Andor iq2

S. pombe strains were grown in YE5S media in the log phase (OD₅₉₅ < 0.8) for 36 h. The cells were harvested at OD₅₉₅ 0.4–0.7 (4–7 × 10⁶ cells/ml), washed twice with EMM5S media, and mounted on EMM5S 2% agarose pads. Images were acquired at room temperature with an Olympus IX-71 microscope with a 100×/NA 1.4 Plan Apo lens (Olympus) and an Andor CSU-X1 spinning disk confocal system with an iXON-EMCCD camera (Andor Technology). Fluorescence was excited using a Coherent OBIS 488-nm LS 20-mW laser with adjustable power and an internal power meter. Time-lapse movies of endocytic actin patches were acquired with Andor iQ2 software (Andor Technology); other images were acquired with the μManager 1.4 plugin (Stuurman et al, 2010 ) for ImageJ (Schneider et al, 2012 (link)).

For quantification analysis, z-stack images covering an entire nuclear signal were acquired on an Axio Observer.Z1 (Zeiss) microscope equipped with a CSU-X1 confocal scanner unit (Yokogawa), iXon3 DU897E-CS0 camera (Andor) and Plan-Apochromat 100×/1.46 oil lens (Zeiss) with a spacing of 0.22 μm using Andor iQ2 software (Andor). Images in Fig. 1C,E and Fig. 8A,C,E were acquired by same method. Other images were acquired on an Axio Observer.Z1 (Zeiss) equipped with a LSM700 scanning module and an Objective Plan-Apochromat 63×/1.46 oil lens (Zeiss) using ZEN 2009 software (Zeiss). z-stack images were acquired with a spacing of 0.38 μm. Images shown are maximum projection of several slices around the ectopic alphoid^tetO integration site or whole nucleus. Counting assays were performed in the same microscopic condition. Visible signals on the ectopic site in interphase cells were counted.

Comparative analysis of Dendra2 protein trafficking was performed using AndorIQ2 software (Andor). The mean fluorescence intensity was quantified in user defined ROIs and exported to excel for further analysis. The data was normalized by subtracting the background signal and subsequently quantified over time. Movies of Dendra2 protein trafficking were generated using IMARIS (Bitplane).