Slide chamber assembly for live cell imaging. Assembly of simple slide-based chambers for live cell imaging for upright (A–C) and inverted (D–F,G–I) microscopes. (A–C) For an upright scope filter-sterilized media containing 2% ultrapure agarose is poured between 2 × 1–2-mm thick spacers that have been fixed 40 mm apart onto a clean microscope slide (A). Once cooled and set, cells are added directly to the surface of the agarose pad (B), which is then covered with a 25 × 50 mm coverslip (C) held in place with wax or tape. (D–F) A simple flow cell. Coverslips are coated with lectin by transiently applying a solution (e.g., 1 mg/mL soybean lectin) before immediately being removed and the coverslip dried (D). The coverslip is inverted and placed carefully onto a slide with appropriately spaced double-sided sticky tape (E). Yeast cells are adhered to the surface of the lectin-coated 25 × 50 mm coverslip and excess cells are washed off with media. The space between coverslip and slide is filled with preconditioned minimal media (F) before the slide is mounted coverslip down onto an inverted microscope. (G–I) Imaging under an agarose pad. A molten solution of 0.5% ultrapure agarose in filter-sterilized medium is poured onto a 2-cm coverslip resting on a smaller diameter coin (G). When the agarose has set, a drop of cells is placed on the oxygen-permeable base of the culture dish and the agarose pad/coverslip is inverted onto the cells. The plate is imaged in a Perspex box containing a damp tissue to maintain a humid atmosphere throughout imaging. After 20 min of settling, cells remain stationary for days, although the flexibility of the membrane demands focusing approximately every 10 min during an imaging period.