Biomedical Engineering Reference
In-Depth Information
Fig. 7
With super resolution, the structural continuity of an organelle can be less apparent. The trans-Golgi
network (TGN46) (
green
) and medial-Golgi network (NAGT-1) (
red
) were labeled with the respective antibodies.
Alexa Fluor 647 (
green
) was used for the trans-Golgi network secondary antibody, and Cy3B was used for the
medial-Golgi network secondary antibody, and samples were imaged via either wide-fi eld microscopy (
top
panel
) or dSTORM (
bottom panel
). It is clear that some structural continuity loss in the Golgi apparatus resulting
from dSTORM is likely due to protein clustering or limitations in the effi ciency of dye photophysics. Investigators
will often superimpose a SIM or confocal image on the dSTORM outcome to reveal a better sense of organelle
structure
5.5 Post-processing
Because most super-resolution techniques rely on mathematical
algorithms to extract frequency data of fl uorescence intensities,
post-processing is extensive. As previously described, SIM records
a series of images by rotating different light grid patterns on the
sample. A Fourier transform-based algorithm transforms grid pat-
terns into frequency space to generate a full, reconstructed image.
Reconstructions from 2D SIM are acquired by rotating the light
pattern grid in the
XY
plane by 3-5 rotations and up to fi ve phase
shifts for up to 25 images, and gaining resolution in the
Z
dimen-
sion requires image stacking. In our experience, the raw data fi les
for a three-color, 10
ΚΌ
m image stack result in approximately
