Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing

In this paper, the Rapid Transfer System (RTS), an attachment to the Leica EMPACT2 high‐pressure freezer, is described as a new tool for special applications within the cryofixation field. The RTS is an automated system that allows for fast processing of samples (<5 s) that make it possible for the first time to use high‐pressure freezing in combination with high time resolution correlative light and electron microscopy. In addition, with a working cycle of 30 s this rapid turn over time allows one to acquire more samples of biopsy material before it deteriorates than with other HPF machines with longer cycle times. With the use of the RTS it was possible to obtain three samples each of four different tissues in 6 min. Together with the finding that 90% of samples of cells grown on sapphire discs were well frozen, the RTS was both fast and reliable. Most important, together with other newly developed accessories, the RTS made it possible to capture specific events occurring live in the cell as observed by light microscopy, to cryofix that sample/event within 4 s, and then to analyze that event at high resolution in the electron microscope with excellent preservation of ultra‐structure. These developments should give us the tools to unravel intracellular processes that can be observed by live cell imaging but are too rare or fast to be picked up by routine EM methods or where the history of a structure is necessary to be able to discern its nature.     

Correlative fluorescence and electron microscopy in tissues: immunocytochemistry

Correlative microscopy is a collection of procedures that rely upon two or more imaging modalities to examine the same specimen. The imaging modalities employed should each provide unique information and the combined correlative data should be more information rich than that obtained by any of the imaging methods alone. Currently the most common form of correlative microscopy combines fluorescence and electron microscopy. While much of the correlative microscopy in the literature is derived from studies of model cell culture systems we have focused, primarily, on correlative microscopy in tissue samples. The use of tissue, particularly human tissue, may add constraints not encountered in cell culture systems. Ultrathin cryosections, typically used for immunoelectron microscopy, have served as the substrate for correlative fluorescence and electron microscopic immunolocalization in our studies. In this work, we have employed the bifunctional reporter FluoroNanogold. This labeling reagent contains both a fluorochrome and a gold-cluster compound and can be imaged by sequential fluorescence and electron microscopy. This approach permits the examination of exactly the same sub-cellular structures in both fluorescence and electron microscopy with a high level of spatial resolution.     

Correlative fluorescence and transmission electron microscopy: an elegant tool to study the actin cytoskeleton of whole-mount (breast) cancer cells

Elucidating the structure and dynamics of lamellipodia and filopodia in response to different stimuli is a topic of continuing interest in cancer cells as these structures may be attractive targets for therapeutic purposes. Interestingly, a close functional relationship between these actin-rich protrusions and specialized membrane domains has been recently demonstrated. The aim of this study was therefore to investigate the fine organization of these actin-rich structures and examine how they structurally may relate to detergent-resistant membrane (DRM) domains in the MTLn3 EGF/serum starvation model. For this reason, we designed a straightforward and alternative method to study cytoskeleton arrays and their associated structures by means of correlative fluorescence (/laser)- and electron microscopy (CFEM).
  CFEM on whole mounted breast cancer cells revealed that a lamellipodium is composed of an intricate filamentous actin web organized in various patterns after different treatments. Both actin dots and DRM's were resolved, and were closely interconnected with the surrounding cytoskeleton. Long actin filaments were repeatedly observed extending beyond the leading edge and their density and length varied after different treatments. Furthermore, CFEM also allowed us to demonstrate the close structural association of DRMs with the cytoskeleton in general and the filamentous/dot-like structural complexes in particular, suggesting that they are all functionally linked and consequently may regulate the cell's fingertip dynamics. Finally, electron tomographic modelling on the same CFEM samples confirmed that these extensions are clearly embedded within the cytoskeletal matrix of the lamellipodium.     

Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching

Fluorescence light microscopy (LM) has many advantages for the study of cell organization. Specimen preparation is easy and relatively inexpensive, and the use of appropriate tags gives scientists the ability to visualize specific proteins of interest. LM is, however, limited in resolution, so when one is interested in ultrastructure, one must turn to electron microscopy (EM), even though this method presents problems of its own. The biggest difficulty with cellular EM is its limited utility in localizing macromolecules of interest while retaining good structural preservation. We have built a cryo-light microscope stage that allows us to generate LM images of vitreous samples prepared for cryo-EM. Correlative LM and EM allows one to find areas of particular interest by using fluorescent proteins or vital dyes as markers within vitrified samples. Once located, the sample can be placed in the EM for further study at higher resolution. An additional benefit of the cryo-LM stage is that photobleaching is slower at cryogenic temperatures (−140°C) than at room temperature.     

High-resolution scanning electron microscopy of frozen-hydrated and freeze-substituted kidney tissue

Inner surfaces and fracture faces of rabbit kidney tissue were investigated with high-resolution scanning electron microscopy using two different cryopreparation techniques: (i) for the observation of fracture faces, cryofixed tissue was fractured and coated in a cryopreparation chamber dedicated to SEM, vacuum transferred onto a cold stage and observed in the frozen-hydrated state; (ii) for the observation of inner surfaces of the nephron, water was removed after freezing and fracturing by freeze substitution and critical-point drying of the tissue. By both methods, macromolecular structures such as intramembranous particles on fracture faces and particles on inner surfaces were imaged. The latter method was used to investigate in more detail surface structures of cells in the cortical collecting duct. These studies revealed a heterogeneity of intercalated cells not described thus far.     

Development of a new bimodal imaging methodology: a combination of fluorescence microscopy and high-resolution secondary ion mass spectrometry

In this paper, we present a new experimental methodology to combine mass spectrometry (NanoSIMS) with fluorescence microscopy to provide subcellular information on the location of small molecules in cultured cells. We demonstrate this by comparing the distribution of 5-bromo-2-deoxyuridine in the same cells given by both NanoSIMS analysis and by fluorescence immunohistochemistry. Fiducial markers in the substrates ensured that the images formed by SIMS mapping of bromine ions could be co-registered exactly with images from fluorescence microscopy. The NanoSIMS was shown to faithfully reproduce the information from fluorescence microscopy, but at a much higher spatial resolution. We then show preliminary SIMS images on the distribution of ATN-224, a therapeutic copper chelator for which there is no fluorescent marker, co-registered with conventional Lysotracker and Hoechst stains on the same cells.     

A simple photographic grid system for the correlative examination of cell smears by light and scanning electron microscopy

A simple inexpensive grid system reproduced photographically on black-and-white film provides a support system that allows the same cells to be examined by light and scanning electron microscopy.     

Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature

A survey of methods combining light microscopy and scanning electron microscopy is presented. A simple correlation is made when two preparations from adjacent parts of one specimen are investigated in two different microscopes. A more sophisticated method is the consecutive investigation of one specimen with two microscopes. A major problem in this method is the relocation of the area of interest. Several authors have presented solutions for this problem. It is preferable when one preparation is investigated in only one instrument, combining the two microscopical (LM and SEM) techniques, thus making relocation redundant.     

Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution

Freezing of bulk biological objects was investigated by X-ray cryodiffraction. Freezing at atmospheric pressure of most microscopic biological samples gives rise to large hexagonal crystals and leads to poor structural preservation of these specimens. High-pressure freezing induces the formation of different ices (hexagonal, cubic and a high-pressure form) consisting of crystals having sizes smaller than those formed at atmospheric pressure. With both freezing methods, a cryoprotectant has to be added to the biological object to avoid the formation of ice crystals. However, special cases can be encountered: some biological objects contain large amounts of natural cryoprotectant or have a low water content. In these cases, vitrification can be achieved, especially using high-pressure freezing. Cryo-sectioning can be performed on vitrified samples, and the sections studied by electron cryomicroscopy. Images and electron diffraction patterns having a resolution better than 2 and 0.2 nm, respectively, can be obtained with such sections. Because samples containing crystalline ices cannot be cryosectioned, their structure has to be studied using cryosubstitution and resin embedding. We show that bacteria, yeast, and ciliate and marine worm elytrum have cellular compartments with an organization that has not been described by classical techniques relying on chemical fixation of the tissues. A high-pressure artefact affecting the Paramecium trichocysts is described. Such artefacts are not general; for example, we show that 70% of high-pressure frozen yeast cells survive successive high-pressure freezing and thawing steps.     

