Opterra multipoint scanning confocal microscope

For photoactivation experiments, helios one-line 405-nm solid state laser (Obis lasers, Coherent), mounted on Bruker Opterra Multipoint Scanning Confocal Microscope (Buđa et al., 2017 (link)), was used to photoactivate MTs in U2OS cells with stable co-expression of photoactivatable-GFP-α-tubulin, CENP-A-GFP and mCherry-α-tubulin. Experiments were performed in Live/Ablation mode, at 80% laser power, by using Prairie View software (Prairie Technologies). In order to visualize GFP and mCherry, 488-nm and 561-nm laser lights were used, respectively, together with 250 ms exposure time. K-fibers belonging to same sister kinetochore pairs were sequentially photoactivated when sister kinetochore pairs were displaced from spindle equator, giving rise to shorter and longer sister k-fibers. Images were acquired at one focal plane with a time interval of 2 s.

All RPE-1 and U2OS fixed cells were imaged using Bruker Opterra Multipoint Scanning Confocal Microscope (Bruker) described above. In experiments where whole spindle stack was imaged, z-stacks were acquired at 30-60 focal planes for immunofluorescence images, and 60-120 focal planes for expanded samples, separated by 0.5 μm with unidirectional xyz scan mode. A 60 μm pinhole aperture was used and the xy-pixel size was 83 nm. For excitation of DAPI, GFP, mCherry or RFP and SiR fluorescence, a 405, 488, 561 and 647 nm diode laser line was used, respectively. The excitation light was separated from the emitted fluorescence by using Opterra Dichroic and Barrier Filter Set 405/488/561/640 nm (DAPI/eGFP/TRITC/Cy5) (Chroma). Images were captured with an Evolve 512 Delta EMCCD Camera using 300ms exposure time (Photometrics, Tucson, AZ, USA) with no binning performed. The frame average was performed 8 times for immunofluorescence images and 16 times for expansion microscopy images. All experiments were carried out using Nikon CFI Plan Apo VC ×100/1.4NA oil objective (Nikon).