Structural analysis of herpes simplex virus by optical super-resolution imaging

Laine, Romain F.; Albecka, Anna; van de Linde, Sebastian; Rees, Eric J.; Crump, Colin M.; Kaminski, Clemens F.

dSTORM imaging. The imaging was performed on inverted total internal reflection fluorescence (TIRF) microscopes (Nikon TE-300 and Olympus IX-71, Japan) custom-built for dSTORM acquisition, as previously described 22, 65, 66 . The Nikon TE-300 microscope was equipped with an iXon3 897 EM-CCD camera (Andor, UK) and the Olympus IX-71 was equipped with two iXon DU897 EM-CCD cameras (Andor). The dyes were irradiated with lasers and their fluorescence collected with appropriate band-pass filters using the following laser-filter combinations: 640 nm diode laser (either iBeam Smart, Toptica, Germany, or Cube 640-100C, Coherent, USA) and 676/37 or 679/41 (Semrock, USA) for AF647, 561/568 nm laser (either 561 DPSS, Oxxius, France, or 568LP, Coherent) and 607/ 70 (Semrock) for AF568, 532 nm Nd:YAG laser (Nano 250-532-100, Linos, Germany) and 582/75 (Semrock) for ATTO532, 491 nm DPSS laser (Cobolt, Sweden) and 530/55 (Semrock) for EYFP and 405 nm diode laser with 530/55 (Semrock) for mTurquoise. For single-colour dSTORM imaging using AF647, the switching buffer was composed of 100 mM mercaptoethylamine (Sigma) in PBS solution at pH 8.2. For each field of view, a time series of up to 30,000 frames was acquired with 15 ms exposure and 1-2 kW cm À 2 irradiance. Subsequently, fluorescence emission from AF568 and mTurquoise were acquired (100 frames and 100 ms exposure).