Examination of the ultrafast SFMI developed here to determine whether it can correctly evaluate hop-diffusion parameters for Cy3-DOPE and TfR in the apical PM, previously found by ultrafast gold-particle imaging (dwell lifetimes were previously determined using additional data from video-rate observations of fluorescently labeled molecules)
| . | Mode of Motion (%) . | Cmpt. Size (L, nm)b . | DMACRO (µm2/s)c . | Dmicro (µm2/s) . | Dwell Lifetime (τ, ms)d . | n (mole-cules) . | |
|---|---|---|---|---|---|---|---|
| . | Suppa . | Simpa . | Median Mean ± SEM Fig. No. . | Median Mean ± SEM Fig. No. . | Median Mean ± SEM Fig. No. . | Mean ± SEM Fig. No. . | . |
| Cy3-DOPE | 76 | 24 | 101 107 ± 6.2#1 Fig. 5 D | 0.30 0.30 ± 0.021#2 Fig. 5, C a | 0.78 0.83 ± 0.056 Fig. 5, C a | 9.2 ± 0.34#3 Fig. 6 A | 50 |
| gold-DOPEe | 100 | 0 | 110 120 ± 9.7 | 0.34 0.35 ± 0.019 (30 Hz Cy3-DOPE, n = 60) | NR | 8.9 | 35 |
| gold-DOPEe (NRK cells) | 85 | 15 | 230 240 ± 11 | 1.1 1.2 ± 0.071 (40 kHz gold-DOPE, n = 90) | 5.4 (median) | 13 | 90 |
| Cy3-TfR | 80 | 20 | 103 107 ± 5.41 Fig. 5 D | 0.11 0.13 ± 0.0142 Fig. 5, C b | 0.51 0.57 ± 0.045 Fig. 5, C b | 23 ± 1.53 Fig. 6 A | 50 |
| gold-TfRe | 97 | 3 | 100 120 ± 10 | 0.17 0.19 ± 0.0081 (30 Hz Cy3-TfR, n = 174) | NR | 15 | 38 |
| gold-TfRe (NRK cells) | 91 | 9 | 260 270 ± 10 | 0.29 0.29 ± 0.011 (30 Hz Cy3-TfR, n = 61) | 5.2 (median) | 58 | 107 |
| Cy3-DOPE (Actin-depleted Blebbed PM) | 10 | 90 | NA | 5.7f 6.0 ± 0.38f Fig. 5, C a | NA | NA | 20 |
| gold-DOPEe (Actin-depleted Blebbed PM of NRK cells) | 13 | 87 | NA | 8.5f 8.9 ± 0.47f | NA | NA | 30 |
| Cy3-TfR (Actin-depleted Blebbed PM) | 5 | 95 | NA | 2.5f 3.3 ± 0.52f Fig. 5, C b | NA | NA | 20 |
| gold-TfRe (Actin-depleted Blebbed PM of NRK cells) | 26 | 74 | NA | 8.1f 8.0 ± 0.71f | NA | NA | 19 |
| . | Mode of Motion (%) . | Cmpt. Size (L, nm)b . | DMACRO (µm2/s)c . | Dmicro (µm2/s) . | Dwell Lifetime (τ, ms)d . | n (mole-cules) . | |
|---|---|---|---|---|---|---|---|
| . | Suppa . | Simpa . | Median Mean ± SEM Fig. No. . | Median Mean ± SEM Fig. No. . | Median Mean ± SEM Fig. No. . | Mean ± SEM Fig. No. . | . |
| Cy3-DOPE | 76 | 24 | 101 107 ± 6.2#1 Fig. 5 D | 0.30 0.30 ± 0.021#2 Fig. 5, C a | 0.78 0.83 ± 0.056 Fig. 5, C a | 9.2 ± 0.34#3 Fig. 6 A | 50 |
| gold-DOPEe | 100 | 0 | 110 120 ± 9.7 | 0.34 0.35 ± 0.019 (30 Hz Cy3-DOPE, n = 60) | NR | 8.9 | 35 |
| gold-DOPEe (NRK cells) | 85 | 15 | 230 240 ± 11 | 1.1 1.2 ± 0.071 (40 kHz gold-DOPE, n = 90) | 5.4 (median) | 13 | 90 |
| Cy3-TfR | 80 | 20 | 103 107 ± 5.41 Fig. 5 D | 0.11 0.13 ± 0.0142 Fig. 5, C b | 0.51 0.57 ± 0.045 Fig. 5, C b | 23 ± 1.53 Fig. 6 A | 50 |
| gold-TfRe | 97 | 3 | 100 120 ± 10 | 0.17 0.19 ± 0.0081 (30 Hz Cy3-TfR, n = 174) | NR | 15 | 38 |
