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Control and manipulation of pathogens with an optical trap for live cell imaging of intercellular interactions.

ABSTRACT: The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

SUBMITTER: Tam JM

PROVIDER: S-EPMC3013098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We illustrate control of a polarized laser optical trapping potential landscape through the nonideal mixing of binary liquids. The inherent trapping potential asymmetry (ITPA) present in the trapping region results from the asymmetric intensity distribution in focal volume due to the high numerical aperture objective lens. Experimentally, we show that this ITPA effect can be modified and/or removed by the use of binary liquid mixtures. From our femtosecond optical tweezers experiments, we determine the topograph of the trapping potential base on the fluctuation-dissipation theorem. Additionally, the Brownian motion of the trapped bead is sensitive to the frictional force (FF) of the surroundings that is exerted by clusters of water and alcohol binary mixture through extended hydrogen bonding. Thus, using these two effects, ITPA and FF of the medium, we have shown that one can indeed modify the effective trapping potential landscape. Water-alcohol binary mixtures display a nonlinear dependence on the microrheological properties of the solvent composition as a result of rigid cluster formation. Volumetrically, at about 30% methanol in water binary mixture, the trapping asymmetry is minimal. In this particular binary mixture composition, the hydrophobic part of the methanol molecule is surrounded by 'cages' of water molecules. Enhanced H-bonding network of water molecules results in higher viscosity, which contributes to the higher frictional force. Increased viscosity decreases the degree of anisotropy due to hindered dipolar rotation. However, at higher methanol concentrations, the methanol molecules are no longer contained within the water cages and are free to move, which decrease their overall bulk viscosity. Thus, for pure solvents, experimentally measured anisotropy matches quite well with the theoretical prediction, but this fails in case of the binary mixtures due to the increased frictional force exerted by binary mixtures that result from the formation of cage-like structures.

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Control and manipulation of pathogens with an optical trap for live cell imaging of intercellular interactions.

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