Scanning electron microscopy as a new tool for diagnostic pathology and cell biology.

Scanning electron microscopy (SEM) use in the biomedical sciences has traditionally been used for characterisation of cell and tissue surface topography. This paper demonstrates the utility of high-resolution scanning electron microscopy (HRSEM) to diagnostic pathology and cell biology ultrastructural examinations. New SEM applications based on the production of transmission electron microscopy-like (TEM-like) images are now possible with the recent introduction of new technologies such as low kV scanning transmission electron microscopy (STEM) detectors, automated scan generators and high-resolution column configurations capable of sub-nanometre resolution. Typical specimen types traditionally imaged by TEM have been examined including renal, lung, prostate and brain tissues. The specimen preparation workflow was unchanged from that routinely used to prepare TEM tissue, apart from replacing copper grids for section mounting with a silicon substrate. These instruments feature a small footprint with little in the way of ancillary equipment, such as water chillers, and are more cost-effective than traditional TEM columns. Also, a new generation of benchtop SEMs have recently become available and have also been assessed for its utility in the tissue pathology and cell biology settings.

The condensation of water on adsorbed viruses.

The wetting and dewetting behavior of biological nanostructures and to a greater degree single molecules is not well-known even though their contact with water is the basis for all biology. Here, we show that environmental electron microscopy (EM) can be applied as a means of imaging the condensation of water onto viruses. We captured the formation of submicrometer water droplets and filaments on single viral particles by environmental EM and by environmental transmission EM. The condensate structures are compatible with capillary condensation between adsorbed virus particles and with known droplet shapes on patterned surfaces. Our results confirm that such droplets exist down to <50 nm. The viruses preserved their shape after a condensation/evaporation cycle as expected from their stability in air and water. Moreover we developed procedures that overcome problems of beam damage and of resolving structures with a low atomic number.

Nanoparticles isolated from blood: a reflection of vesiculability of blood cells during the isolation process.

BACKGROUND: Shedding of nanoparticles from the cell membrane is a common process in all cells. These nanoparticles are present in body fluids and can be harvested by isolation. To collect circulating nanoparticles from blood, a standard procedure consisting of repeated centrifugation and washing is applied to the blood samples. Nanoparticles can also be shed from blood cells during the isolation process, so it is unclear whether nanoparticles found in the isolated material are present in blood at sampling or if are they created from the blood cells during the isolation process. We addressed this question by determination of the morphology and identity of nanoparticles harvested from blood. METHODS: The isolates were visualized by scanning electron microscopy, analyzed by flow cytometry, and nanoparticle shapes were determined theoretically. RESULTS: The average size of nanoparticles was about 300 nm, and numerous residual blood cells were found in the isolates. The shapes of nanoparticles corresponded to the theoretical shapes obtained by minimization of the membrane free energy, indicating that these nanoparticles can be identified as vesicles. The concentration and size of nanoparticles in blood isolates was sensitive to the temperature during isolation. We demonstrated that at lower temperatures, the nanoparticle concentration was higher, while the nanoparticles were on average smaller. CONCLUSION: These results indicate that a large pool of nanoparticles is produced after blood sampling. The shapes of deformed blood cells found in the isolates indicate how fragmentation of blood cells may take place. The results show that the contents of isolates reflect the properties of blood cells and their interaction with the surrounding solution (rather than representing only nanoparticles present in blood at sampling) which differ in different diseases and may therefore present a relevant clinical parameter.

Post-prandial rise of microvesicles in peripheral blood of healthy human donors.

BACKGROUND: Microvesicles isolated from body fluids are membrane - enclosed fragments of cell interior which carry information on the status of the organism. It is yet unclear how metabolism affects the number and composition of microvesicles in isolates from the peripheral blood. AIM: To study the post - prandial effect on microvesicles in isolates from the peripheral blood of 21 healthy donors, in relation to blood cholesterol and blood glucose concentrations. RESULTS: The average number of microvesicles in the isolates increased 5 hours post - prandially by 52%; the increase was statistically significant (p = 0.01) with the power P = 0.68, while the average total blood cholesterol concentration, average low density lipoprotein cholesterol concentration (LDL-C) and average high density lipoprotein cholesterol concentration (HDL-C) all remained within 2% of their fasting values. We found an 11% increase in triglycerides (p = 0.12) and a 6% decrease in blood glucose (p < 0.01, P = 0.74). The post - prandial number of microvesicles negatively correlated with the post - fasting total cholesterol concentration (r = - 0.46, p = 0.035) while the difference in the number of microvesicles in the isolates between post - prandial and post - fasting states negatively correlated with the respective difference in blood glucose concentration (r = - 0.39, p = 0.05). CONCLUSIONS: In a population of healthy human subjects the number of microvesicles in isolates from peripheral blood increased in the post - prandial state. The increase in the number of microvesicles was affected by the fasting concentration of cholesterol and correlated with the decrease in blood glucose.