Evos fl auto imaging microscope

Separate C6/C7 coronal joint tissue sections were stained with Safranin O/Fast Green to visualize the joint’s cartilage and bone or Picrosirius Red/Alcian Blue to visualize collagen fibers in the ligament (Ita et al., 2020 (link)); stained sections were imaged with the 20x objective of an EVOS FL Auto Imaging microscope (Thermo Fisher) (Ita et al., 2020 (link)). Stained Safranin O/Fast Green articular surfaces (n = 3–6/rat) were scored by blinded graders using the modified Mankin score (Yeh et al., 2007 (link); Ita et al., 2020 (link)). Regions of interest (ROIs; n = 2–4/image) throughout the images (n = 3-9 images/rat) were analyzed by Fourier transform (Sander and Barocas, 2009 (link)) to compute the principal orientation vectors of the image. The anisotropy index was calculated from the ratio of the principal axes to describe orientation on a continuous scale from isotropic (random; 0) to aligned (1) (Sander and Barocas, 2009 (link)) and averaged across ROIs for each rat to quantify collagen fiber orientation.

We established an in vitro setup that interfaced seminal fluid and a higher-viscosity medium (Supplementary Fig. 1b). The microchannel was first filled with a higher-viscosity medium (15, 40, 65, or 100 cP) by capillary action. The inlet was then loaded with raw semen (20 μL) that interfaced with the medium at the entrance of the channel. This setup was incubated at 37 °C for 20 min and subsequently analyzed using an EVOS FL Auto Imaging Microscope (Thermo Fisher Scientific, Canada) at 20× magnification. Sperm groups and individual sperm were located, and short videos of their swimming trajectories were recorded. The proportion of sperm in a group was computed as the number of sperm belonging to a group out of the total between sperm belonging to a group and individual sperm. The straight-line swimming velocity of sperm groups and individual sperm were calculated by evaluating their straight swimming distances achieved in 5 s, obtained through recordings. These recordings were converted to image sequences and then imported to ImageJ for velocity analyses.