Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level

Multi-modal particle manipulator to enhance bead-based bioassays

By sequentially pushing micro-beads towards and away from a sensing surface, we show that ultrasonic radiation forces can be used to enhance the interaction between a functionalized glass surface and polystyrene micro-beads, and distinguish those that bind to the surface, ultimately by using an integrated optical waveguide implanted in the reflector to facilitate optical detection. The movement towards and immobilization of streptavidin coated beads onto a biotin functionalized waveguide surface is achieved by using a quarter-wavelength mode pushing beads onto the surface, while the removal of non-specifically bound beads uses a second quarter-wavelength mode which exhibits a kinetic energy maxima at the boundary between the carrier layer and fluid, drawing beads towards this surface. This has been achieved using a multi-modal acoustic device which exhibits both these quarter-wavelength resonances. Both 1-D acoustic modelling and finite element analysis has been used to design this device and investigate the spatial uniformity of the field. We demonstrate experimentally that 90% of specifically bound beads remain attached after applying ultrasound, with 80% of non-specifically bound control beads being successfully removed acoustically. This approach overcomes problems associated with lengthy sedimentation processes used for bead-based bioassays and surface (electrostatic) forces, which delay or prevent immobilisation. We explain the potential of this technique in the development of DNA and protein assays in terms of detection speed and multiplexing

Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis

Continuous cultivation in a glucose-limited chemostat was used to det. the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain. Maintenance coeffs. of 0.45 and 0.66 mmol of glucose g-1 h-1 were detd. for the wild-type and recombinant strains, resp. However, the max. molar growth yield of 82 to 85 g (cell dry wt.)/mol of glucose was found to be almost identical in both strains. A nonlinear relationship between the specific riboflavin prodn. rate and the diln. rate was obsd., revealing a coupling of product formation and growth under strict substrate-limited conditions. Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1. For mol. characterization of B. subtilis, the total amino acid compn. of the wild type was exptl. detd. and the complete building block requirements for biomass formation were derived. In particular, the murein sacculus was found to constitute approx. 9% of B. subtilis biomass, three- to five-fold more than in Escherichia coli. Estn. of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway. Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation. Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle. [on SciFinder (R)