Plan apo λ 100x oil objective

Cells were grown in MgM pH 5.6 or pH 7.2 and fixed with 1.5% PFA for 30 mins. They were then pelleted and permeabilized with 0.1% Triton X100 in PBS for 30 mins. The cells were washed three times with PBS via centrifugation, followed by staining with DRAQ5 for 15 mins. They were then washed 3 times with PBS and placed onto a 2% agarose pad and subsequently sealed with a clean glass coverslip. Imaging was performed using structured-illumination microscopy (SIM) on a W1 spinning Disk microscope (CSU-W1 Nikon, Japan) combined with the Live-SR system (Roper scientific) and equipped with a Plan-Apo λ 100x oil objective (1.45 NA, Nikon, Japan) as previously described (Gao et al., 2017 (link)). The area of the nucleoid per cell was quantified as previously described (Gao et al., 2017 (link)).

Sperm samples were fixed in absolute ethanol for at least 1 h at −20°C, spread on glass slides and air-dried. Next, they were incubated with TRITC-phalloidin 80 nM in PBS for 50 min followed by incubation with DAPI 0.2 mM in PBS for 15 min and then FITC-conjugated PSA at 50 μg/ml in PBS for 20 min. Finally, they were washed three times with tap water and mounted with Vectashield mounting medium (Vector H-1000).
The acquisition of triple staining was realized with Nikon A1r laser confocal scanning microscope, equipped with a Plan Apo λ 100X Oil objective, detector Galvano, with a pinhole size of 69 μM and a pixel size 0.04 um. We used an averaged 2 mode in channels series as follows:

Channel 1: FITC: λ_exc = 488 nm; λ_em = 525/50 nm, at 6.6% of the maximum laser power

Channel 2: DAPI: λ_exc = 404 nm; λ_em = 450/50 nm at 3% of the maximum laser power

Channel 3: TRITC: λ_exc = 561.5 nm; λ_em = 595/50 nm at 1.8% of the maximum laser power