Background: Controlled restriction of cellular movement using microfluidics allows one to study individual cells to\r\ngain insight into aspects of their physiology and behaviour. For example, the use of micron-sized growth channels\r\nthat confine individual Escherichia coli has yielded novel insights into cell growth and death. To extend this approach\r\nto other species of bacteria, many of whom have dimensions in the sub-micron range, or to a larger range of growth\r\nconditions, a readily-fabricated device containing sub-micron features is required.\r\nResults: Here we detail the fabrication of a versatile device with growth channels whose widths range from 0.3�µm\r\nto 0.8�µm. The device is fabricated using electron beam lithography, which provides excellent control over the shape\r\nand size of different growth channels and facilitates the rapid-prototyping of new designs. Features are successfully\r\ntransferred first into silicon, and subsequently into the polydimethylsiloxane that forms the basis of the working\r\nmicrofluidic device. We demonstrate that the growth of sub-micron scale bacteria such as Lactococcus lactis or\r\nEscherichia coli cultured in minimal medium can be followed in such a device over several generations.\r\nConclusions: We have presented a detailed protocol based on electron beam fabrication together with specific dry\r\netching procedures for the fabrication of a microfluidic device suited to study submicron-sized bacteria. We have\r\ndemonstrated that both Gram-positive and Gram-negative bacteria can be successfully loaded and imaged over a\r\nnumber of generations in this device. Similar devices could potentially be used to study other submicron-sized\r\norganisms under conditions in which the height and shape of the growth channels are crucial to the experimental\r\ndesign.
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