Environmental scanning electron microscopy in cell biology.

Environmental scanning electron microscopy (ESEM) (1) is an imaging technique which allows hydrated, insulating samples to be imaged under an electron beam. The resolution afforded by this technique is higher than conventional optical microscopy but lower than conventional scanning electron microscopy (CSEM). The major advantage of the technique is the minimal sample preparation needed, making ESEM quick to use and the images less susceptible to the artifacts that the extensive sample preparation usually required for CSEM may introduce. Careful manipulation of both the humidity in the microscope chamber and the beam energy are nevertheless essential to prevent dehydration and beam damage artifacts. In some circumstances it is possible to image live cells in the ESEM (2).In the following sections we introduce the fundamental principles of ESEM imaging before presenting imaging protocols for plant epidermis, mammalian cells, and bacteria. In the first two cases samples are imaged using the secondary electron (topographic) signal, whereas a transmission technique is employed to image bacteria.

The development of anisotropic behaviours of 3T3 fibroblasts on microgrooved patterns.

3T3 fibroblasts cultured on microgrooved polydimethylsiloxane (PDMS) surfaces of two different widths (25 µm and 55 µm) were individually tracked using confocal microscopy with a novel live-cell staining technique over several hours without noticeable cytotoxic effects. By quantifying the cell morphology, orientation, and migration over time, we identified the timescale (about 2-4 h after seeding) over which cell behaviours transitioned from isotropy to anisotropy, where the preference is in the direction parallel to the pattern. The development of anisotropy occurred more rapidly and distinctly when a narrower ridge width was used, suggesting that it is the ridge width that imposed a physical barrier on the cells' morphology and motility. Furthermore, while we found a weak but statistically significant correlation between cell orientation and morphology on the single-cell level, there is a lack of correlation on the same level between cell orientation and migratory direction. This suggests that while morphology and migration are affected anisotropically by topographical patterns in a similar way, the underlying processes giving rise to the anisotropy is slightly different in the two cases.

Tracking the heterogeneous distribution of amyloid spherulites and their population balance with free fibrils.

The analysis of amyloidogenic systems reveals the appearance of distinct states of aggregation for amyloid fibrils. For different proteins and under specific experimental conditions, amyloid spherulites are recognized as a significant component occurring in several protein model systems used for in vitro fibrillation studies. In this work we have developed an approach to characterize solutions containing a mixture of amyloid spherulites and individual fibrils. Using bovine insulin as the model system, sedimentation kinetics for the amyloid aggregates were followed using a combination of UV-Vis spectroscopy and cross-polarized optical microscopy. Spherulites were identified as the species undergoing sedimentation. A simple mathematical approach allows the description of the kinetics in terms of decay time/rate distribution. Moreover, based on the sedimentation kinetics, a rough estimate of the balance between amyloid spherulites and individual fibrils can be provided. Fitting the experimental data with the proposed physico-chemical approach shows self-consistent results in reasonable agreement with quantitative imaging analysis previously reported. Our results provide new physical insights into the analysis of amyloidogenic systems, providing a method to characterize the heterogeneous distribution of amyloid spherulites and simultaneously distinguish spherulites and free fibril populations. Importantly, the method can be generally applied to the characterization of polydisperse solutions containing optically traceable spherical particles in the micrometric range.

ESEM imaging of dynamic biological processes: the closure of stomatal pores.

Historically, electron microscopy of dynamic biological processes has been impossible to achieve in real time because conventional electron microscopy requires specimen fixation, dehydration and metallic coating. The advent of the environmental scanning electron microscope removes these restrictions, allowing fully hydrated samples to be imaged in their native state. We explore the possibility of secondary electron imaging of biological systems undergoing natural morphological changes in the microscope chamber and present a proof of principle study on the closure of stomatal pores in Tradescantia andersonia leaf tissue. An imaging protocol is developed and the advantages and limitations of this high-resolution imaging technique are considered, including a discussion of potential beam damage mechanisms.

Investigating the inner structure of irregular beta-lactoglobulin spherulites.

When beta-lactoglobulin in low p H aqueous solutions is exposed to high temperature for extended time, spherulites composed of amyloid fibrils of the beta-lactoglobulin protein form. Many of these spherulites have fibrils that radiate out from a centre and, under crossed polarisers, exhibit a symmetric Maltese Cross structure. However, a significant fraction (50 of the 101 observed spherulites) of beta-lactoglobulin spherulites formed under these conditions demonstrate various forms of irregularity in apparent structure. The irregularities of spherulites structures were qualitatively investigated by comparing optical microscopy images observed under crossed polarisers to computationally produced images of various internal structures. In this way, inner spherulite structures are inferred from microscopy images. Modelled structures that were found to produce computed images similar to some of the experimentally viewed images include fibrils curving as they radiate from a single nucleation point; multiple spherulites nucleating in close proximity to one another; and fibrils curving in opposite directions above and below a single nucleation point.

