Figure 5. 2P Super FRAP verifies Qdot... | Rockefeller University Press

Figure 5.

$Figure 5. 2P Super FRAP verifies Qdot tracking results. (A) Schematic showing the geometries of a 300-nm-diameter cilium membrane and the psf of the Ti:S laser focused to the diffraction limit by the 1.2-NA water-immersion objective. The red ellipsoid is an iso-intensity surface representing the psf at 1/e Imax (Imax being at the center of the ellipsoid). (B) Heat map of the relative psf intensity at the ciliary membrane, and thus the pattern of photoconverted GPCRs produced by a brief laser pulse. Yellow lines show the pattern of corral boundaries used in the ensemble diffusion model (see Theory). (C) Confocal image of an SSTR3-EGFP cilium used in a 2P Super FRAP experiment. Red circle indicates the bleach position. (D) Mean of 25 bleach-recovery curves acquired at 200 kHz (black line). A 10-ms bleach pulse began at time 0. Red symbols represent 10-fold downsampled recovery phase (decimation). (E) Fitted ensemble diffusion model (line) and error residuals (gray line). Lower green line is 0. Best fit was determined by varying Dm and finding the minimum RMSE, as described in Calvert et al. (2007), Materials and methods. (F) Time image series of SSTR3-PAGFP equilibration. PAGFP was photoconverted by a 10-ms exposure from the Ti:S laser tuned to 820 nm (Calvert et al., 2007). Equilibration was monitored with serial 1P confocal scans (488 nm, 1.31 s/frame) at a single z plane. (G) Time course of PAGFP relaxation from the photoconversion site (black trace). Smooth green line is the best fit of the ensemble diffusion model where Dm = 0.11 µm2/s. (H) Time series images of the ensemble diffusion model where the source at time 0 represents the distribution of photoconverted molecules after a brief 2P laser exposure. Circumferential corral boundaries were present in the model membrane every 270 nm along the axis, as shown in B. (I) Mean membrane diffusion coefficients obtained with 2P Super FRAP, Dsf, and long-term equilibration by PAGFP FRAPa, Dpa. Error bars are SEM. Mean diffusion coefficients were significantly different, P < 0.05, as determined from pairwise t test. 2P Super FRAP n = 7 cilia obtained from three independent experiments. PAGFP FRAPa n = 8 cilia obtained from five independent experiments.$

2P Super FRAP verifies Qdot tracking results. (A) Schematic showing the geometries of a 300-nm-diameter cilium membrane and the psf of the Ti:S laser focused to the diffraction limit by the 1.2-NA water-immersion objective. The red ellipsoid is an iso-intensity surface representing the psf at 1/e I_max (I_max being at the center of the ellipsoid). (B) Heat map of the relative psf intensity at the ciliary membrane, and thus the pattern of photoconverted GPCRs produced by a brief laser pulse. Yellow lines show the pattern of corral boundaries used in the ensemble diffusion model (see Theory). (C) Confocal image of an SSTR3-EGFP cilium used in a 2P Super FRAP experiment. Red circle indicates the bleach position. (D) Mean of 25 bleach-recovery curves acquired at 200 kHz (black line). A 10-ms bleach pulse began at time 0. Red symbols represent 10-fold downsampled recovery phase (decimation). (E) Fitted ensemble diffusion model (line) and error residuals (gray line). Lower green line is 0. Best fit was determined by varying D_m and finding the minimum RMSE, as described in Calvert et al. (2007), Materials and methods. (F) Time image series of SSTR3-PAGFP equilibration. PAGFP was photoconverted by a 10-ms exposure from the Ti:S laser tuned to 820 nm (Calvert et al., 2007). Equilibration was monitored with serial 1P confocal scans (488 nm, 1.31 s/frame) at a single z plane. (G) Time course of PAGFP relaxation from the photoconversion site (black trace). Smooth green line is the best fit of the ensemble diffusion model where D_m = 0.11 µm²/s. (H) Time series images of the ensemble diffusion model where the source at time 0 represents the distribution of photoconverted molecules after a brief 2P laser exposure. Circumferential corral boundaries were present in the model membrane every 270 nm along the axis, as shown in B. (I) Mean membrane diffusion coefficients obtained with 2P Super FRAP, D_sf, and long-term equilibration by PAGFP FRAPa, D_pa. Error bars are SEM. Mean diffusion coefficients were significantly different, P < 0.05, as determined from pairwise t test. 2P Super FRAP n = 7 cilia obtained from three independent experiments. PAGFP FRAPa n = 8 cilia obtained from five independent experiments.