Biomedical Engineering Reference
In-Depth Information
A distinct concern with regard to all super-resolution techniques is
that alignment of fi elds must be precise. Given the nature of super-
resolution microscopy, movement of fi eld by just a few nanometers
can deliver compromised results. There are a variety of fi ducial and
non-fi ducial markers on the market to compensate for drift, and
these ultimately need to be optimized for each scientist's system.
SIM specifi cally comes with additional problems because grid
background remains unless software applications are designed to
remove it. This is illustrated in comparison with a wide-fi eld and
SIM image of the Golgi, as shown in Fig.
6
. Note the distinct grid
appearance in Fig.
6d
. There is some concern that increased reso-
lution reveals decreased organelle continuity. As shown in Fig.
7
,
some loss in Golgi continuity is apparent in the image collected
from dSTORM techniques. Whether this loss is due to the failure
to collect enough image frames or the inherent clustering of the
labeled protein requires further experimentation. It is therefore
very important to use appropriate controls and optimize dye con-
ditions for the imaging system.
5.4 Reducing
Background, Drift,
and Alignment Issues
Fig. 6
3D SIM can produce artifacts. Clathrin (
red
) and the trans-Golgi network (
green
) were labeled with anti-
bodies and imaged by wide-fi eld microscopy (
a
), confocal microscopy (
b
), and 3D SIM (
c
). The blowup of a
region of the image reconstruction (
d
) reveals a grid pattern which is an artifact
