The Aaron Klug Centre for Imaging and Analysis

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Method for making holey grids

  • rinse glass slides with dilute teepol and wipe dry
  • 50 ml 0.5% formvar in chloroform in a beaker (dont leave around to evaporate, nb parafilm dissolves in chloroform vapour)
  • add 10 drops (5 drops for more sparse holes, for trapping microcrystals on the grid) of 50% glycerol from a long Pasteur while stirring.
  • sonicate chloroform mixture at full power for 1-3 min (probe sonicator, large probe)
  • dip prepared slides into the sonicated mixture immediately (ie next to sonicator, not after returning to lab). Drain on tissue. Holes may vary with position along the slide as a function of drainage.
  • Assess hole size and density on slide by phase contrast microscopy. Adjust sonication procedure if not right.
  • Fill a crystallizing dish with distilled H2O, skim dust off surface with velin tissue. Score the dipped slide around the edges with a diamond glass cutter or sharp implement, making sure the score marks intersect at the corners. Float the formvar film off onto the water by dipping the slide in, at a steep angle at first, slowly, and then easing it under the surface as the film lifts off (this does not always work!). Drop grids onto film dull side down, scoop up grids with filter paper, allowing the film to stick first. Let the filter paper dry.
  • soak filter paper with methanol ½ hr in petri dish (use paper as wick) to remove glycerol
  • Check formvar grids individually by phase contrast, if to be used for cryo work.
  • Thick carbon coat on grids
  • fresh filter paper - soak in chloroform to remove formvar, in closed petri dish.
  • When grids become hydrophobic, glow discharge on day of use.
  • apply sample, stain
  • For some samples, a very light post carbon coat could be used to stabilize the film of stain in the holes (although John Finch said for viruses torn holes are best, because otherwise the film is too strained, and he doesnt do a final coat)
  • For microcrystals: try thinner carbon and fewer holes.
  • Neg stain test for cryo samples - blot from back and see how many particles left in holes.
  • Try embedding specimens in glucose or tannin.