Fig 2: Adhesion asymmetry in rolling cells revealed by molecular adhesion footprinting.(a) Schematic of the molecular adhesion footprint assay. A surface is coated with P-selectin-functionalised DNA molecular sensors (Step 1). Adhesion of PSGL-1 and application of force (Step 2) unzips the DNA sensor (Step 3), leaving single-stranded (ss)DNA at the adhesion site on the surface (Step 4). Complementary Cy3-labeled DNA flowed into the chamber (Step 5) hybridizes with unzipped sensors (Step 6), marking their location for fluorescence imaging. (b) Representative molecular adhesion footprint of a rolling cell (image A) by Cy3-fluorescence imaging of the surface under TIRF illumination. Cell rolling was performed under a calculated shear stress of 0.12 Pa. (c) Normalized 2D cross-correlation of image A with its subset image (image B). Color map displays the numerical value of the cross-correlation. Arrows indicate the horizontal positions of the local cross-correlation maxima and reveal the periodicity in the adhesion footprint. (d) Images cropped from image A, with width corresponding to the period from the cross-correlation in (c) and centered about the positions of the local cross-correlation maxima. (e) Average of the fluorescence images from (d) over a single-period (0–2p) and a linear contours plot of fluorescence intensity from background (value = 0) to maximum (value = 1). (f) Map of the 2D molecular adhesion footprint from (e) onto the surface of a sphere, representing the contact circumference of the cell. Color scales linearly with fluorescence intensity in (e). (g) Normalized autocorrelations of the fluorescence footprint along the x axis for individual cell tracks (N = 20; gray), and their average (red). The x axis is normalized by the individual cell circumference (pd). Inset shows the histogram of cell diameter. (h) Average cell diameter from video rotation tracking (14.1 ± 1.0 µm) and fluorescent footprint (14.2 ± 1.2 µm) agree with each other.

Fig 3: Bead rolling adhesion with adhesion footprint assay.(A) Schematic of the rolling adhesion footprint assay. A PSGL-1–coated bead rolls on a surface by binding to P-selectin and eventually rupturing the conjugated DNA duplex, leaving a single-stranded DNA (ssDNA) on the surface corresponding to the precise locations of each adhesion event. This adhesion footprint assay leaves a trail of ssDNA on the surface that can be fluorescently labeled with a complementary strand and further imaged with total internal reflection fluorescence (TIRF) microscopy to observe the tracks left by the rolling beads. (B) Dark-field imaging of bead rolling under altering steps of shear stress. The top image imposes snapshots of a bead every 2.5 s in response to the different shear stresses. The bottom panel shows the corresponding instantaneous velocity (black) and mean velocity (red). (C). A stitched image showing the fluorescence adhesion footprint tracks (left) that are observed after PSGL-1 beads roll on a P-selectin–TGT surface. Individual tracks (green lines) are detected (right), enabling isolation and analysis of the fluorescence intensity trajectory of individual beads. (D) Diffraction-limited (left) and DNA-PAINT super-resolution (right) image of a single fluorescence track.

Fig 4: Shear stress dictates bead rolling velocity and fluorescence track intensity.(A) Syringe pump–controlled flow series applied to the rolling beads. (B) Population mean (n = 9) rolling velocity of PSGL-1–coated beads over time resulting from the applied flow series shown in (A). (C) Mean bead velocity from tracking as a function of shear stress. Bead velocities were determined through single-particle tracking. Velocity data (n = 9, 12, 11, 10, and 45) from three independent experiments were pooled together. (D) Representative fluorescence track imaged with TIRF after rolling beads on the TGT/P-selectin surface with the flow series shown in (A). (E) Intensity trace along the track’s length showing raw data in gray and the mean intensity at each shear stress from (A) in red. a.u., arbitrary units. (F) Mean fluorescence bead velocity as a function of shear stress. Fluorescence bead velocity was determined by the length of each segment seen in a fluorescence track and the duration corresponding to the segments (n = 78, 72, 75, 42, and 269). (G) Scatter plot of fluorescence velocity versus tracking bead velocity showing a positive correlation (r = 0.99, P < 0.001), with error bars representing SEM. (H) Fluorescence track intensity as a function of shear stress (n = 78, 72, 75, 42, and 269).

Fig 5: Effect of TGT-P-selectin system on bead rolling.(A) Control experiments showing how different combinations of surface functionalization affect bead surface interaction behavior (n = 102, 96, 126, 118, 84, 104, 75, and 180). (B) Force-dependent bond lifetime of P-selectin:PSGL-1 (red dashed), TGT (blue dashed), and the combined TGT-P-selectin:PSGL-1 system (green solid, also illustrated in the inset schematic). (C) Box plot showing that bead rolling velocity is consistently higher on the TGT-P-selectin surface than on the P-selectin–only surface over a range of shear stress values and under otherwise identical conditions. Each data point indicates the mean velocity of a single bead under the specific shear stress. The number of independent data points in each group is n = 36, 22, 13, 28 16, and 14 (from left to right). Red lines in the box plots indicate the median value; boxes indicate the first to the third quartile, and whiskers indicate the 1.5× interquartile range extended beyond the first and third quartile. Statistical analysis was performed by Student’s two-tailed t test, where * indicates P < 0.05.