(a) Bright field images of one imaging chamber captured in two fields of view taken after 24 hours growth in synthetic media. Image taken using a 40× long working distance air objective (NA=0.6) (b) Growth curve of cells in a chamber over 24 hours showing growth to high density (3.5×10⁹ cells/ml) under periodic diffusive exchange of nutrients and metabolites. (c) Compatibility of imaging platform with high-resolution microscopy. Fluorescent images taken with a 40× air objective (NA=0.6) show localization of a STE20-GFP fusion protein at the shmoo tip after exposure to pheromone. Inset shows a separate brightfield image taken using a 100× oil immersion objective (NA=1.3), demonstrating that the device is compatible with high resolution microscopy. Scale bar is 8 μm.

(a) Bright field image of colonies of bar1Δ cells after 40 minute exposure to 5nM α-factor in microfluidic chamber. (b) Close-up of upper two colonies from inset of panel a after 320 minute exposure to 5nM α-factor. Automated cell-tracking assigns a unique label to each cell to allow correlation of lineage, morphology, budding pattern and gene expression. (c) Fluorescent image of GFP expression in colonies from panel b showing lineage-dependent sensitivity to pheromone. (d) Time-lapse sequence of colonies highlighted in panel a showing the evolution of variability in morphological response to pheromone response. The red population undergoes growth arrest and displays an elongated phenotype while the green keeps budding over multiple generations. (e) EGFP concentration in each cell reporting the pheromone pathway activity. New lines starting at a zero value for GFP correspond to newborn cells. Plotted gene expression is normalized to cell volume and therefore represents an estimate of GFP concentration. (f) Lineage tree with time scale for both populations.

(a) Image of microfluidic device. A Canadian penny has been included for scale. (b) Working area of microfluidic device showing (1) array of 128 imaging chambers (8 columns * 16 rows), (2) column inlets for loading different strains, (3) 8 chemical inlets controlled by independent valves, (4) outlet ports, (5) fluidic multiplexer to deliver reagents to specified rows, (6) integrated peristaltic pump for on-chip formulation of stock reagents. (c) Cell loading and immobilization. Top: Cell suspension containing low melting temperature liquid agarose gel and media are introduced through column flow channels (blue) which are separated by the actuation of column valves (red) to prevent cross contamination of strains. Diffusion valves (yellow) are closed while the row valves (pink) are open. Middle: Actuation of row valves stops flow of cells and isolates each chamber. Column valves are released to flow media along rows, flushing excess cells and agarose to the outlet. Bottom: The chip is cooled for 3 min at 4 C to gel the agarose. Diffusion valves are opened to initiate diffusive exchange between medium in row channels and imaging chambers. Each row has a dedicated waste channel above the chambers for the priming of rows prior to initiating diffusion, thereby eliminating possible contamination from adjacent rows. (d) Optical micrograph showing the programmable and independent sequential perfusion of adjacent rows with 4 solutions of food dye. (e) Programmable on-chip mixing of 3 fluorescein stock solutions (1×, 10×, 100×) to create 16 concentrations. Each data point corresponds to the mean fluorescence of the 8 chambers on each row. Y-error bars represent standard deviations across the 8 chambers of each row. Measurements were taken at edge of the chamber furthest away from the diffusion channel and therefore reflect the longest possible diffusion time within chambers. (f) Temporal modulation of the chemical environment demonstrated by constant perfusion with repeated pulses alternating between medium and fluorescein. Greater than 95% equilibration is achieved within 9 minutes. Note that data presented in panels 1e and 1f was taken from chambers filled with polymerized agarose as in biological experiments.

(a) Data from one of 128 chambers showing variation of GFP expression for wild-type cells subject to constant 10 nM α-factor stimulation. Each data point represents one time-point for a single cell measured every 20 min over 9 h. (b) Mean expression data from one column of the device presented as a surface plot of GFP concentration as a function of time and α-factor concentration. (c)-(i) GFP expression surface plots for seven mutants studied on the same device. Each surface response plot represents approximately 200,000 individual data points.