Volume 97, Issue 9, Pages 2613-2622 (November 2009) Homo-FRET Imaging Enables Quantification of Protein Cluster Sizes with Subcellular Resolution  Arjen N. Bader, Erik G. Hofman, Jarno Voortman, Paul M.P. van Bergen en Henegouwen, Hans C. Gerritsen  Biophysical Journal  Volume 97, Issue 9, Pages 2613-2622 (November 2009) DOI: 10.1016/j.bpj.2009.07.059 Copyright © 2009 Biophysical Society Terms and Conditions

Figure 1 Native PAGE analysis of dimerization constructs. Cells expressing FKBP-mGFP or 2xFKBP-mGFP, either incubated with AP20187 or mock-treated, were lysed under nonreducing and nondenaturing conditions. After size separation on a NativePAGE gel and blotting to PVDF membrane, proteins were detected with anti-GFP antibodies. Biophysical Journal 2009 97, 2613-2622DOI: (10.1016/j.bpj.2009.07.059) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 2 (A) Confocal steady-state intensity and fluorescence anisotropy images of cells expressing GFP-FKBP and GFP-2xFKBP, in both the presence and absence of AP20187 (AP). (B) Histograms of the average anisotropy of the samples in A. From each sample, five cells were imaged. The intensity values of all significant pixels were summed, and the overall anisotropy/cell was calculated. The error bars give the standard deviation between the cells. Here, r0 = 0.382, as indicated by the top of the gray area in the histogram. Biophysical Journal 2009 97, 2613-2622DOI: (10.1016/j.bpj.2009.07.059) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 3 Confocal time-gated fluorescence anisotropy imaging. (A) A schematic representation of a typical r decay due to homo-FRET. The limiting anisotropy (rinf) is independent of the homo-FRET efficiency for E > 0.5. (B) Time-resolved anisotropy decays of cells expressing FKBP-mGFP and 2xFKBP-mGFP, in both the presence and absence of AP20187 (AP). For all images in each sample, the intensities/gate in the significant pixels were summed to create the average r decays. (C) The average anisotropy for each sample is plotted in a histogram. The dynamic range (Δr) is larger than for rss (compare Fig. 2B). Here, r0 = 0.382, indicated by the top of the gray area in the histogram. Biophysical Journal 2009 97, 2613-2622DOI: (10.1016/j.bpj.2009.07.059) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 4 Two-photon excitation fluorescence anisotropy imaging. Similar to Figs. 2B and 3C, the average anisotropy/sample was measured using TPE. For both steady-state (A) and time-gated detection (B), the dynamic range (Δr) increases compared to that found using OPE. Here, r0 = 0.505, indicated by the top of the gray area in the histogram. Biophysical Journal 2009 97, 2613-2622DOI: (10.1016/j.bpj.2009.07.059) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 5 (A) Anisotropy reduction due to homo-FRET is not altered by the presence/absence of a flexible linker or dimerization domain in the fluorophore. (B) Schematic diagram of possible relative orientations of the GFP fluorophores in FKBP-induced dimers. Here, r0 = 0.382, indicated by the top of the gray area in the histogram. Biophysical Journal 2009 97, 2613-2622DOI: (10.1016/j.bpj.2009.07.059) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 6 Significance of direct cluster size determination from anisotropy data. The solid curves represent the measured rinf/rmono of GFP in glycerol/buffer solution (Nav = 1), in cells expressing FKBP-mGFP in the presence of AP20187 (Nav = 2), or in cells expressing 2xFKBP-mGFP in the presence of AP20187 (Nav ≥ 3). The dashed curves are the theoretical standard deviations plotted as a function of the intensity, as formulated by Lidke et al. (26). Note that the standard deviations are also dependent on the factor that corrects for differences in transmission between the parallel and perpendicular channels (the G-factor). Here, an average G-factor of 0.35 was used. Biophysical Journal 2009 97, 2613-2622DOI: (10.1016/j.bpj.2009.07.059) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 7 Confocal time-gated anisotropy imaging shows nanoscale clustering of GPI-GFP in the plasma membrane. (A) Time-resolved anisotropy decay, obtained by summing the intensities/gate in the significant pixels. (B–D) Intensity (B), limiting anisotropy (C), and cluster-size (D) images of cells expressing GPI-GFP. The latter two images are binned to 80 × 80 pixels. Threshold = 1200 counts, rmono = 0.330. Biophysical Journal 2009 97, 2613-2622DOI: (10.1016/j.bpj.2009.07.059) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 8 Confocal time-gated anisotropy imaging shows 40% clustering of EGFR-mGFP in the plasma membrane. (A–C) Intensity (A), limiting anisotropy (B), and fraction of clusters (C) in a cell expressing EGFR-mGFP. The images are binned to 80 × 80 pixels. Threshold = 1200 counts, rmono = 0.381. (D) Histogram of Fcluster for one image. The width of the histogram is predominantly determined by photon statistics (26). Biophysical Journal 2009 97, 2613-2622DOI: (10.1016/j.bpj.2009.07.059) Copyright © 2009 Biophysical Society Terms and Conditions