Developing dual-beam methodologies for the study of heterogeneous polymer-based systems.

The use of a combined focused ion beam/environmental scanning electron microscope (FIB/ESEM) offers new possibilities for imaging the internal structure of complex heterogeneous polymeric samples. The use of the focused ion beam, using positively charged gallium ions in conjunction with a measured 'defocused' low-energy primary electron beam, has permitted milling through the heterostructure to be achieved in a controlled way, exposing the inner structure, without introducing significant ion beam damage/destruction into the sample. The subsequent use of the environmental scanning electron microscope for imaging the revealed internal structure has then enabled insulating polymer structures to be imaged, without charging problems. Cross-sections of a 900-nm-thick spun cast film of phase-separated polystyrene-polybutadiene blends have been successfully milled and imaged; the morphology agreeing with previous results produced using ultramicrotomy and transmission electron microscopy.

Particle tracking microrheology of gel-forming amyloid fibril networks.

Microrheology is a technique that is increasingly used to investigate the local viscoelastic properties of complex fluids non-invasively, by tracking the motion of micron-sized probe spheres. In this work, passive Particle Tracking Microrheology (PTM) is used to study network formation in the milk protein beta-lactoglobulin at 80 degrees C and pH 2. In these conditions the protein aggregates to form thread-like structures known as amyloid fibrils, which can further aggregate into elastic networks. Using PTM, gels were observed to form at significantly lower concentrations than determined by bulk rheometry, where the oscillatory shear forces may disrupt either fibril or network formation. During incubation, the Mean Square Displacement (MSD) of the probe particles exhibited time-cure superposition, allowing the critical relaxation exponent to be calculated as approximately 0.63, consistent with other biopolymer gels. Combined with the gel-like appearance of the complex modulus at long incubation times, this confirms that a true gel is forming, with physical or chemical crosslinks forming between the fibrils, refining the conclusions of other workers in the field.

Application of environmental scanning electron microscopy to determine biological surface structure.

The use of environmental scanning electron microscopy in biology is growing as more becomes understood about the advantages and limitations of the technique. These are discussed and we include new evidence about the effect of environmental scanning electron microscopy imaging on the viability of mammalian cells. We show that although specimen preparation for high-vacuum scanning electron microscopy introduces some artefacts, there are also challenges in the use of environmental scanning electron microscopy, particularly at higher resolutions. This suggests the two technologies are best used in combination. We have used human monocyte-derived macrophages as a test sample, imaging their complicated and delicate membrane ruffles and protrusions. We have also explored the possibility of using environmental scanning electron microscopy for dynamic experiments, finding that mammalian cells cannot be imaged and kept alive in the environmental scanning electron microscopy. The dehydration step in which the cell surface is exposed causes irreversible damage, probably via loss of membrane integrity during liquid removal in the specimen chamber. Therefore, mammalian cells should be imaged after fixation where possible to protect against damage as a result of chamber conditions.

Improvements to a cryosystem to observe ice nucleating in a variable pressure scanning electron microscope.

The variable pressure scanning electron microscope (VPSEM) has expanded the scope of the SEM to allow the imaging of dynamic, electrically insulating systems. The use of water vapor as the imaging gas present in the chamber allows the successful imaging of hydrated samples. As awareness of the system capabilities becomes more well known, greater pressure has been put onto the microscopist to push the boundaries of both temperature and resolution for the study of diverse hydrated samples whose dynamics may not occur at the usual room temperatures in a VPSEM. In this article we discuss the stages in the development of a cryosystem that has led to the successful observation of the nucleation of ice from a solution in situ. This investigation also leads to further possibilities of imaging hydrated samples in the little explored temperature range of 188-238 K (from -85 to-35 degrees C). This study includes the exploration of how the temperature of various surfaces inside the microscope will change the system's ability to keep a sample hydrated or in its native state.

An environmental scanning electron microscopy study of aqueous gibbsite suspensions.

Synthetic gibbsite has been used as a model system for study in the environmental scanning electron microscopy (ESEM), to probe its utility as a tool to study clay dispersions under different conditions of aggregation. We have been able to show that we can study the nature of the platelet interactions as the pH is altered, by imaging the dispersions after water evaporation from the surface to permit the surface of the platelets to be clearly seen. It has been possible to show that at alkaline pH there are very few face-edge contacts between the platelets, consistent with what is known about the charges at high pH on the faces and edges of the gibbsite. In contrast, at lower pHs, when faces and edges have opposite sign charges, there are significantly more platelets touching with edge and faces in contact. Finally, when the salt lithium chloride is added to a dispersion at approximately neutral pH, the plates appear to stack suggesting face-face interactions in the dispersion. Thus, ESEM has been able to demonstrate the variability of packing in gibbsite dispersions and to correlate the structures observed with the known charge distribution on the gibbsite platelets.

