Plan apo

Live and fixed cells were imaged under Olympus IX83 inverted wide-field microscope system equipped with an oil ×100/NA 1.40 objective (Plan Apo), an oil ×63/NA 1.40 objective (Plan Apo), and an oil ×40/NA 1.20 objective (fluorite), a motorized stage, a focus drift correction device, a 37 °C enclosed environment chamber, motorized filter cubes, a scientific complementary metal oxide semiconductor camera (Neo; Andor), and a 200-W metal-halide excitation light source (Lumen Pro 200; Prior Scientific). Filters and dichroic mirrors were optimized for GFP/Alexa Fluor 488, mCherry/Alexa Fluor 594, and Alexa Fluor 680. The microscope system was controlled by MetaMorph (Molecular Devices).

Cells were transfected with Lifeact-GFP and keratin 18-mCherry and seeded on PRIMO micropatterned PDMS, as described previously. Images were taken every 4 s using a Nikon TiE inverted microscope with a spinning-disc confocal unit (CSU-WD, Yokogawa) and a Zyla scientific complementary metal–oxide–semiconductor camera (Andor) controlled by µManager^{71 (link),72 (link)} using a ×60 objective (Plan Apo; NA, 1.2; water-immersion type). The local velocity fields of the actin and keratin fluorescence signals were measured by comparing each frame and its previous time point with a custom-made PIV software in MATLAB. A mask of each cell was drawn with respect to the F-actin signal in ImageJ^{73 (link)}. A radial coordinate, centred in the mask centroid, was assigned to each PIV data point, and normalized by the local radius of the cell-mask contour. Likewise, the local velocity fields were decomposed into their radial and tangential components. The distributions of the total and radial velocities inside each cell were then binned into equal-sized intervals of the normalized radial coordinate. The average total and radial velocity for each radial bin was then calculated.

After incubation in the appropriate medium, cells were fixed in 0.1% glutaraldehyde and 1.5% formaldehyde in PBS for 1 h and collected by centrifugation at 1300g for 5 min. The sperm pellet was immediately resuspended and incubated with 50 mM NH₄Cl in PBS during 15 min and washed twice by resuspension/centrifugation in PBS and once in distilled water. Water-resuspended cells were used to prepare smears on glass slides, which were air-dried at room temperature. Sperm were rinsed with PBS for 7 min and then permeabilized using acetone at −20 °C for 7 min and washed three times in PBS. Slides were then incubated with TRITC-phalloidin (1:30) in PBS for 1 h at room temperature in humid conditions, in the dark. Sperm were washed three times with PBS, once in distilled water and air-dried at room temperature. Finally, they were mounted under coverglass slides using Vectashield mounting media. Nonspecific staining was determined by incubating the sperm in the absence of TRITC-phalloidin. Slides were examined using a fluorescence confocal microscope (Olympus IX83-DSU), and images were captured at 60x magnification (Plan Apo, NA = 1.42 [oil]), with a sCMOS camera (Andor, Zyla). The fluorescence intensity was quantified using ImageJ software1.47 V (National Institute of Health) and was calculated in regions of interest localized in the sperm head. The background intensity was subtracted.