Uplanfl 100 1.3 oil objective

For imaging, the cells were immobilized on 1% agarose [30 (link)] and photographed with a CoolSnap fx (Photometrics) charge-coupled device (CCD) camera mounted on an Olympus BX-60 fluorescence microscope through an UPLANFl 100×/1.3 oil objective (Olympus, Tokyo, Japan). Images were taken using modified acquisition software that used the program ImageJ by Wayne Rasband and analyzed using Object-J’s Coli-Inspector [17 (link)]. Statistical testing of normalized DAPI localization patterns was done using Kolmogorov–Smirnov testing available on http://www.physics.csbsju.edu/stats/ [31 ].

Unless stated otherwise, the following procedure was used to perform phase-contrast microscopy: Cells were first fixed by incubation in PBS containing 2.8% formaldehyde and 0.04% glutaraldehyde at room temperature for 15 min. The cells were kept cold during all subsequent procedures. To remove residual formaldehyde and glutaraldehyde, the cells were collected by low-speed centrifugation, transferred to a new tube and resuspended in fresh PBS. After three additional washings in PBS, the cells were resuspended in PBS before immobilization on 1% agarose in water slab-coated object glasses as described [66 (link)]. Samples were photographed with a CoolSnap fx (Photometrics) CCD camera mounted on an Olympus BX-60 fluorescence microscope through a UPLANFl 100 × /1.3 oil objective. Images were acquired with Micro-Manager (http://www.micro-manager.org/) with direct output of the desired hyperstack structure for ImageJ by Wayne Rasband (http://imagej.nih.gov/ij/).

For immunolocalization imaging, the cells were immobilized on 1% agarose [51 (link)] and photographed with a CoolSnap fx (Photometrics) charge-coupled device (CCD) camera mounted on an Olympus BX-60 fluorescence microscope through an UPLANFl 100×/1.3 oil objective (Olympus, Tokyo, Japan). Images were taken using modified acquisition software that used the program ImageJ by Wayne Rasband and analyzed using Object-J’s Coli-Inspector [47 (link)] (see for details supporting experimental procedures).