Atomic force microscopy as a tool for study of human hair

Atomic force microscopy (AFM) is a newly developed microscopic technique that offers high-resolution power, less intrusive measurement, and requires little sample pretreatment for elucidating structures of biological materials in three dimensions and in their natural environment. In this study, AFM has been used not only as an imaging technique for examining human hair structure at high resolution, but also as a tool for quantitative assessment of the effect of treatment in 10 mM phosphate buffered saline of pHs 3.0, 7.0, and 11.0 and heating on human hair structure. It is observed that the hair cuticle is a sensitive indicator of external influences on hair structure, and that its height can be used as a parameter for quantitative assessment. The experimental results obtained show that the swelling of hair caused by the incubation in the buffer decreases with the increase of the pH values and that, depending on the duration of heating, the hair undergoes structural expansion and shrinkage. This study demonstrates that AFM can be used as a valuable alternative to conventional microscopic techniques for hair research.     

High-resolution cryo-scanning electron microscopy study of the macromolecular structure of fibronectin fibrils

High-resolution cryo-scanning electron microscopy was used to examine fibronectin fibrils formed in culture by human skin fibroblasts and in a cell-free system by denaturing soluble plasma fibronectin with guanidine. These studies indicate that the conformation of fibrils formed in culture and in a cell-free system appeared to be similar and that fibronectin fibrils have at least two distinct structural conformations. Fibronectin fibrils can be very straight structures with smooth surfaces or highly nodular structures. The average diameter of the nodules in these fibrils is 12 nm. Both conformations can be seen within an individual fibril indicating that they are not different types of fibronectin fibrils but rather different conformational states. Immunolabeling studies with a monoclonal antibody, IST-2, to the heparin II binding domain of fibronectin revealed that the epitope was buried in highly smooth fibrils, but it was readily exposed in nodular fibrils. We propose, therefore, that fibronectin fibrils are highly flexible structures and, depending on the conformation of the fibril, certain epitopes on the surface may be buried or exposed.     

Scanning electron microscopy and transmission electron microscopy study of hot-deformed γ-TiAl-based alloy microstructure

The aim of this work was to assess the changes in the microstructure of hot‐deformed specimens made of alloys containing 46–50 at.% Al, 2 at.% Cr and 2 at.% Nb (and alloying additions such as carbon and boron) with the aid of scanning electron microscopy and transmission electron microscopy techniques. After homogenization and heat treatment performed in order to make diverse lamellae thickness, the specimens were compressed at 1000 °C. Transmission electron microscopy examinations of specimens after the compression test revealed the presence of heavily deformed areas with a high density of dislocation. Deformation twins were also observed. Dynamically recrystallized grains were revealed. For alloys no. 2 and no. 3, the recovery and recrystallization processes were more extensive than for alloy no. 1.     

Reflection electron microscopy methods for the study of surface structure

The techniques of reflection electron microscopy (REM) using TEM instruments and scanning reflection electron microscopy (SREM) using STEM instruments have been explored as means for the observation of surface structure with high spatial resolution, better than 1 nm in each case. Under the ordinary environment of a commercial TEM instrument, we have studied the contrast in REM images of atomic steps and made comparison with the calculated results from the multi-slice dynamical diffraction theory. Comparison has also been made between the REM images of defects and the calculated images based on the column approximation. The influence of surface resonances on the contrast has been investigated. By SREM performed in a modified HB5 STEM with attached high vacuum preparation chamber, we have observed the formation of periodically distributed Pd particles on the surface of cleaved MgO.     

