Deltavision deconvolution microscope

Prometaphase samples cells were obtained as described above. Fixed cells were dropped on cover slides and then dried. We added probes against the centromere of chromosome 8 (XCE 8 ORANGE, MetaSystems Probes) and shielded the cells with a coverslip and rubber cement. The slides were incubated for 2 min at 75 °C, followed by overnight incubation at 37 °C. The cells were washed with 0.4× SSC at 72 °C for 2 min, followed by washing at room temperature with 2× SSC, 0.05% Tween-20, for 30 s. The slides were washed with water and stained with DAPI, followed by mounting with Prolong Gold (Invitrogen).
G2 samples were collected by treating the cells for 18 h with RO-3306. We verified that the cells were synchronized in G2 by incubation in Nicoletti buffer followed by flow cytometry (BD LSRFortessa). Plots were generated with FlowJo (v.10). G2-synchronized cells were spun down and resuspended with fixative solution (methanol/acetic acid, 3:1), followed by the same protocol as described above.
Images were taken using a DeltaVision deconvolution microscope (Applied Precision), and images were acquired using Softworx (Applied Precision) and ImageJ. The fluorescence signal was categorized as singlet (distance between the two highest intensity signals ≤300 nm) or doublet (distance between the two highest intensity signals >300 nm), as described previously^{32 (link)}.

Brightfield images were collected on a Stemi 2000C and an Axio Zoom V16 equipped with a 1× and 2.3× objective (Carl Zeiss MicroImaging, Thornwood, NY). Images were captured using an AxioCam MRc digital microscope camera (Carl Zeiss MicroImaging, Thornwood, NY) or a Rebel T3i digital SLR camera (Canon U.S.A., Inc., Melville, NY). DIC images were captured on an Axiovert 200M microscope (Carl Zeiss MicroImaging, Thornwood, NY) equipped with 10× 0.22 NA and 40×0.75 NA objectives with an AxioCam MRm digital microscope camera (Carl Zeiss MicroImaging, Thornwood, NY). Fluorescence images were collected on a Deltavision deconvolution microscope (Applied Precision, Issaquah, WA) equipped with 10×0.4 NA, 20×0.5 NA, and 100× oil 1.4 NA objectives using a CoolSnap HQ (Photometrics, Tucson, AZ) digital microscope camera. Immunofluorescence images are Z-stacks taken with 2 µm step sizes for 20× images and 0.2 µm step sizes for 100× images. Images of cells that were too large to fit into a single image were manually stitched using the cortical rows to align the two images, and the seam is indicated with a yellow dashed line.

HFF-hTERT cells transduced with retrovirus expressing Flag-RRM2B were seeded onto glass coverslips, fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 in PBS. Cells were stained for 1 h with affinity-purified rabbit anti-RRM2B antibody followed by a 30-min exposure to anti-rabbit IgG-AlexaFluor 647, mouse anti-PYCR2 antibody followed by a 30-min exposure to anti-mouse IgG-AlexaFluor 594 and DAPI before mounted in ProLong® Gold Antifade Mountant (Thermo Fisher Scientific Inc.). Fluorescence images of RRM2B and PYCR2 were captured with wide-field DeltaVision deconvolution microscope (Applied Precision Inc., GE Healthcare Life Science, Pittsburgh PA.), equipped with 60x/1.42 N.A. oil-immersion objective lens. Both microscope and camera were controlled by SoftWorX application suite software. Stacks of optical section images, with image size of 512 × 512, were collected for all fluorochromes. All images were deconvolved by using SoftWorX software (Applied Precision Inc.), and later analyzed with VoloCITY software (PerkinElmer).

All fluorescence microscopy was performed on a DeltaVision deconvolution microscope (Applied Precision, Issaquah, WA), based on a Nikon TE200 (Melville, NY) with an inverted 100X NA 1.4 objective, a 50-W mercury lamp, and a Photometrics Cool Snap HQ CCD camera (Photometrics, Tucson, AZ). Image pixel size is 49.2 × 49.2 nm. All images were deconvolved using the Applied Precision SoftWoRx imaging software.
For nuclear fusion assay microscopy (fixed, DAPI-stained cells), we acquired large z-stacks that captured the entire cell (typically 19 slices separated by 0.2 μm). The remaining imaging experiments used live cells in growth medium with smaller z-stacks (typically ~9 slices separated by 0.2 μm) to avoid photobleaching.