An introduction to cryo-FIB-SEM cross-sectioning of frozen, hydrated Life Science samples.

The introduction of cryo-techniques to the focused ion-beam scanning electron microscope (FIB-SEM) has brought new opportunities to study frozen, hydrated samples from the field of Life Sciences. Cryo-techniques have long been employed in electron microscopy. Thin electron transparent sections are produced by cryo-ultramicrotomy for observation in a cryo-transmission electron microscope (TEM). Cryo-TEM is presently reaching the imaging of macromolecular structures. In parallel, cryo-fractured surfaces from bulk materials have been investigated by cryo-SEM. Both cryo-TEM and cryo-SEM have provided a wealth of information, despite being 2D techniques. Cryo-TEM tomography does provide 3D information, but the thickness of the volume has a maximum of 200-300 nm, which limits the 3D information within the context of specific structures. FIB-milling enables imaging additional planes by creating cross-sections (e.g. cross-sectioning or site-specific X-sectioning) perpendicular to the cryo-fracture surface, thus adding a third imaging dimension to the cryo-SEM. This paper discusses how to produce suitable cryo-FIB-SEM cross-section results from frozen, hydrated Life Science samples with emphasis on 'common knowledge' and reoccurring observations. LAY DESCRIPTION: Life Sciences studies life down to the smallest details. Visualising the smallest details requires electron microscopy, which utilises high-vacuum chambers. One method to maintain the integrity of Life Sciences samples under vacuum conditions is freezing. Frozen samples can remain in a suspended state. As a result, research can be carried out without having to change the chemistry or internal physical structure of the samples. Two types of electron microscopes equipped with cryo-sample handling facilities are used to investigate samples: The scanning electron microscope (SEM) which investigates surfaces and the transmission electron microscope (TEM) which investigates thin electron transparent sections (called lamellae). A third method of investigation combines a SEM with a focused ion beam (FIB) to form a cryo-FIB-SEM, which is the basis of this paper. The electron beam images the cryo-sample surface while the ion beam mills into the surface to expose the interior of the sample. The latter is called cross-sectioning and the result provides a way of investigating the 3rd dimension of the sample. This paper looks at the making of cross-sections in this manner originating from knowledge and experience gained with this technique over many years. This information is meant for newcomers, and experienced researchers in cryo-microscopy alike.

Cryo-FIB preparation of whole cells and tissue for cryo-TEM: use of high-pressure frozen specimens in tubes and planchets.

The desire to study macromolecular complexes within their cellular context requires the ability to produce thin samples suitable for cryo-TEM (cryo-transmission electron microscope) investigations. In this paper, we discuss two similar approaches, which were developed independently in Utrecht (the Netherlands) and Albany (USA). The methods are particularly suitable for both tissue samples and cell suspensions prepared by a high-pressure freezer (HPF). The workflows are explained with particular attention to potential pitfalls, while underlying principles are highlighted ('why to do so'). Although both workflows function with a high success rate, full execution requires considerable experience and remains demanding. In addition, throughput is low. We hope to encourage other research groups worldwide to take on the challenge of improving the HPF- cryo-FIB-SEM - cryo-TEM workflow. We discuss a number of suggestions to this end. LAY DESCRIPTION: Life is ultimately dictated by the interaction of molecules in our bodies. Highly complex equipment is being used and further developed to study these interactions. The present paper describes methods to prepare small, very thin lamellae (area of 5×5 µm2 , thickness 50-300 nm) of a cell to be studied in a cryo-transmission electron microscope (cryo-TEM). Special care must be taken to preserve the natural state of molecules in their natural environment. In the case of cryo-TEM, the samples must be frozen and kept frozen to be compatible with the vacuum conditions in the microscope. The frozen condition imposes technical challenges which are addressed. Two approaches to obtain the thin lamellae are described. Both make use of a focused ion beam (FIB) microscope. The FIB allows removal of material with nanometre precision by focusing a beam of ionised atoms (gallium ions) onto the sample. Careful control of the FIB allows cutting out of the required thin lamellae. In both strategies, the thin lamellae remain attached to the original sample, and the ensemble of sample with section and sample holder is transported from the FIB microscope to the TEM while being kept frozen.

Predicting intraparticle diffusivity as function of stationary phase characteristics in preparative chromatography.

