Images of spread spermatocytes were acquired on a Zeiss Axio Observer Z1 Marianas Workstation, equipped with an ORCA-Flash 4.0 camera and DAPI, CFP, FITC, TEXAS red and Cy5 filter sets, illuminated by an X-Cite 120 PC-Q light source, with either 63×/1.4 NA oil immersion objective or 100×/1.4 NA oil immersion objective. Marianas Slidebook 5.0 (Intelligent Imaging Innovations) software was used for acquisition.
Structured illumination microscopy (3D-SIM) was performed at the Bio-Imaging Resource Center in Rockefeller University using an OMX Blaze 3D-SIM super-resolution microscope (Applied Precision), equipped with 405 nm, 488nm and 568 nm lasers, and 100×/1.40 NA UPLSAPO oil objective (Olympus). Image stacks of several μm thickness were taken with 0.125 μm z-steps, and were reconstructed in Deltavision softWoRx 6.1.1 software with a Wiener filter of 0.002 using wavelength specific experimentally determined OTF functions. Slides were prepared and stained as described above, except that chromosomes were spread only on the central portion of the slides, and the slides mounted using 18 × 18 mm coverslips (Zeiss).
Structured illumination microscopy (3D-SIM) was performed at the Bio-Imaging Resource Center in Rockefeller University using an OMX Blaze 3D-SIM super-resolution microscope (Applied Precision), equipped with 405 nm, 488nm and 568 nm lasers, and 100×/1.40 NA UPLSAPO oil objective (Olympus). Image stacks of several μm thickness were taken with 0.125 μm z-steps, and were reconstructed in Deltavision softWoRx 6.1.1 software with a Wiener filter of 0.002 using wavelength specific experimentally determined OTF functions. Slides were prepared and stained as described above, except that chromosomes were spread only on the central portion of the slides, and the slides mounted using 18 × 18 mm coverslips (Zeiss).