Large scan area high-speed atomic force microscopy using a resonant scanner.

A large scan area high-speed scan stage for atomic force microscopy using the resonant oscillation of a quartz bar has been constructed. The sample scanner can be used for high-speed imaging in both air and liquid environments. The well-defined time-position response of the scan stage due to the use of resonance allows highly linearized images to be obtained with a scan size up to 37.5 mum in 0.7 s. The scanner is demonstrated for imaging highly topographic silicon test samples and a semicrystalline polymer undergoing crystallization in air, while images of a polymer and a living bacteria, S. aureus, are obtained in liquid.

Immobilizing live bacteria for AFM imaging of cellular processes.

Coccoid cells of the bacterial species Staphylococcus aureus have been mechanically trapped in lithographically patterned substrates and imaged under growth media using atomic force microscopy (AFM) in order to follow cellular processes. The cells are not perturbed as there is no chemical linkage to the surface. Confinement effects are minimized compared to trapping the cells in porous membranes or soft gels. S. aureus cells have been imaged undergoing cell division whilst trapped in the patterned substrates. Entrapment in lithographically patterned substrates provides a novel way for anchoring bacterial cells so that the AFM tip will not push the cells off during imaging, whilst allowing the bacteria to continue with cellular processes.

The role of mononuclear-cell recruitment to the lungs in the development and expression of immunity to Schistosoma mansoni.

The role of pulmonary cellular responses in the induction and expression of immunity to Schistosoma mansoni has been evaluated. From experiments in which mice were challenged at intervals after vaccination, we conclude that the resistance mechanism operating in the lungs develops between days 11 and 20. Injection of 51Cr-labelled splenocytes revealed that mononuclear cell recruitment to the lungs, stimulated by the arrival of attenuated schistosomula, intensified progressively between days 10 and 16 post-vaccination. The increased cellularity was reflected in a 19.5% augmentation in the wet-weight index (WW). The period of enhanced cell recruitment therefore coincided with the build-up of resistance. By day 22 post-vaccination infiltration had declined, whilst WW remained elevated. This indicates the persistence of recruited cells, which include schistosome-reactive T lymphocytes, in the lungs. We were unable to demonstrate augmented recruitment of 51Cr-labelled cells after challenge of vaccinated mice, but WW rose slightly, peaking on day 12. Although clearly of a lower order than the primary response, the secondary response was more rapid, implying the existence of immunological memory. These results accord with the concept that schistosome-reactive T lymphocytes recruited after vaccination 'arm' the lungs against the arrival of challenge parasites.

The magnitude and kinetics of delayed-type hypersensitivity responses in mice vaccinated with irradiated cercariae of Schistosoma mansoni.

A footpad assay was used to measure the DTH of mice to soluble worm antigens (SWAP), and to living day 7 lung schistosomula, following vaccination and challenge infections with Schistosoma mansoni. DTH to SWAP was first observed on day 10, and reached its maximum on day 17 post-vaccination. Treatment of mice with anti-CD4 antibody on the 3 days prior to footpad challenge completely abrogated this response. Reactivity to living parasites was of a lower order than that to SWAP; it also peaked earlier, on day 10 post-vaccination. By day 35, responsiveness to both sets of antigens had declined almost to control levels. There was no correlation between the level of DTH to living schistosomula, at any time, and the degree of resistance subsequently developed. Percutaneous challenge of vaccinated mice was followed by a resurgence of reactivity to SWAP. This secondary response occurred more rapidly than the primary response, peaking on day 7 post-challenge, and was of a similar magnitude. We were unable to detect a similar recall of DTH to living schistosomula, possibly because the assay was insufficiently sensitive. We conclude that the intensity and kinetics of DTH responsiveness are crucial features of the irradiated vaccine model, and suggest that further investigation of cell-mediated immune reactions, particularly those occurring in the lungs, is vital to a better understanding of events underlying the development and expression of immunity.