Electron beam fabrication of a microfluidic device for studying submicron-scale bacteria

Abstract

BackgroundControlled 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.ResultsHere 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.ConclusionsWe 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.

Zooming in to see the bigger picture: Microfluidic and nanofabrication tools to study bacteria

“…Although arranging individual bacterial cells in single file is conceptually simple, the ability to monitor the same cell for unlimited periods of time while having accurate temporal control over its chemical environment is a powerful new tool in microbiology (21)(22)(23)(24). In particular, studies of phenotypes that exhibit temporal 1251821-2 24 The scales at which nanofabrication and microfluidics can shape and control the environment matches well to the characteristic size scales of a bacterium (0.3 to 10 mm) and its subcellular components (for example, flagella and pili are~20 and~7 nm wide, respectively).…”

Review of methods to probe single cell metabolism and bioenergetics

“…The inclusion of media carrying trenches next to the tracks (Norman et al, 2013), as well as the use of channels of an orthogonal – asymmetric – geometry have also been reported (Long et al, 2013). Advanced microfabrication techniques based on electron beam lithography allowing the fabrication of much narrower tracks have been recently reported, primarily for the 1D confinement of much smaller bacteria (Moolman et al, 2013). …”

Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

“…Because cells absorb violet light strongly, when photoactivating fluorophores in live cells using UV or violet light (λ = 350–450 nm), one must minimize laser powers and exposure times to avoid damaging the cells. At high power densities, even visible radiation can generate reactive species that can damage or kill cells [118,120,121]. Wagner et al .…”