Advantages of indium-tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells.

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium-tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.

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.

A technique for improved focused ion beam milling of cryo-prepared life science specimens.

The combination of focused ion beam and scanning electron microscopy with a cryo-preparation/transfer system allows specimens to be milled at low temperatures. However, for biological specimens in particular, the quality of results is strongly dependent on correct preparation of the specimen surface. We demonstrate a method for deposition of a protective, planarizing surface layer onto a cryo-sample, enabling high-quality cross-sectioning using the ion beam and investigation of structures at the nanoscale.

The effect of burn injury on adolescents autobiographical memory.

Autobiographical memory recall was investigated in two female adolescent groups; one group who had experienced a burn injury and a matched control group. The Burn group was not currently depressed or anxious, but scored significantly higher on the intrusion subscale of the impact of event scale compared to controls. Two autobiographical memory tasks, the autobiographical memory cueing task and the Children's Autobiographical Memory Inventory (CAMI), were used. For the cueing task, the Burn group was significantly slower to recall specific memories. This group also recalled significantly fewer specific memories and significantly more extended overgeneral memories. For the CAMI, the burns group produced significantly lower semantic and episodic recall. The Burn group also produced significant correlations between sub-scales of the impact of event scale and selected measures on the autobiographical memory tasks. Higher intrusion scores were associated with less detailed episodic recall. Higher avoidance scores were associated with longer latencies to recall memories to negative cue words and fewer specific memories to all cue words. These results are discussed from the perspective that the Burn group experienced intrusive thoughts which interfered with normal autobiographical functioning.

Static and dynamic experiments in cryo-electron microscopy: comparative observations using high-vacuum, low-voltage and low-vacuum SEM.

We carried out a unique comparative study between three modes of cryo-scanning electron imaging: high-vacuum, low-voltage and low-vacuum, using ice cream as a model system. Specimens were investigated both with and without a conductive coating (Au/Pd) and at temperatures for which ice either remains fully frozen (< -110 degrees C) or undergoes sublimation (-110 to -90 degrees C). At high magnification, high-vacuum imaging of coated specimens gave the best results for 'static' specimens (i.e. containing fully frozen ice). Low voltages, such as 1 kV, could be used for imaging uncoated specimens at high vacuum, although slight 'classical' charging artefacts remained an issue, and the reduced electron beam penetration tended to decrease the definition between different microstructural features. However, this mode was useful for observing in situ sublimation from uncoated specimens. Low-vacuum mode, involving small partial pressures of nitrogen gas, was particularly suited to in situ sublimation work: when sublimation was carried out in low vacuum in the absence of an anti-contaminator plate, sublimation rates were significantly reduced. This is attributed to a small partial pressure of sublimated water vapour remaining near the specimen surface, enhancing thermodynamic stability.

Electron microscopy of mammalian cells in the absence of fixing, freezing, dehydration, or specimen coating.

Human osteoblast-like (bone-forming) cells were imaged using environmental scanning electron microscopy (ESEM). The cells were hydrated, unfrozen, and uncoated. Specimens were cooled to 3 degrees C and imaged in water vapor, with partial pressures varying from saturated conditions to a humidity of approximately 50%, relative to pure water. The ESEM images show the presence of cell nuclei, nucleoli, and cytoplasmic membranes. Comparisons between chemically fixed and unfixed specimens (neither dried nor coated) show that cell morphologies are similar in both cases. These results are compared with a fixed, dried, carbon-coated specimen. Thermodynamic and kinetic arguments are used to show that humidities significantly lower than 100% correspond to metastable states suitable for stabilizing hydrated biological tissues and cells. The ability to perform observations with minimal specimen preparation is potentially useful for studying interactions between mammalian cells and biomaterials that are developed for tissue engineering. The methods employed are equally applicable to the study of specimens in the biological, materials, and physical sciences where careful control over specimen stability is required.

Dynamic secondary electron contrast effects in liquid systems studied by environmental scanning electron microscopy.

We report an investigation into a dynamic contrast phenomenon in water-oil emulsions imaged in the environmental scanning electron microscope. Secondary electron contrast between oil and water phases is shown to change with scan rate, even inverting in extreme cases. This effect is attributed to the fact that charge carriers in liquids have intermediate mobilities compared with those in metallic conductors and solid insulators. Thus, increasing the electron energy flux density (via slower scan rates) results in the temporary accumulation of excess charge, which in turn gives rise to increased secondary electron emission. Excess charge dissipates between frames, however, such that classical charging of the specimen is not observed. The oils used here have conductivities lower than that of water, making them more susceptible to the effect. However, the material within the primary electron interaction volume is a conductive medium. We demonstrate that charging effects are not seen in regions of the oil where the interaction volume is in contact with the more conductive continuous water phase. Secondary electron emission from these regions therefore approximates to the intrinsic yield.