Above left: Culture of human bronchial cells (~ 40 µm tall). Center: Spots of uncaged fluoresciene being transported in normal HBE cultures. Upper culture with, lower culture without mucus. Each pair of images collected at 30 sec intervals. Note lack of transport in mucus-free culture. In transported spot , note the lack of smearing, indicating fluoresceine in PCL being transported along with mucus. Right: XZ images at edge of transporting spot. Red is dextran; green is uncaged fluoresciene. Top row is before uncaging, 2nd - 4th rows, row t=0, 15, 30 sec, resp. Note green fluorescence transport, left to right. Vertical uniformity indicates PCL transport with mucus.

Behavior and fate of periciliary liquid during mucociliary clearance: Cilia, slender µm-diameter projections of the plasma membrane with cores containing microtubule-based motors, rise approximately 7µm from the cell surface, and beat in a whip-like fashion in a layer of mucus-free PCL. Previous theoretical work had predicted that the PCL was more or less stationary during the transport of mucus, which is propelled forward by the power stroke of the cilium. In a vertical profile, it was only the outer about 2µm of PCL that was predicted to undergo net movement, a narrow zone which experiences the cilium's power stroke but which lies above the level of the return stroke [Fulford1986].

What we observed in a recent study [Matsui1998a], however, was contrary to this prediction, namely that within the z-axis resolution of the confocal microscope the entire PCL was transported during mucus transport, and it was transported uniformly at the same rate as the overlying mucus gel. We hypothesized that ciliary mixing of the PCL effectively distributes the momentum imparted to the PCL by its frictional interactions with mucus.

We are testing this hypothesis by using the 3DFM to track the movements of single microspheres added at low concentration to the ASL of human airway epithelial cell cultures [Matsui1998a]. We predict that individual particles will suffer turbulent-like movements throughout the height of the PCL. The 3-D magnetic force capabilities of the instrument should enable us to determine the vertical distribution of propulsive forces generated by the cilia on individual cells. By placing the system under position feedback, we should be able to measure the forces needed to maintain the position of the particle, and therefore determine the forces experienced during the cilium beating cycle.

Effects of volume reduction on mucus viscoelasticity: Mucus is a gel whose scaffolding is made of mucin polymers. The airway mucins are linear polymers of mucin subunits with lengths of at least 30µm. [Sheehan1991; Gendler1995] The mucin monomers are polymerized through end-to-end disulfide bonds and each monomer is a glycoconjugate, 80 - 90 % carbohydrate, with molecular weights exceeding 10 MD. The gels are tangled networks with no inter-molecular crossbridges. [Verdugo1990; Bansil1995] In the ASL of normal humans, the concentrations of mucins in the mucus is approximately 1% by weight, but in cystic fibrosis (CF), an inherited disease characterized by a hyperabsorption of Na+ and liquid from the ASL, the mucin concentra-tion increases to as high as 3 - 4% [Matsui1998b]. We hypothesize that the changes in physical properties of the concentrated mucus in CF (increased viscosity, reduced elasticity, increased adhesivity) impede its transport, creating an environment favorable for bacterial colonization and biofilm formation.

Past investigations of mucus viscoelastic properties [Litt1970; Litt1973; King1974] relied primarily upon cone and plate, or vibrating microball viscometers, techniques that shear and/or align mucin molecules in the gel during the measurement. We propose to use the sub-micron single particle tracking and magnetic force capabilities of the new 3DFM to map the viscoelastic properties of the mucus at high resolution. In samples with low viscosity, the tracking of the Brownian motion of the particles can map the frequency-dependent storage and loss moduli [Gittes1997b; Mason1997]. At high viscosities, it will be necessary to drive the system with the magnetic force capabilities.

In parallel experiments, we are presently studying the growth behavior and adherence of bacteria in mucus subjected to the same series of experimental manipulations. In the final analysis, we hope to provide quantitative data on the changes in mucus viscoelastic properties caused by clinically relevant volume depletion, as well as the effects of these changes on the growth properties of the bacteria that infect CF patients.

Implications for 3DFM: These applications are the first being tested with the system. The viscoelastic property measurements require only particle tracking as a start, as the bead position fluctuations are related to the local viscosity through the fluctuation dissipation theorem. The second measurement requires the local measurement of viscoelastic properties and will require some manipulation of the magnetic particle and long-range tracking.