title: Electron beam fabrication of a microfluidic device for studying submicron-scale bacteria.
abstract: BACKGROUND
  Controlled restriction of cellular movement using microfluidics allows one to study individual cells to gain insight into aspects of their physiology and behaviour. For example, the use of micron-sized growth channels that confine individual Escherichia coli has yielded novel insights into cell growth and death. To extend this approach to other species of bacteria, many of whom have dimensions in the sub-micron range, or to a larger range of growth conditions, a readily-fabricated device containing sub-micron features is required.
  
  
  RESULTS
  Here we detail the fabrication of a versatile device with growth channels whose widths range from 0.3 μm to 0.8 μm. The device is fabricated using electron beam lithography, which provides excellent control over the shape and size of different growth channels and facilitates the rapid-prototyping of new designs. Features are successfully transferred first into silicon, and subsequently into the polydimethylsiloxane that forms the basis of the working microfluidic device. We demonstrate that the growth of sub-micron scale bacteria such as Lactococcus lactis or Escherichia coli cultured in minimal medium can be followed in such a device over several generations.
  
  
  CONCLUSIONS
  We have presented a detailed protocol based on electron beam fabrication together with specific dry etching procedures for the fabrication of a microfluidic device suited to study submicron-sized bacteria. We have demonstrated that both Gram-positive and Gram-negative bacteria can be successfully loaded and imaged over a number of generations in this device. Similar devices could potentially be used to study other submicron-sized organisms under conditions in which the height and shape of the growth channels are crucial to the experimental design.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, Delft, The Netherlands.
authors.forename: M Charl
authors.initials: MC
authors.lastname: Moolman
authors.forename: Zhuangxiong
authors.initials: Z
authors.lastname: Huang
authors.forename: Sriram Tiruvadi
authors.initials: ST
authors.lastname: Krishnan
authors.forename: Jacob W J
authors.initials: JW
authors.lastname: Kerssemakers
authors.forename: Nynke H
authors.initials: NH
authors.lastname: Dekker
mesh_terms: D004129:Dimethylpolysiloxanes; D004583:Electrons; D004926:Escherichia coli / Q000166:cytology* / Q000254:growth & development; D005456:Fluorescent Dyes / Q000378:metabolism; D006046:Gold; D007734:Kymography; D013294:Lactococcus lactis / Q000166:cytology* / Q000254:growth & development; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008855:Microscopy, Electron, Scanning; D055616:Microtechnology / Q000295:instrumentation*; D012825:Silicon; D013997:Time Factors
chemical_list: D004129:Dimethylpolysiloxanes; D005456:Fluorescent Dyes; D006046:Gold; D012825:Silicon