Uplanapo objective

For analysis of localisation of tagged proteins or soluble fluorescent protein by native fluorescence, cells were harvested from mid-log phase cultures, washed twice in phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄) and allowed to settle for 5 min onto glass slides at ~2x10⁷ cell ml^-1 density. Cells were fixed for 5 min in 2% (w/v) formaldehyde, permeabilised in -20°C methanol for 10 min, re-hydrated in PBS and incubated with 15 ng ml^-1 4′,6-diamidino-2-phenylindole for 5 min, before mounting in 1% (w/v) 1,4-diazabicyclo[2.2.2]octane, 90% (v/v) glycerol, 50 mM sodium phosphate, pH 8.0. Counter-staining of cells expressing ESP14-sfGFP was by incubation with 25 μg ml^-1 tomato lectin conjugated to AlexaFluor 594 (Invitrogen) for 20 min. Images were captured on an Olympus BX51 microscope equipped with a 100x UPlanApo objective (1.35 NA; Olympus) and Retiga R1 CCD camera (Qimaging) without binning. All images of fluorescent proteins were captured at equal exposure settings without prior illumination. Images for level comparison were also processed in parallel with the same alterations to minimum and maximum display levels, except where stated. Image acquisition was controlled by μManager open source software [58 (link)]. Processing and analysis were performed in ImageJ [59 (link)].