1. A Simple Marker-Assisted 3D Nanometer Drift Correction Method for Superresolution Microscopy 
Hongqiang Ma, Jianquan Xu, Jingyi Jin, Yi Huang, Yang Liu 
Biophysical Journal 
Volume 112, Issue 10, Pages (May 2017)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. Figure 1
High-precision superresolution imaging system. (a) Shown here is the schematic of the optical setup. (b) Shown here is the measured 3D point spread function distribution of the eight fiducial markers attached to the surface of the coverslip. (c) Shown here is the estimated joint 3D position and (d) the corresponding precision of the eight fiducial markers at 17 axial positions ranging from −400 to 400 nm. (e) Shown here is the tracking 3D position of our high-precision imaging system for 40,000 frames or ∼15 min, with the SD of 1.3 nm in the lateral direction and 3.0 nm in the axial direction. Exposure time for each image: 20 ms. Drift correction rate: every 200 frames. (f) Shown here is the reconstructed superresolution image of microtubules (labeled with Alexa647) obtained with our high-precision imaging system. (g) A magnified region in (f) clearly shows the hollow structure of the microtubules. (h) Shown here is the intensity profile of the hollow structures of microtubules. The full width half-maximum of each microtubule shown in (g) is 21 and 19 nm, with a separation of 37 nm. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

3. Figure 2
(a) Wide-field and reconstructed superresolution images of replication structure of a normal cell nucleus immediately after the initiation of new DNA synthesis, labeled with EdU-Alexa Fluor 647 after exposing to EdU for 1 min. (b) The magnified wide-field image and superresolution images of the region is indicated by the box in (a). (c) Shown here is the corresponding superresolution image with only MA-based axial drift correction. (d) Shown here is the superresolution image after MA-based axial drift correction, RCC-based lateral drift correction, and MA-based axial drift correction and (e) the superresolution image after MA-based 3D drift correction. The image resolution defined by FRC is 84.5 ± 1.1 nm, 75.8 ± 1.4 nm, and 67.3 ± 1.9 nm for (c)–(e), respectively. The white arrows indicate the structure differences before and after drift correction. (f) The distribution of localization events along the vertical direction in the region is indicated by the dotted box in (c)–(e). (g) Shown here is a comparison of the lateral drift estimated by (f) our marker-assisted method and RCC. (h) Shown here is the axial drift estimated by our method. Please note that given that RCC-based drift correction can only compensate the drift in the lateral dimension, we have corrected the axial drift using our online MA-based drift correction for all images presented. Here, we only compare the performance of lateral drift correction between the MA-based method and posterior image cross-correlation method. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

4. Figure 3
(a) Shown here are the wide-field and (b)–(g) reconstructed superresolution images of cancer cell nuclei labeled with histone protein H2B-Alexa Fluor 647, (b and e) after MA-based axial drift correction but before lateral drift correction, and after MA-based axial drift correction and the lateral drift correction (c and f) using RCC and (d) and (g) our marker-assisted methods. Boxes 1 and 2 indicate two regions of the cell nuclei that exhibit the clusters with different shapes from RCC-based and MA-based drift correction methods. The image resolution defined by FRC is 60.6 ± 2.1, 49.4 ± 1.7, 46.1 ± 1.2, 61.0 ± 1.9, 50.1 ± 1.6, and 56.3 ± 1.7 nm for (b)–(g), respectively. Please note that we have corrected the axial drift using our online MA-based drift correction for all images presented here. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

5. Figure 4
Superresolution images of replication foci at four different time points (1, 2, 5, and 10 min) of (a1)–(d1) normal-like, (e1)–(h1) precancerous, and (i1)–(l1) tumorigenic cells. The figure inset shows the corresponding wide-field fluorescence image. (a2)–(l2) Here we have the corresponding higher zoom of the regions inside the boxes. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

6. Figure 5
Given here is the average histogram distribution of (a)–(c) spot number per cluster and (d)–(f) cluster size of normal, precancerous, and tumorigenic cells at 1, 2, 5, and 10 min of exposure to EdU. To eliminate the effect of nucleus size, the y axis uses cluster density, calculated as the number of clusters per bin divided by the area of the cell nucleus. The number of cells averaged per group is shown in Table 1. (g–i) Shown here is the statistical mean of cluster size, spot number per cluster, and cluster density at 1, 2, 5, and 10 min of exposure to EdU in normal, precancerous, and tumorigenic cells, respectively. The error bar is 95% confidence interval of the mean. The detailed statistical analysis is shown in Tables 1 and S3–S5. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions