Sub-diffraction Limit Localization of Proteins in Volumetric Space Using Bayesian Restoration of Fluorescence Images from Ultrathin Specimens
Figure 6
Comparison of Synapsin puncta imaged using CWSTED versus deconvolution.
(A) Image of a single thin section imaged in CWSTED, ATW and ATD image. (B) Overlay of CWSTED (red) to ATW (green), CWSTED (red) to ATD (green) and ATW (red) to ATD (green). Note the high correspondence between the images, especially that of the CWSTED and ATD. The arrows in B point to apparent points in the ATD image (green) that is not in the CWSTED image (red). These few discrepancies are most likely due to the loss of primary antibody in a viscous mounting media overnight, because the CWSTED images were taken the day after the AT images, mostly because of the tendency of the CWSTED to bleach the sample during imaging. R value is the calculated Pearson's correlation coefficient between the two images. (C–E) Max projection of identical tissue volumes (ten 70 nm sections) imaged in CWSTED, ATW and ATD. Note that the CWSTED images were acquired using a 100× objective with 50 nm pixels whereas the AT images were acquired using a 63× objective with 100 nm pixels, thus the images were scaled and aligned to maintain the correct aspect ratios. Scale Bar = 5 um. (F) A distance histogram of centers of mass calculated from the CWSTED volume as compared to the ATW volume or the ATD volume. (G) A bar graph representing the total number of object centers found in the CWSTED volume, the ATW volume and the ATD volume.