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Analysis of cellular responses to chemicals at high spatiotemporal resolution is required for precise understanding of intracellular signal transduction. Here, we demonstrated a novel method for applying different solutions to a part of or all of a cell at high spatiotemporal resolution. We fabricated a microfluidic device using polydimethylsiloxane, and the sharp interface between the two solution streams flowing in the channel was used for the application of different solutions. We constructed a computer-controlled system to control the interface movement precisely, rapidly, and reproducibly during positioning, and spatial and temporal resolutions attained were 1.6 Â¿m and 189 ms, respectively. We then applied the present system to the analysis of intracellular responses to chemicals. We were able to measure [Ca2+]i increases within 500 ms, when one laminar stream covered a part of the cell. This method can be used as a generic platform to investigate responses against drugs at the single cell level.