Particle tracking to reveal gelation of hectorite dispersions.

Passive microrheological techniques using particle tracking have been developed for the study of the gelation of hectorite suspensions. By following the Brownian motion of the particles, it is possible to determine the increasing caging of the particles with time, as the system gels. Since only the Brownian motion is followed, the gelation process itself should not be affected by the measurement. As gelation proceeds the increasing heterogeneity of the particle environments can be monitored by a variety of measures, including kurtosis. An effective viscosity can be extracted from the measurements and used to indicate the gelation process.

Protein particulates: another generic form of protein aggregation?

Protein aggregation is a problem with a multitude of consequences, ranging from affecting protein expression to its implication in many diseases. Of recent interest is the specific form of aggregation leading to the formation of amyloid fibrils, structures associated with diseases such as Alzheimer's disease. The ability to form amyloid fibrils is now regarded as a property generic to all polypeptide chains. Here we show that around the isoelectric point a different generic form of aggregation can also occur by studying seven widely different, nonrelated proteins that are also all known to form amyloid fibrils. Under these conditions gels consisting of relatively monodisperse spherical particulates are formed. Although these gels have been described before for beta-lactoglobulin, our results suggest that the formation of particulates in the regime where charge on the molecules is minimal is a common property of all proteins. Because the proteins used here also form amyloid fibrils, we further propose that protein misfolding into clearly defined aggregates is a generic process whose outcome depends solely on the general properties of the state the protein is in when aggregation occurs, rather than the specific amino acid sequence. Thus under conditions of high net charge, amyloid fibrils form, whereas under conditions of low net charge, particulates form. This observation furthermore suggests that the rules of soft matter physics apply to these systems.

Mechanisms of structure formation in particulate gels of beta-lactoglobulin formed near the isoelectric point.

Particulate gels are known to be formed by bovine beta-lactoglobulin near the isoelectric point when partial unfolding is allowed to occur under heating. The aggregation process of the protein has been investigated within the context of a nucleation and growth process by preparing gels under precisely controlled thermal histories. This was achieved using a Differential Scanning Calorimeter (DSC) to provide controlled heating rates, and known final temperatures and incubation times. The resulting particulate gels were characterized by their particle size and polydispersity using Environmental Scanning Electron Microscopy (ESEM), which permits hydrated samples to be observed. Particle size was found to decrease with increasing final temperature, with the aggregation taking longer to reach completion for lower temperatures. Particle size was also found to decrease with increasing heating rate. This system could be modelled as evolving via nucleation and growth by taking into account the fact that the concentration of the aggregating species was varying as a function of temperature as well as time. The intrinsic tryptophan fluorescence as a function of temperature was used as a guide to the fraction of unfolded protein in solution, thereby permitting successful comparisons between the model predictions and the particle sizes to be made.-1.

Microrheological characterization of anisotropic materials.

We describe the measurement of anisotropic viscoelastic moduli in complex soft materials, such as biopolymer gels, via video particle tracking microrheology of colloid tracer particles. The use of a correlation tensor to find the axes of maximum anisotropy without prior knowledge, and hence the mechanical director, is described. The moduli of an aligned DNA gel are reported, as an application of the technique; this may have implications for high DNA concentrations in vivo. We also discuss the errors in microrheological measurement, and describe the use of frequency space filtering to improve displacement resolution, and hence probe these typically high modulus materials.

Investigating the permanent electric dipole moment of beta-lactoglobulin fibrils, using transient electric birefringence.

Amyloid fibrils, which are polymeric assemblies of protein molecules, have been intensively studied on a structural level, yet due to complications such as the disorder within the molecules, several aspects of their structure remain mysterious. Similarly, the kinetics of assembly are not well understood. Here we investigate the electric dipole moment of beta-lactoglobulin fibrils, a model amyloid fibril system, by applying the technique of transient electric birefringence. This moment appears to be large, and comparable to the total moment of the constituent protein monomers if they were joined in a chain, head-to-tail, without changing conformation, suggesting an ordered joining of monomers in the fibril. Such an ordered assembly may have implications for the assembly mechanism of beta-lactoglobulin fibrils in particular, and amyloid fibrils in general.

Electric birefringence study of an amyloid fibril system: the short end of the length distribution.

