Al(3+) and Zn(2+)-induced structural changes and localization in a fully-hydrated live eukaryotic cellular model.

This study combined techniques that did not require preparation protocols that were potentially harmful to the cell, making it possible to investigate cells at, or close to, their natural physiological state. We used the freshwater protozoon Chilomonas paramecium as a eukaryotic cellular model to locate sites of Al(3+) or Zn(2+) accumulation and quantify the associated structural changes. Cells were fully hydrated throughout the study, which used a combination of differential interference contrast light microscopy, confocal laser scanning microscopy and transmission X-ray microscopy. The latter technique allowed high resolution (50 nm) and high contrast imaging of live cells in solution. For confocal laser scanning microscopy the relatively new fluorochrome Newport Green was used. This made fluorescent complexes with intracellular Al(3+) and Zn(2+), allowing localisation of metal-containing granules and vesicles. After long term exposure a previously unreported annular-shaped site of metal accumulation was found, signifying a vesicle with metal accumulated in the periphery only. After exposure to Al(3+) and Zn(2+), the cell pellicle was thinner and the majority of rounded-up cells had a concentric layering of organelles. By combining a variety of techniques it was possible to gain high resolution structural and chemical information on cells minimally exposed to potentially artefact-inducing procedures.

Dark field X-ray microscopy: the effects of condenser/detector aperture.

In order to visualize the functionality of a biological cell, it is often desirable to label specific proteins. In this work we concentrate on the optical theory of visualizing colloidal gold labels with soft X-ray microscopes, where scattering from small gold spheres used as labels dominates the image. Using numerical simulations of bright and dark field imaging, we compare different configurations of condenser and objective lenses in transmission X-ray microscopes, and configurations of detector and objective lens in scanning transmission X-ray microscopes. It is verified that the contrast of small, closely spaced features is strongly affected by changes in these configurations; the optimum situation is to have the condenser aperture (in TXM) or detector aperture (in STXM) equal to 3/2 that of the objective numerical aperture.

Effect of cooling on the motility and function of human spermatozoa.

Human spermatozoa were cooled from 37 to 0 degrees C at 10 degrees C min(-1) in 5 degrees C steps with 1 min equilibration at each step, the temperature control was +/- 0.1 degrees C. Spermatozoa were held at 0 degrees C for 5 min and then rewarmed at the same rate. No significant effect of cooling on the straight-line velocity was found using computer-aided semen analysis. The physiological function of spermatozoa was also examined before and after cooling using hypoosmotic swelling, ionophore-provoked acrosome reaction, and binding to fragments of human zonae pellucidae. Spermatozoa were cooled either in seminal plasma or in conventional IVF medium with or without fractionation by centrifugation through a discontinuous Percoll gradient. When spermatozoa were cooled and rewarmed in seminal plasma there was no significant change in either the ionophore-induced acrosome reaction or the binding to zona pellucida fragments. When spermatozoa were fractionated by centrifugation through Percoll an increased response in both was seen. However, following cooling and rewarming, a significant decline in the response of both occurred. We suggest that motility alone is not a reliable predictor of changes in other physiological functions of spermatozoa following cooling. Furthermore, short-term cooling appears to have no significant detrimental effect on normozoospermic samples and cold shock may be avoided in the clinical context by controlled cooling and warming.

X-ray microscopy of human spermatozoa shows change of mitochondrial morphology after capacitation.

Using X-ray microscopy two morphologically distinct states were observed of the human spermatozoan mitochondria: (i) compact and tightly wrapped around the axoneme, and (ii) morphologically transformed, i.e. with circular areas of high X-ray transmission, either loosely wrapped around the axoneme or distended. The spermatozoa were examined at two stages of their post-ejaculation maturation process, i.e. as present in fresh ejaculated semen and after in-vitro capacitation. X-ray microscopy allowed sample preparation that was as simple as for conventional light microscopy whilst giving high resolution (30 nm) imaging of samples in liquid media compatible with the requirements of live biological specimens. The specimens were not fixed, stained or metal coated. These features make X-ray microscopy useful in the study of cells, particularly cells in suspension. The relative frequencies of the two morphological states of the mitochondria in seminal plasma and after in-vitro capacitation were compared. In seminal plasma, almost all spermatozoa had compact and tightly wrapped mitochondria. After harvesting by swim-up technique, an increase in the morphologically transformed state had occurred. However, the greatest increase in the morphologically transformed state occurred when the sample had been incubated under capacitating conditions. In this case almost all spermatozoa had morphologically transformed mitochondria.

Production of ultra thin silicon foils.

Ultra thin silicon foils with thicknesses below 100 nm and large diameters have been produced. Foils with a thickness of 50-60 nm have been used as supporting foils for zone plates and as vacuum windows withstanding a pressure difference of 1 atm.