Diffusion inside pores is the rate limiting step in many preparative chromatographic separations and a key parameter for process design in weak interaction aqueous chromatographic separations employed in food and bio processing. This work aims at relating diffusion inside porous networks to properties of stationary phase and of diffusing molecules. Intraparticle diffusivities were determined for eight small molecules in nine different stationary phases made from three different backbone materials. Measured intraparticle diffusivities were compared to the predictive capability of the correlation by Mackie and Meares and the parallel pore model. All stationary phases were analyzed for their porosity, apparent pore size distribution and tortuosity, which are input parameters for the models. The parallel pore model provides understanding of the occurring phenomena, but the input parameters were difficult to determine experimentally. The model predictions of intraparticle diffusion were of limited accuracy. We show that prediction can be improved when combining the model of Mackie and Meares with the fraction of accessible pore volume. The accessible pore volume fraction can be determined from inverse size exclusion chromatographic measurements. Future work should further challenge the improved model, specifically widening the applicability to greater accessible pore fractions (> 0.7) with corresponding higher intraparticle diffusivities (Dp/Dm  > 0.2). A database of intraparticle diffusion and stationary phase pore property measurements is supplied, to contribute to general understanding of the relationship between intraparticle diffusion and pore properties.

FIB-SEM cathodoluminescence tomography: practical and theoretical considerations.

Focused ion beam-scanning electron microscope (FIB-SEM) tomography is a powerful application in obtaining three-dimensional (3D) information. The FIB creates a cross section and subsequently removes thin slices. The SEM takes images using secondary or backscattered electrons, or maps every slice using X-rays and/or electron backscatter diffraction patterns. The objective of this study is to assess the possibilities of combining FIB-SEM tomography with cathodoluminescence (CL) imaging. The intensity of CL emission is related to variations in defect or impurity concentrations. A potential problem with FIB-SEM CL tomography is that ion milling may change the defect state of the material and the CL emission. In addition the conventional tilted sample geometry used in FIB-SEM tomography is not compatible with conventional CL detectors. Here we examine the influence of the FIB on CL emission in natural diamond and the feasibility of FIB-SEM CL tomography. A systematic investigation establishes that the ion beam influences CL emission of diamond, with a dependency on both the ion beam and electron beam acceleration voltage. CL emission in natural diamond is enhanced particularly at low ion beam and electron beam voltages. This enhancement of the CL emission can be partly explained by an increase in surface defects induced by ion milling. CL emission enhancement could be used to improve the CL image quality. To conduct FIB-SEM CL tomography, a recently developed novel specimen geometry is adopted to enable sequential ion milling and CL imaging on an untilted sample. We show that CL imaging can be manually combined with FIB-SEM tomography with a modified protocol for 3D microstructure reconstruction. In principle, automated FIB-SEM CL tomography should be feasible, provided that dedicated CL detectors are developed that allow subsequent milling and CL imaging without manual intervention, as the current CL detector needs to be manually retracted before a slice can be milled. Due to the required high electron beam acceleration voltage for CL emission, the resolution for FIB-SEM CL tomography is currently limited to several hundreds of nm in XY and up to 650 nm in Z for diamonds. Opaque materials are likely to have an improved Z resolution, as CL emission generated deeper in the material is not able to escape from it.

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).

Gridded Aclar: preparation methods and use for correlative light and electron microscopy of cell monolayers, by TEM and FIB-SEM.

Aclar, a copolymer film with properties very similar to those of tissue culture plastic, is a versatile substrate to grow cells for light (including fluorescence) and electron microscopic applications in combination with both chemical fixation and cryoimmobilization. In this paper, we describe complete procedures to perform correlative light and electron microscopy using Aclar as substrate for the culture of cell monolayers to be finally embedded in plastic. First, we developed straightforward, efficient and flexible ways to mark the surface of the Aclar to create substrates to locate cells first at the light microscopy and then the electron microscopy level. All the methods enable the user to self-design gridded Aclar pieces, according to the purpose of the experiments, and create a large number of substrates in a short time. Second, we confirmed that marked Aclar supports the normal growth and morphology of cells. Third, we validated the correlative light and electron microscopy procedure using Aclar. This validation was done for the high-resolution analysis of endothelial cells using transmission electron microscopy and focused ion beam-scanning electron microscopy in combination with the use of fluorescence, phase contrast and/or bright field microscopy to map areas of interest at low resolution. The methods that we present are diverse, easy to implement and highly reproducible, and emphasize the versatility of Aclar as a cell growth substrate for diverse microscopic applications.

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.