In this article, a system of amyloid fibrils, based on the protein beta-lactoglobulin, is studied by transient electric birefringence. Single pulses of an electric field were applied to the solution, and the initial rise and subsequent decay of birefringence analysed. The decay takes place on a range of relaxation times, and therefore contains information about the length distribution of fibrils in the system. The information can be extracted using theories of the electric polarisability of polyelectrolyte rods, since the fibrils are an example of these. Despite the long-standing complications of such theories, useful quantitative information about the system can still be obtained. Using the Fixman model of polyelectrolyte polarisability, we obtain a measurement of the short end of the length distribution which shows the fibril concentration as a function of length rising linearly from 0.02-2 microm. The short end of the length distribution was unobtainable in our previous study using rheo-optics (S.S. Rogers et al., Macromolecules 38, 2948 (2005)), but reasonable agreement between the two techniques shows they are complementary.

The binding of thioflavin-T to amyloid fibrils: localisation and implications.

Amyloid fibrils are a polymeric form of protein, involving a continuous beta-sheet with the strands perpendicular to the long axis of the fibril. Although typically implicated in diseases such as Alzheimer's disease and the transmissible spongiform encephalopathies, non disease-associated protein can also be converted into amyloid fibrils. Traditionally, amyloid fibrils are identified via the use of specific dyes such as Congo red and thioflavin-T, although their specificity is ill understood. Recently, solutions of bovine insulin and bovine beta-lactoglobulin have been found to form spherulites, micron-sized spherical structures containing radially arranged amyloid fibrils. When studied by confocal microscopy using polarised laser light and thioflavin-T, a consistent pattern of emission, rather than a uniform disc, was observed. This suggests the dye binds in a specific, regular fashion to amyloid fibrils. Confocal microscopy studies of thioflavin-T aligned in stretched poly-vinyl alcohol films showed that the dye dipole excitation axis lies parallel to the long molecular axis. Therefore, thioflavin-T binds to amyloid fibrils such that their long axes are parallel. We propose binding occurs in 'channels' that run along the length of the beta-sheet. Steric interactions between dye molecules and side chains indicate why thioflavin-T fluoresces more intensely when bound to amyloid fibrils and can explain why this interaction with amyloid fibrils is specific, but with varying efficiency.

The mechanism of amyloid spherulite formation by bovine insulin.

The formation of amyloid-containing spherulite-like structures has been observed in some instances of amyloid diseases, as well as in amyloid fibril-containing solutions in vitro. In this article we describe the structure and kinetics of bovine insulin amyloid fibril spherulites formed in the presence and absence of different salts and at different salt concentrations. The general spherulite structure consists of radially oriented amyloid fibrils, as shown by optical microscopy and environmental scanning electron microscopy. In the center of each spherulite, a "core" of less regularly oriented material is observed, whose size decreases when the spherulites are formed in the presence of increasing concentrations of NaCl. Similarly, amyloid fibrils form faster in the presence of NaCl than in its absence. A smaller enhancement of the rate of formation with salt concentration is observed for spherulites. These data suggest that both amyloid fibril formation and random aggregation occur concurrently under the conditions tested. Changes in their relative rates result in the different-sized cores observed in the spherulites. This mechanism can be likened to that leading to the formation of spherulites of polyethylene, in agreement with observations that polypeptide chains under partially denaturing conditions can exhibit behavior not dissimilar to that of synthetic polymers.

Investigation of quantitative secondary electron imaging of semiconducting polymer materials using environmental scanning electron microscopy.

The development of environmental scanning electron microscopy has opened the way for the examination of a wide variety of new sample types that were previously inaccessible to conventional scanning electron microscope techniques. With the advent of such a new methodology comes also the potential for new contrast mechanisms. We investigated the use of environmental scanning electron microscopy on semiconducting organic polymer materials. We observed contrast from these materials in secondary electron images, this contrast being attributed to differences in electron yield due to the polymer's electronic structure. Further study of these materials, and specifically the influence of film thickness on signal, has indicated a significant effect as the secondary electrons move through the sample. Systematic studies such as these are needed for a full understanding of the relationship between electronic properties and signal and, hence, the ability to probe structure-property relationships in greater depth.

An improved model for analyzing the small angle x-ray scattering of starch granules.

The structure of starch was studied using small-angle x-ray scattering (SAXS). The scattering data was modeled by considering a finite stack of alternating lamellae that are allowed to fluctuate both along the layer repeat direction and along the transverse layer direction. Analysis in this way of the SAXS data from starch allowed fresh insights into the native structure of several starch species, particularly potato starch. The novel model presented in this work was able to capture the experimentally observed SAXS patterns much better than previous models, which did not incorporate transverse layer fluctuations.