Dcc1545m

Oocytes, placed in individual drops of modified human tubal fluid media, were measured, and individually tracked for outcomes. A micropipette was placed on the zona pellucida directly opposite to the location of the polar body (see Fig 3) to standardize the location of measurement in an area, a procedure performed similarly to the previous study [15 (link)]. Given the larger size of a human oocyte (~110 μm) as compared to that of a mouse embryo (~80 μm), 50-μm inner diameter of micropipettes (custom-made by Origio) and an applied pressure (-0.1 p.s.i.), which was modified to be slightly larger than the 40-μm inner diameter of micropipettes in Yanez et. al [15 (link)] and the applied pressure of ~0.5 kPa (~0.073 p.s.i.) in Young’s modulus for mouse embryo [17 (link)], were used and applied. The pressure was applied through a micropipette for 2 seconds and the movement of the zona pellucida inside the micropipette was recorded at 70 frames per second with a CMOS camera (Thorlabs DCC1545M). The pressure was regulated back to the balance pressure after each measurement to release the oocyte from the micropipette tip. The operator could easily navigate through the measurement steps displayed on the computer screen or pressure log file. This included monitoring pressure values both before and during measurements, allowing them to assess the success of each measurement.

All experimental observations were performed on a homemade inverted bright-field microscope enclosed in a custom-made environmental chamber (Okolab) with temperature control (T = 22 ± 0.5 °C). The microscope was mounted on a floated optical table for vibration dampening. The bacteria were tracked by digital video microscopy using the image projected by a microscope objective (×20, NA = 0.5, Nikon CFI Plan Fluor) on a monochrome CMOS camera (1280 × 1024 pixels, Thorlabs DCC1545M) at 10 f.p.s.^{57 (link)}. The magnification of our imaging path allowed us to achieve a conversion of 0.22 μm per pixel, corresponding to a field of view of ≈280 × 225 μm². The incoherent illumination for the tracking of the bacteria was provided by a red LED (λ = 660 nm, Thorlabs M660L3-C2) employed in a Köhler configuration to control and improve coherence and contrast of the illumination at the sample plane. The typical duration of an experiment was ≈60 min before bacteria motility started to decrease considerably. In total, we recorded over 3500 individual bacterial trajectories of variable duration. The data shown in the figures are obtained from the analysis of segments of these trajectories.