Spectral, biochemical and photochemical properties of the purified PAmCherry variants. (a) Absorbance, excitation and emission spectra of PAmCherry1 before (pre) and after (post) photoactivation with the 399 nm laser line. (b) Maturation kinetics for the indicated proteins at 37 °C. (c) Equilibrium pH dependence for the red fluorescence of the indicated proteins. (d) Rate and efficiency of PAmCherry1 photoactivation at different physiological pH values, normalized to the fluorescence intensities at the respective pH (data shown in c). (e,f) Photoactivation (e) and photobleaching (f) kinetics for the indicated proteins using arc lamp illumination through ×60 oil objective with 390/40 nm (e) and 570/30 nm (f) filters. (g–i) Cycling illumination of PAmCherry1, rsCherry and rsCherryRev using an arc lamp, ×100 oil objective, 570/30 filter for 12 s and one of the following filters: 390/40 nm for 0.6 s (g), 436/20 for 0.5 s (h) and 480/40 for 0.4 s (i). The maxima of the fluorescence signals shown in g–i were normalized per the relative brightness for each protein indicated in Table 1 to achieve 100%. The ground states of the proteins are indicated with the respective symbols.

Comparison of PAmCherry1 and tdEosFP fusions in fixed cells. (a) Distribution of the number of photons collected from molecules in cells expressing TfR-PAmCherry1 (mean, 724; median, 413) and TfR-tdEosFP (mean, 940; median, 349). (b) Distribution of σ in cells expressing TfR-PAmCherry1 (mean, 15.18; median, 14.99) and TfR-tdEosFP (mean, 16.75; median, 16.69). (c) Distribution of the duration of the molecular fluorescence (0.1-s frames) in cells expressing TfR-PAmCherry1 (mean, 2.34; median, 1) and TfR-tdEosFP (mean, 3.26; median, 1). Two-tailed Wilcoxon-Mann-Whitney tests indicated differences between the TfR-PAmCherry1 and TfR-tdEosFP data medians for all three distributions (P < 0.0001, n = 757,180 from five TfR-PAmCherry1–expressing cells and n = 656,290 from five TfR-tdEosFP–expressing cells). (d) Distribution of the number of photons collected from molecules in cells expressing VSVG-PAmCherry1 (mean, 657; median, 348) and VSVG-tdEosFP (mean, 1,057; median, 444). (e) Distribution of σ in cells expressing VSVG-PAmCherry1 (mean, 20.96; median, 19.68) and VSVG-tdEosFP (mean, 19.91; median, 19.04). (f) Distribution of the duration of the molecular fluorescence (0.1-s frames) in cells expressing VSVG-PAmCherry1 (mean, 1.82; median, 1) and VSVG-tdEosFP (mean, 2.37; median, 1). Two-tailed Wilcoxon-Mann-Whitney tests indicated differences between the VSVG-PAmCherry1 and VSVG-tdEosFP data medians for all three distributions (P < 0.0001, n = 377,010 from three VSVG-PAmCherry1 cells and n = 768,040 from three VSVG-tdEosFP cells).

Plasma membrane distribution of TfR observed using PALM. (a) Cells expressing TfR-PAmCherry1 were subjected to low-level 405-nm laser photoactivation, and we simultaneously collected 561-nm-laser-light–excited PAmCherry1 single-molecule fluorescence. Data were collected at 10 frames per second for 15,000 frames. The fluorescence collected during the 15,000 frames is shown in a and represents a diffraction-limited TIRF microscopy image. (b) The position and uncertainty of the molecules plotted as Gaussian-normalized spots to form a PALM image. (c) Cluster analysis was performed on the molecules localized over the entire PALM image. The threshold used for clusters required the local molecular density to be fivefold that of the average molecular density. Individual clusters are plotted in different colors with nonclustered molecules represented in black. (d–f) Magnified views of the boxed region in a are shown as TIRF microscopy (d), PALM (e) and cluster analysis images (f). PALM data in these images were limited to molecules that localized to <25 nm uncertainty. Scale bars, 5 μm (a,b) and 1 μ (d,e).

Distributions of TfR and CLC by two-color PALM. (a–i) COS-7 cells expressing TfR-PAmCherry1 and PAGFP-CLC were subjected to low-level 405-nm laser photoactivation while we alternately collected 561-nm-light–excited PAmCherry1 (a–c) and 488-nm-light–excited PAGFP (d–f) single-molecule fluorescence. TIRF microscopy images of PAmCherry1 fluorescence (a), PAGFP fluorescence (d) and the merge (g) are shown. Position and uncertainty for TfR-PAmCherry1 (b,c) and PAGFP-CLC (e,f) are plotted as Gaussian-normalized spots. Magnified views of the boxed area in b are shown in c, f and i. Merge images (g–i) show the relative distributions of TfR-PAmCherry1 (red) and PAGFP-CLC (green). (j) Pair correlation analysis (top) and normalized results (bottom) for the presence and size of TfR-PAmCherry1 and PAGFP-CLC clusters. (k) Cluster analysis of the molecules localized for the PALM images in h (k) and i (l). The threshold used for clusters required the local molecular density to be fivefold that of the average molecular density. TfR-PAmCherry1 clusters are plotted in red, PAGFP-CLC clusters in green and nonclustered molecules in black. PALM data in these images are limited to molecules that localized to <25 nm σ. Scale bars, 2 μm (a,b,d,e,g,h) and 0.5 μm (c,f,i).