Cfi plan apo vc 100 1.4 numerical aperture oil objective

Live RPE1 and HeLa cells were imaged using Bruker Opterra Multipoint Scanning Confocal Microscope⁷⁵ (link) (Bruker Nano Surfaces, Middleton, WI, USA). The system was mounted on a Nikon Ti-E inverted microscope equipped with a Nikon CFI Plan Apo VC ×100/1.4 numerical aperture oil objective (Nikon, Tokyo, Japan). During imaging, cells were maintained at 37 °C in Okolab Cage Incubator (Okolab, Pozzuoli, NA, Italy). A 22 μm slit aperture was used for RPE1 and 60 μm pinhole for HeLa cells. The xy-pixel size was 83 nm. For excitation of GFP and mCherry fluorescence, a 488 and a 561 nm diode laser line was used, respectively. For SiR-dyes, a 640 nm diode laser line was used. The excitation light was separated from the emitted fluorescence by using Opterra Dichroic and Barrier Filter Set 405/488/561/640. Images were captured with an Evolve 512 Delta EMCCD Camera (Photometrics, Tucson, AZ, USA) with no binning performed. To cover the whole metaphase spindle, z-stacks were acquired at 30–60 focal planes separated by 0.5 μm with unidirectional xyz scan mode. The system was controlled with the Prairie View Imaging Software (Bruker Nano Surfaces, Middleton, WI, USA).