Kyle Campbell

MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.

The mating pathway in Saccharomyces cerevisiae has been the focus of considerable research effort, yet many quantitative aspects of its regulation still remain unknown. Using an integrated approach involving experiments in microfluidic chips and computational modelling, we studied gene expression and phenotypic changes associated with the mating response under well-defined pheromone gradients. Here we report a combination of switch-like and graded pathway responses leading to stochastic phenotype determination in a specific range of pheromone concentrations. Furthermore, we show that these responses are critically dependent on mitogen-activated protein kinase (MAPK)-mediated regulation of the activity of the pheromone-response-specific transcription factor, Ste12, as well as on the autoregulatory feedback of Ste12. In particular, both the switch-like characteristics and sensitivity of gene expression in shmooing cells to pheromone concentration were significantly diminished in cells lacking Kss1, one of the MAP kinases activated in the mating pathway. In addition, the dynamic range of gradient sensing of Kss1-deficient cells was reduced compared with wild type. We thus provide unsuspected functional significance for this kinase in regulation of the mating response.

Self-Organization in High-Density Bacterial Colonies: Efficient Crowd Control

Colonies of bacterial cells can display complex collective dynamics, frequently culminating in the formation of biofilms and other ordered super-structures. Recent studies suggest that to cope with local environmental challenges, bacterial cells can actively seek out small chambers or cavities and assemble there, engaging in quorum sensing behavior. By using a novel microfluidic device, we showed that within chambers of distinct shapes and sizes allowing continuous cell escape, bacterial colonies can gradually self-organize. The directions of orientation of cells, their growth, and collective motion are mutually correlated and dictated by the chamber walls and locations of chamber exits. The ultimate highly organized steady state is conducive to a more-organized escape of cells from the chambers and increased access of nutrients into and evacuation of waste out of the colonies. Using a computational model, we suggest that the lengths of the cells might be optimized to maximize self-organization while minimizing the potential for stampede-like exit blockage. The self-organization described here may be crucial for the early stage of the organization of high-density bacterial colonies populating small, physically confined growth niches. It suggests that this phenomenon can play a critical role in bacterial biofilm initiation and development of other complex multicellular bacterial super-structures, including those implicated in infectious diseases.

Femtosecond laser-drilled capillary integrated into a microfluidic device

Recent growth in microfluidic technology is, to a large extent, driven by soft lithography, a high-throughput fabrication technique where polymer materials, such as poly(dimethyl) siloxane (PDMS), are molded to form microscopic channel networks. Nevertheless, the channel architectures that can be obtained by molding are limited. We address this limitation by using femtosecond laser micromachining to add unmoldable features to the microfluidic devices. We apply laser ablation to drill microcapillaries, with diameters as small as 0.5μm and aspect ratios as high as 800:1, in the walls of molded PDMS channels. Finally, we use a laser-drilled microcapillary to trap a polystyrene bead by suction and hold it against a shear flow.

On-chip microfluidic tuning of an optical microring resonator

We describe the design, fabrication, and operation of a tunable optical filter based on a bus waveguide coupled to a microring waveguide resonator located inside a microchannel in a microfluidic chip. Liquid flowing in the microchannel constitutes the upper cladding of the waveguides. The refractive index of the liquid controls the resonance wavelengths and strength of coupling between the bus waveguide and the resonator. The refractive index is varied by on-chip mixing of two source liquids with different refractive indices. We demonstrate adjustment of the resonance by 2nm and tuning the filter to an extinction ratio of 37dB.

Bound attractant at the leading vs. the trailing edge determines chemotactic prowess

We have analyzed chemotaxis of neutrophil-differentiated HL60 cells in microfluidic devices that create exponential gradients of the chemoattractant, f-Met-Leu-Phe (fMLP). Such gradients expose each cell to a difference in fMLP concentration (ΔC) across its diameter that is directly proportional to the ambient concentration (C) at that cells position in the gradient, so the ratio ΔC/C is constant everywhere. Cells exposed to ambient fMLP concentrations near the constant of dissociation (Kd) for fMLP binding to its receptor (≈10 nM) crawl much less frequently when ΔC/C is 0.05 than when it is 0.09 or 0.13. Hence, cells can detect the gradient across their diameter without moving and, thus, without experiencing temporal changes in attractant concentration. At all ΔC/C ratios tested, the average chemotactic prowess of individual cells (indicated by the distance a cell traveled in the correct direction divided by the length of its migration path) is maximal for cells that start migrating at concentrations near the Kd and progressively decreases at higher or lower starting concentrations.

Generation of complex concentration profiles in microchannels in a logarithmically small number of steps

We describe the principles of design and the architecture of planar microfluidic networks producing concentration gradients with the shape of any given monotonic function. Each microfluidic network is fed by two separate source solutions and delivers to its outlet a set of N solutions that all differ in concentration. Inside the network, the source solutions flow through a series of k = log(2)(N-1) stages, where they are repeatedly split and mixed. Streams of the solutions emerging from the network are combined to create a single stream with the desired shape of the concentration profile across the direction of flow. To demonstrate the functionality of the proposed architecture, we have built and tested three networks with k = 4 and N = 17 that generate an exponential concentration profile, a linear profile, and a profile with a shape of two fused branches of a parabola.

A microfluidic 2×2 optical switch

A 2×2 microfluidic-based optical switch is proposed and demonstrated. The switch is made of an optically clear silicon elastomer, Polydimethylsiloxane (PDMS), using soft lithography. It has insertion loss smaller than 1 dB and extinction ratio on the order of 20 dB. The device is switching between transmission (bypass) and reflection (exchange) modes within less than 20 ms

Set of two orthogonal adaptive cylindrical lenses in a monolith elastomer device

A microfluidic device that operates as a set of two adaptive cylindrical lenses focusing light along two orthogonal axes is designed, fabricated and characterized. The device is made out of a silicon elastomer, polydimethylsiloxane, using soft lithography, and consists of a few chambers separated by flexible membranes and filled with liquids of different refractive indices. The cylindrical lenses can be both converging and diverging; their focal lengths are varied independently and continuously adjusted between -40 and 23 mm by setting pressure in the chambers. Applications of the device to shaping of a laser beam, imaging and optical signal processing are demonstrated.

Spherical aberration correction in nonlinear microscopy and optical ablation using a transparent deformable membrane

We describe the design and utilization of a deformable membrane to minimize the negative spherical aberration that occurs when a standard water-dipping objective is used to focus within a higher-index sample. In connection with two-photon laser scanning microscopy, we demonstrate twofold improved axial resolution of structures as deep as 1mm in gels and brain tissue. In conjunction with plasma-mediated ablation, we demonstrate enhanced production of optical damage deep within a glass substrate. The present method provides a simple and inexpensive correction for a limited yet important class of optical aberrations.

Pneumatically actuated adaptive lenses with millisecond response time

We describe the design and operation of two pneumatically actuated adaptive lenses with refractive power adjustable by >4 diopters. The refractive element in the lenses is a transparent flexible membrane. When vacuum is applied, the membrane is deformed and becomes a diverging meniscus lens. The two lenses have membranes with 18 and 12mm diameters and 5 and 2ms transition times after sudden changes in pressure. Both lenses can be driven at 500Hz with 4 diopter variation in the refractive power. The adaptive lenses can be used for longitudinal scanning in three-dimensional imaging and for fast focusing.