells and thereby to cell death. The question is; what are the key events during ischemia which cause this transition from reversible to irreversible injury. In this article we discuss whether acidosis may play a crucial role by inducing Ca2+ release from the sarcolemma and reorganization of membrane components especially the membrane lipids, i.e. lateral phase separation, resulting in membrane protein clustering and changes in lipid asymmetry.

Aggregation of myocardial sarcolemmal transmembrane proteins is not hindered by an interaction with the cytoskeleton. Possible implications for ischemia and reperfusion

Heart myocytes subjected to ischemia show a clustering of the sarcolemmal proteins. In the erythrocyte membrane, a system in which intramembranous particle (IMP) aggregation is extensively studied, it is found that an IMP aggregation can in principle only occur upon removal of the membrane skeleton of spectrin and actin by rather drastic experimental conditions. With regard to phospholipid composition and topology the sarcolemma and the erythrocyte membrane show large similarities and therefore it was proposed that a loss of the interaction of the IMPs and the cytoskeleton is also a prerequisite for the sarcolemmal IMP aggregation (Verkleij et al., 1990). Freezing myocardial tissue, both from adult and neonatal rat, from temperatures lower than 37 degrees C resulted in an aggregation of the sarcolemmal IMPs. The aggregation is proportional to the degree of lowering of the temperature at which the tissue is cryofixed. This in contrast to the erythrocyte membrane, where lowering the temperature only induces moderate IMP aggregation. The IMP aggregation in the sarcolemma is reversible upon a subsequent increase in incubation temperature. The results clearly demonstrate that the interaction between the sarcolemmal proteins does not hinder aggregation of the IMPs, as proposed previously, and suggest that loosening of this complex does not have to proceed the aggregation of the sarcolemmal intramembranous particles during ischemia.

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.