Emccd 9100 13

FRAP analyses was carried out with fungi on cellophane under a spinning disk confocal microscope (UltraVIEW VoX, Perkin Elmer, Beaconsfield, Buckinghamshire, UK) equipped with a Yokogawa Nipkow CSU-X1 spinning disk scanner, Hamamatsu EMCCD 9100–13, and Nikon TiE inverted microscope with the Perfect Focus System. We used the UltraVIEW PK Device to photobleach GFP. For the FRAP analyses, the specific region of interest (ROI) covering the entire fluorescence in the ring was selected for bleaching. Twenty bleaching iterations were performed using a 488 laser power of 60%. Image scans were obtained with 15% 488 laser power before and after bleaching. For quantitative analyses, the GFP fluorescence recovery curves were measured as the mean intensity of the ROI pixels, normalized using the using Volocity software (Perkin Elmer), and graphed using Microsoft Excel.

Live-cell imaging was carried out under a spinning disk confocal microscope (UltraView VoX, Perkin Elmer, Beaconsfield, Buckinghamshire, UK) equipped with the Yokogawa Nipkow CSU-X1 spinning disk scanner, Hamamatsu EMCCD 9100-13, Nikon TiE inverted microscope with the Perfect Focus System as described previously (Liu et al., 2014 (link)). Acquired images were processed and analyzed using Volocity (Perkin Elmer), Image J (http://rsbweb.nih.gov/ij), MetaMorph (Molecular Devices, Sunnyvale, CA, United States).
The run-length distribution and the velocity distribution of GFP-KCBP were calculated in Origin software (OriginLab) by frequency counts. The mean values and 95% confidence interval were calculated in SAS (SAS Software), as described previously (Kong et al., 2015 (link)).