Analytical Chemistry 2006 78 5987 - 5993.
Parallel Chemical Dosing of Subcellular Targets
R. Nielson and J. B. Shear
To characterize the role of spatially heterogeneous signaling in cellular function, methods are required for differentially exposing distinct regions of individual cells to externally applied reagents. Although a range of standard approaches exists for generating localized chemical gradients in culture, including puffer pipet spritzing and photolytic release of caged effectors, each is limited in key respects. Here, we report development of a cell-dosing strategy that addresses these limitations, providing the means to create steep gradients of any aqueous-miscible compound at essentially unlimited numbers of sites in parallel. In this approach, cells are cultured on a micrometer-thick polymer membrane that serves as a barrier between two stacked laminar-flow channels: one containing the cell culture and the other serving as a reagent flow cell. By focusing a pulsed laser beam onto one or more selected membrane positions, micrometer-diameter pores can be ablated upstream of desired cellular targets. Nascent pores thus serve as ports of entry into the culture environment for reagent streams capable of modifying subcellular features at positions potentially hundreds of micrometers from ablation sites. Importantly, individual reagent streams also can be rapidly eliminated by photo-cross-linking a protein plug over a selected pore. This versatile strategy for dynamically reshaping the chemical microenvironments in which cells reside should be useful in a variety of cell biology applications, ranging from neurotrophic modulation of neurite pathfinding to stimulation of cellular networks.