Apo tirf 100 oil objective

Time-lapse TIRFM movies were taken on at least two different days for each strain and condition. Cells were first grown in shaking flasks or U-bottom 96-well cell culture plates (CellStar) at 37 °C to reach early exponential phase (OD₆₀₀∼0.1). One microlitre of the liquid culture was spotted onto a thin agarose pad (1%), topped by a coverslip and immersion oil, and mounted immediately in the temperature-controlled microscope stage. All experiments were done inside the incubation chamber at 37 °C, within 10 min after taking the sample. For all GFP fusions exposure time was 100 ms. Inter-frame intervals were 1 s over 2-min movies. Imaging was performed on an inverted microscope (Nikon Ti-E) with an Apo TIRF × 100 oil objective (Nikon, NA 1.49), with either diode-pumped solid-state lasers (Cobolt Calypso, 50 mW, 491 nm and Cobolt Jive, 50 mW, 561 nm) or an iLas2 laser coupling system from Roper Scientific (150 mW, 488 nm and 50 mW, 561 nm). Images were collected with an electron-multiplying charge-coupled device camera (iXON3 DU-897, Andor) at maximum gain setting (300) attached to a × 2.5 magnification lens. Final pixel size was 64 nm. Image acquisition was controlled by the NIS-Elements (Nikon) or Metamorph v.7 software packages.

Immediately prior to imaging, 10% sodium hydroxide (w/v) was mixed with pure 200-proof ethanol for 45 minutes to prepare a mild sodium ethoxide solution. Glass-bottom dishes with ultrathin retina sections were immersed for 30–45 minutes for chemical etching of the resin. Etched sections were then washed and dried on a 50°C heat block. The following STORM imaging buffer was prepared: 45 mM Tris (pH 8.0), 9 mM NaCl, and oxygen scavenging system: 0.7 mg/mL glucose oxidase (Amresco) + 42.5 μg/mL catalase (MilliporeSigma), 10% (w/v) glucose + 100 mM MEA (i.e., l-cysteamine, Chem-Impex) + 10% VECTASHIELD (Vector Laboratories). Imaging buffer was added onto the dried, etched sections and sealed with a second number 1.5 coverslip for imaging.
Imaging was performed on the Nikon N-STORM system, which features a CFI Apo TIRF 100× oil objective (NA1.49) on an inverted Nikon Ti Eclipse microscope. STORM image acquisition was controlled by NIS-Elements Ar software.
To begin a STORM acquisition, both the 561 nm and 647 nm laser lines were increased to maximum power to photobleach the fluorescence and initiate photoswitching. Imaging frames were collected at approximately 56 frames per second. A total of 50,000 frames were collected for each imaging experiment.