Biomedical Engineering Reference
In-Depth Information
as is the case with confocal microscopy; much live cell work is done
with spinning disk systems in which light from many pinholes are
collected in parallel.
PALM/STORM systems present an inherent challenge in
terms of acquisition speed. By their nature, these systems require
thousands of frames to produce a stochastic image. For now, it is
our opinion that these techniques are unlikely to have general
applicability in live cell imaging. That said, PALM has been used to
study adhesion-complex dynamics [
35
]. In this case, the move-
ment being studied was slower than most biological movements.
7
Conclusions and Comments
New techniques are developed in the hope of obtaining concrete
answers to biological questions and to be able to ask better ques-
tions. Ideally, super-resolution microscopy approaches provide
both. However, at present, there is no single ideal approach to
super-resolution co-localization studies. The techniques differ
signifi cantly in resolution and in practicality in their application
to individual research problems. In addition, convenience, time,
and cost must also be considered in making any decision. For many
uses, SIM provides advantages over PALM/STORM or STED.
SIM is practical using conventionally available dyes, while PALM/
STORM relies on a subset of blinking dyes that work well indi-
vidually and less so for two-color co-localization studies. Robust
dye choices will need to be added to the repertoire for PALM/
STORM techniques to be a truly valuable technique for a general
cell biology community. One can imagine a repertoire of dyes that
all switch under the same buffer conditions, allowing for easily
obtained multicolor readouts. Additionally, SIM systems are being
optimized and further developed for the important biological
applications of live cell and multicolor imaging. SIM also lends
itself easily to multicolor 3D imaging, whereas PALM/STORM
techniques are less adept at multicolor 3D. The primary benefi t of
PALM/STORM and STED strategies is the further improvement
in resolution. This gain is typically available only for fi xed samples.
A trend within the industry appears to be the implementation of a
SIM and a 3D PALM/STORM system in the same microscope
stand. For many, that choice may give the best of both worlds.
For our own study of platelets, SIM has been very helpful in
addressing questions of platelet
-granule structure and protein
packaging [
36
], and we look forward to implementing this
method for live cell imaging. In part, this is because platelet gran-
ules are relatively large cylinders, about 250 nm in diameter and
500 nm in length. We consider 3D SIM to be an easily applicable
and quickly implementable technique to augment our current
studies using electron microscopy. Likely, the use of a combined
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