Sectionable microcarrier beads: An improved method for the preparation of cell monolayers for electron microscopy

Cross-linked dextran beads provide an excellent surface for tissue-cultured cell monolayers, and can be processed for transmission (TEM) and scanning (SEM) electron microscopy, as well as light microscopy (LM). Cells are grown to confluency on the surface of the microcarriers, where at any point aliquots can be removed and experimentally treated as desired (e.g. immunocytochemistry) providing a representative sample. Sample preparation for TEM follows standard procedures for any cell monolayer, but infiltration times must be at least doubled to allow penetration of the beads. The polymerized blocks can then be sectioned for TEM or LM with no additional steps required. SEM sample preparation involves attaching the fixed bead/cell suspension to a glass coverslip with poly-1-lysine, dehydration, critical point drying, and coating for conductivity. The fixed and dried sample can also be attached directly to the SEM stub as free beads and subsequently gold coated. These beads provide (1) an increased surface area of cells visible per area of thin section, (2) eliminates the careful orientation required for flat substrate methods of embedding, (3) decreases the amount of sample manipulation in the forms of re-embedding and gluing, and (4) decreases the amount of drying artifact seen as cracking in SEM monolayer preparations.     

The preparation of leaf surfaces for scanning electron microscopy: a comparative study

Cryopreservation is the superior technique for viewing leaf surfaces in the SEM. Epidermal cells become distorted when freeze dried and disrupt the orientation of epicuticular wax structures. The latter are largely lost during critical point drying. Nevertheless, the appearance of surface structures after subjecting them to each drying method is valuable in interpreting the features observed by cryopreservation.     

Comparative study of a block copolymer morphology by transmission electron microscopy and scanning force microscopy

Using transmission electron microscopy (TEM) and scanning force microscopy (SFM) together, it was possible to verify important structural features of a nanostructured bulk material such as the kp‐morphology in an ABC triblock copolymer. By applying suitable imaging techniques during the SFM measurements it was possible to determine the morphology without additional manipulation steps in between. In comparison, TEM investigations on this type of material usually require selective staining procedures prior to the measurement. Also electron beam damage is often encountered during TEM measurements especially if components such as poly(methacrylates) are present. In contrast, SFM measurements can be assumed not to significantly change the phase dimensions of the components.     

Determination of the size distribution of lecithin liposomes: a comparative study using freeze fracture, cryoelectron microscopy and dynamic light scattering

The size distribution of liposomes is often determined using freeze fracture, cryoelectron microscopy or dynamic light scattering. However, the resulting size distributions do not directly coincide owing to the different weighting of the techniques. We present several methods which correct for these effects and allow a comparison of liposome size distributions as obtained by freeze fracture, cryoelectron microscopy or dynamic light scattering. These methods are based on theoretical models for the weighting of the size distribution of liposomes, which result from the preparation procedure for freeze fracture electron microscopy and from the measurement by dynamic light scattering. The proposed transformation methods are then experimentally tested with a sample of lecithin liposomes, whose size distribution was determined by dynamic light scattering, freeze fracture and cryoelectron microscopy. Furthermore, the weaknesses of the experimental techniques and hence of the resulting size distributions are discussed.     

Low-energy electron microscopy (LEEM) and mirror electron microscopy (MEM) of biological specimens: Preliminary results with a novel beam separating system

Low-energy electron microscopy (LEEM) and mirror electron microscopy (MEM) utilize a parallel beam of slow-moving electrons backscattered from the specimen surface to form an image. If the electrons strike the surface an LEEM image is produced and if they are turned back just before reaching the surface an MEM image results. The applications thus far have been in surface physics. In the present study, applications of LEEM and MEM in the biological sciences are discussed. The preliminary results demonstrate the feasibility of forming images of uncoated cultured cells and cellular components using electrons in the threshold region (i.e. 0–10 V). The results also constitute a successful test of a novel beam-separating system for LEEM and MEM.     

Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes

Critical-point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells. Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO₄ fixed, the critical-point dried and the freeze dried states. Critical-point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO₄ fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO₄ fixed volume. Transmission and scanning electron micrographs of the critical-point dried cells showed well-preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals. Double-fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical-point drying. For low magnification scanning electron microscopy (up to about 3000 times) both critical-point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical-point drying is superior to freeze drying.