Orca flash4.0 lt plus camera

Images were acquired under a Zeiss AxioObserver Z1 epifluorescence microscopy system with 40×/1.3 oil Plan-Apochromat, 63×/1.4 oil Plan-Aprochromat and 100×/1.4 oil Plan-Aprochromat objectives and a Hamamatsu ORCA-Flash4.0 LT Plus camera. The system is calibrated and aligned by using 200 nm-diameter TetraSpeck microspheres (T7280, ThermoFisher). Ten to fifty z-stacking images were acquired at 200 nm intervals covering a range from 2 to 10 μm by using ZEN Blue software.
Deconvolution was carried out using Huygens Professional deconvolution software (SVI) with a measured point-spread-function generated by 200 nm diameter TetraSpeck microspheres. Classical maximum likelihood estimation method with iterations of 40–60 and signal-to-noise of 20–60 was applied.

Cells were seeded on 2-well or 4-well tissue culture chambers coverglass II (Sarstedt). SiR-DNA (Spirochrome) was added for at least 5 h prior to live-cell imaging. Images were acquired under a Zeiss AxioObserver Z1 epifluorescence microscopy system equipped with a heating and CO₂ chamber (Digital Pixel) by using 40×/0.6 Plan-Neofluar or 40×/1.3 oil Plan-Apochromat objectives and a Hamamatsu ORCA-Flash4.0 LT Plus camera. For mitotic progression analysis, 5–10 z-stacking images with 2 μm intervals were taken with the indicated time intervals by using ZEN Blue software. Images were processed using ImageJ software and in-focus z-plane images were manually extracted to make image montages. For imaging of DNA thread formation in live cells, 40×/1.3 oil Plan-Apochromat objective was used to capture eight z-stack images with 800 nm intervals and in-focus z-plane images were extracted using ImageJ software.

Most of the experiments were done with an epifluorescence system (Ti2 Nikon inverted microscope equipped with a Hamamatsu Orca Flash 4.0 LT Plus Camera). The following objectives were used: Plan Fluor 10X DIC and S Plan Fluor ELWD 20X DIC. Time lapse were acquired with the NIS elements software (version 4.60).
Z‐stacks of microwells were performed with a confocal spinning disk system (EclipseTi‐E Nikon inverted microscope equipped with a CSUX1‐A1 Yokogawa confocal head), an Evolve EMCCD camera (Photometrics), Plan Fluor 60X objective. Z‐stacks were acquired with Metamorph software (Universal Imaging).
FRAP, k + estimation and visualization of branched actin network formation was done on a total internal reflection fluorescence (TIRF) microscopy instrument composed of a Nikon Eclipse Ti, an azimuthal iLas² TIRF illuminator (Roper Scientific), a × 60 NA1.49 TIRF objective lens and an Evolve EMCCD camera (Photometrics). Time lapse and FRAP were done with Metamorph software (Universal Imaging).