Similar protocols

Pipeline publication

[…] o/n). 2.5 × 105 cells, from a log phase culture, were added to a glass-bottom dish (Iwaki; coated with 5 mg/mL lectin from Bandeiraea simplicifolia BS-I (Sigma)) for live cell imaging. A stage top incubator with lens heater (HoKai Hit) was used to maintain sample temperature at 30 °C. Each sample was used for a maximum three hours of imaging before cells reach stationary phase., Microscopic images of asynchronous cells were obtained using an iXon3 897 EMCCD camera (Andor) connected to Yokokawa CSU-W1 spinning-disc scan head (Yokokawa Electric Corporation) and an OlympusIX83 microscope (Olympus) with an UPlanSApo 100× NA 1.4 objective lens (Olympus). Pictures were captured and analyzed using MetaMorph Software (Molecular Devices). Optical section data (41 focal planes with 0.2 μm spacing every 30 sec.) were collected. Time-lapse sequences were deconvolved using Huygens image analysis software (Scientific Volume Imaging)., Microscope images of the interphase cells were used to examine the distance between SPB and LacO-labeled genomic loci. Deconvolved time lapse sequences for each locus were analyzed using the IMARIS software (Bitplane). The center of the fluorescent focus was tracked over 30 minutes at 30 second intervals for each strain. In order to remove the movement of the nucleus, the distance between SPB and LacO-labeled genomic loci was calculated. The distribution of the distance was obtained using the following number of data points: n = 530 for lys1, n = 2877 for ade6, n = 3595 for his2, n = 2645 for ade3, n = 2507 for ade8, n = 2249 for sod2., G1 phase S. pombe chromosomes were modeled as worm-like polymer chains confined to lie within the nuclear space, excluded from the nucleolus, and subjected to both tethering restraints (i.e. centromere and telomere positioning) and inter- and int […]

Pipeline specifications

Software tools MetaMorph, Huygens, Imaris