| gold-TfRe (NRK cells) | 91 | 9 | 260 270 ± 10 | 0.29 0.29 ± 0.011 (30 Hz Cy3-TfR, n = 61) | 5.2 (median) | 58 | 107 |
| Cy3-DOPE (Actin-depleted Blebbed PM) | 10 | 90 | NA | 5.7f 6.0 ± 0.38f Fig. 5, C a | NA | NA | 20 |
| gold-DOPEe (Actin-depleted Blebbed PM of NRK cells) | 13 | 87 | NA | 8.5f 8.9 ± 0.47f | NA | NA | 30 |
| Cy3-TfR (Actin-depleted Blebbed PM) | 5 | 95 | NA | 2.5f 3.3 ± 0.52f Fig. 5, C b | NA | NA | 20 |
| gold-TfRe (Actin-depleted Blebbed PM of NRK cells) | 26 | 74 | NA | 8.1f 8.0 ± 0.71f | NA | NA | 19 |
Supp = Suppressed diffusion. Simp = Simple-Brownian diffusion.
Cmpt. Size = Compartment size (L), determined by the hop-diffusion fitting, using frame rates at 10 and 40 kHz for fluorescently tagged and gold-tagged molecules, respectively. Fig. No. = The figure in which the histogram (distribution) is shown.
DMACROs for Cy3-DOPE and Cy3-TfR were determined by the hop-diffusion fitting. DMACROs for gold-DOPE and gold-TfR were generally underestimated, due to the crosslinking effect of the gold probes. Therefore, previously they were mostly determined by the observations of Cy3-DOPE and Cy3-TfR at 30 Hz (Fujiwara et al., 2016). DMACRO of gold-DOPE in NRK cells was determined for the diffusion in the time window of 30 ms (using the data obtained at 40 kHz). In the apical PM of the NRK cell, the PM exhibited nested double compartments, and the DMACRO of DOPE over smaller compartments could not be determined using fluorescent probes (Fujiwara et al., 2002; Fujiwara et al., 2016).
Dwell lifetime within a compartment (τ) for Cy3-labeled molecules was determined by the exponential fitting of the distributions of the residency duration for each molecule in each compartment, obtained by the TILD analysis (Fig. S5). SEM was determined from the fitting error of the 68.3% confidence interval. τ for gold-tagged molecules was calculated from the median values of L and DMACRO, using the equation .
Gold-DOPE and gold-TfR data are from Fujiwara et al. (2002), Fujiwara et al. (2016).
Since DOPE and TfR labeled with Cy3 or gold undergo simple-Brownian diffusion in the actin-depleted blebbed PM, the MSD-∆t plot was fitted by a linear function, and the diffusion coefficient was obtained by the slope/4.
Y, and N. Results of statistical tests. The distributions selected as the basis for the comparison are shown by the superscript, e. Different numbers (1–3) indicate different bases for the Brunner–Munzel test (1, 2) and the log-rank test (3). The superscript Y (or N) with numbers indicates that the distribution is (or is not) significantly different from the base distribution indicated by the number after e, with P values smaller (or greater) than 0.05. N1: P = 0.85, Y2: P = 4.3 × 10−14, Y3: P = 1.2 × 10−29.
NR: Not Reported. NA: Not Applicable because most molecules exhibited simple-Brownian diffusion.