Figure 3.

Analysis of a 1 μm bead’s fluctuation data while approaching the membrane of a HT29 cell. (A) Distance-dependent position trajectories b_x(d,t), b_y(d,t), b_z(d,t) calculated from the QPD data after calibration. y data correspond to scan direction. The regions d ≤ R, in which the membrane is already in contact with the bead, are grayed out. The three-dimensional QPD data are divided into subsequent parts with duration τ_mes = 150 ms as indicated by the black box; each spatial dimension is analyzed separately. (B) Upon contact to the membrane, i.e., d = R, the histogram width σ_y(d) of the bead’s fluctuations perpendicular to the cell membrane decreases (circles: data points, solid line: running average over 10 data points). The values at 50% of σ₀ and σ_min are used to determine the likely and the latest membrane contact point. The shaded region right of the vertical d = 0 line represents the cell. (C) The parts of the trajectory that correspond to one specific cell-focus distance d are analyzed by means of AC functions. (D) The AC is approximately linear on a τ ≤ 30 μs timescale, which is used to obtain κ_j(d) and γ_j(d). To see this figure in color, go online.