Fig. 1.

Gelatin-based micro-3D printing in the presence of bacteria. (A) Schematic depicting in situ microfabrication around cells encapsulated in thermally set gelatin. The red and blue arrows indicate steps performed at 37 °C or 18–22 °C, respectively. (B) Confocal fluorescence isosurfaces show isolated Pseudomonas aeruginosa microcolonies within a surface-anchored 2-pL pyramid (Top; two cells initially) and an untethered 3-pL torus (Bottom; partially transparent on the right views; one cell initially; see also Fig. S1). (C) Partially transparent and cut-out views of confocal fluorescence isosurfaces illustrate six physically segregated P. aeruginosa populations organized in three dimensions within a series of spheroid cavities (2–15 pL in volume) tethered to the glass surface by two cylindrical posts, where the right-most spheroid is vacant and the others initially contained either one or two cells. A side view of these clusters is shown in the lower image in which the upper portion of the red channel is digitally removed to reveal bacterial clusters. The upper image provides a top-down view of the same community, with the transparency of the red channel adjusted to reveal bacterial clusters. (D) A bright-field image acquired 10 min postfabrication (Left), and side-on and partially transparent top-down views of a confocal fluorescence isosurface (Center and Right, respectively) showing colony growth at 18 h. The spheroidal chambers are physically connected by channels such that motile P. aeruginosa cells can distribute throughout the 5-pL structure according to preference. Cylindrical posts extend from the base of each spheroid to tether the structure to the glass surface. See also Fig. S2. Cells are false-colored green in B–D for visualization. (Scale bars, 20 μm.)