Four channel easy3d super resolution sted optics module

For confocal and STED microscopy, coverslips mounted on microscopy slides were imaged with a four-channel easy3D super-resolution STED optics module (Abberior Instruments) combined with an Olympus IX83 confocal microscope (Olympus) using an UPlanSApo 100× (1.4 numerical aperture) objective (Olympus). GFP and Vybrant DiO were excited with a 485 nm laser and recorded with combined 500 to 520 nm and 532 to 558 nm filters. Alexa594 and Rhodamine Phalloidin were excited with a 561 nm laser and recorded with a 580 to 630 nm filter. Atto647N and STAR RED were excited with a 640 nm laser and detected with a 650 to 720 nm filter. For STED microscopy, a pulsed 775 nm STED laser was used for depletion of Alexa594, STAR RED, and Atto647N.
A pinhole size of 60 μm was used, and the pixel size was 25 nm for all images. Confocal images were recorded with time-gated detection with 0.78 ns delay and 8 ns gate width. STED micrographs were recorded with six line accumulations and time-gated detection with 0.75 ns delay and 8 ns gate width.

Epididymal sperm cells after swim out were diluted into PBS (1:10); 400 μl of diluted sperm were loaded on poly-L-lysine (CAS 25988-63-0, Sigma Aldrich)-coated coverslips in a six-well plate and air dried for 30 min at 37°C. After removing the PBS, sperm cells were fixed in 4% PFA followed by quenching with 50 mM NH₄Cl for 15 min. Sperm cells were permeabilized by 0.02% Triton X-100 for 3 min followed by washing with PBS, incubation for 1 h at RT with Phalloidin ATTO647 (1/1,000 in 3% BSA, ab176759, Abcam), and mounting with ProLong Gold Antifade (#P36930, Life Technologies). STED micrographs were acquired using a four-channel easy3D super-resolution STED optics module (Abberior Instruments, Göttingen, Germany) coupled with an Olympus IX73 confocal microscope (Olympus, Tokyo, Japan) and equipped with an UPlanSApo × 100 (1.4 NA) objective (Olympus, Tokyo, Japan) (Mikuličić et al., 2019 (link)), available in the LIMES Imaging Facility.