Biomedical Engineering Reference
In-Depth Information
Table 1
(continued)
SIM
PALM
STORM
STED
bsDronpa
X
rsCherry
X
rsCherryRev X
Note that not every dye and probe listed has been tested for every technique. Thus, while ATTO 488 has been tested
as effective for SIM and STORM, this does not mean it will not work for STED, merely that it may not have been tested
for this application
Fig. 5
Three-dimensional views are helpful in interpreting STORM images. Clathrin (
green
) and the trans-Golgi
network (
red
) were labeled with antibodies and imaged by dSTORM. A region of interest was cropped, and
different angles were manipulated to observe co-localization of molecules in the
Z
dimension. Viewing struc-
tures in three dimensions provides added information helpful for drawing co-localization conclusions from
super- resolution data
few commercialized primary antibodies are directly conjugated to
these dyes. Moreover, optimization of antibody labeling density is
required to ensure high quantum yield without background [
31
].
Expression of photoactivatable or photoswitchable proteins
(PA-GFP, PA-mCh, etc.) is an enticing option; however, not all
biological systems can be optimized for expressing these proteins.
For example, platelets lack a transcriptional system and hence
cannot be productively transfected with DNA. Another specifi c
example is overexpression of certain proteins within cells.
Overexpression of certain proteins can lead to alteration of signal-
ing pathways or induction of apoptosis. Indirect immunofl uores-
cence is still the cornerstone of co-localization and imaging
techniques. The microscopy fi eld must focus on ways to tag mol-
ecules that prevent potential steric hindrance and hence effective
