1. Quantitative Localization Microscopy Reveals a Novel Organization of a High-Copy Number Plasmid 
Yong Wang, Paul Penkul, Joshua N. Milstein 
Biophysical Journal 
Volume 111, Issue 3, Pages (August 2016)
DOI: /j.bpj
Copyright © 2016 Biophysical Society Terms and Conditions

2. Figure 1
Characterization of probe hybridization efficiency. (A) An intensity trace showing stepwise photobleaching, which yielded the number of FISH probes hybridized to a single plasmid. (B) The resulting distribution was assembled and fitted by a binomial distribution giving a hybridization efficiency of ∼15% (dark blue bars, experimental measurement; yellow bars, fitted data). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

3. Figure 2
Conventional and superresolved fluorescence images of FISH-labeled plasmids in E. coli. (A) Immobilized bacteria were viewed under bright field illumination. (B) Conventional fluorescence microscopy showed smFISH-labeled plasmids as fluorescent foci on top of a weak fluorescent background. (C) The same bacteria were imaged by localization microscopy. (D–F) The magenta area in (A–C) is shown enlarged. Green and orange arrows indicate fluorescent foci (E), or clusters of localizations (F). (G and H) The localization precision (σx,y) for the superresolved reconstruction was estimated based on Mortensen et al. (35). We achieved an average precision of ≈10 nm for both σx and σy. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

4. Figure 3
Specificity of FISH probe labeling in bacteria. (A and B) The same superresolved image of the bacterium in Fig. 2, D–F, is displayed at two different color scales. (C and D) Negative controls (i.e., bacteria without target plasmids) labeled with FISH probes to assess nonspecific labeling. An example negative control bacterium is shown at the same color scale as the positive sample in (A) and (B), respectively. Nonspecific labeling is virtually absent (C) unless the color scale is set very low (D). (E) Nonspecific labeling was estimated quantitatively by comparing the numbers of localizations per cell for the positive samples and negative controls (mean ± SE = 2284 ± 304 [n = 30] vs ± 3.5 [n = 10]). Nonspecific labeling accounted for 1.3% of the total localizations. The scale bar in the images is 1 μm. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

5. Figure 4
The spatial distribution of localizations in bacteria. (A) The origin was defined as the center-of-mass of all localizations (blue dots) in a cell. The long and short axes were defined as shown, along with the angle θ. (B) The 2D probability map of localizations averaged over 19 bacteria. (C and D) The averaged probabilities of localizations (black solid lines; error bars: mean ± SE) along the long and short axes obtained from 19 cells (colored dashed lines), showing clear plateaus. (E) Probability of localizations as a function of the absolute value of position along the long axis. (F) Angular probability of localizations. (G) Radial probability of localizations. Distributions are presented for the data (○, purple) and, for comparison, a model of random localizations (△, olive). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

6. Figure 5
Steric exclusion of plasmids by chromosomal DNA. (A and D) Two example cells shown as superresolved images. Scale bars = 500 nm. (B and E) Intensity profiles of the purple and olive regions in (A) and (D) are drawn along the short axis of the bacteria in the corresponding colors. (C and F) Smoothed intensity profiles of the cyan lines in (A) and (D) (window = 30 pixels). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

7. Figure 6
NND distributions (1st–45th orders) of localizations (purple, n = 19 bacteria) were compared to those of spatially random localizations (olive) in bacteria. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

8. Figure 7
Clustering analysis of localizations. Localizations in each bacterial cell were sorted into clusters by DBSCAN (minPts = 30, eps = 15). (A and B) Center-of-mass distribution of the clusters along the long and short axes of the bacteria (27 cells). The clusters were found distributed fairly uniformly along both axes. (C–E) The spread/dispersion (σc,x,σc,y,andσc=σc,x2+σc,y2) of the clusters, and (F) the distribution of the radius of gyration (Rg).
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

9. Figure 8
Clustering of plasmids. (A) The bacterium in Fig. 2 F is enlarged with two bright spots highlighted (green and orange arrows). Scale bar = 500 nm. The radii of the spots were manually determined as indicated by the green (r = 100 nm) and orange (r = 75 nm) circles. (B) Localizations inside the two bright spots were counted: 827 (purple) localizations inside the green circle, and 310 (blue) localizations inside the orange circle. (C) Probability distribution of the expected number of plasmids, in the large bright spot indicated by the green arrow in (A), assuming a hybridization efficiency of 15% (green) or 10% (purple). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions