Biomedical Engineering Reference
In-Depth Information
5-10 Gb per stack. Image reconstruction based on these stacks
requires complementary alignment and grid background removal
software. Collectively, this means that SIM requires a high-end
off-microscope workstation with high computing power. Acquisition
of thousands of image frames is required for generation of one full
image in PALM/STORM techniques, and each of those frames
must be aligned and corrected for drift. By necessity, processing of
these images is time-consuming and again requires a high-end
workstation computer. In concept, STED is not a computationally
dependent technique and hence holds an advantage over other
super-resolution techniques. Nevertheless, deconvolution software
is available specifi cally to enhance fi nal STED resolution (e.g.,
Scientifi c Volume Imaging, Hilversum, the Netherlands).
6
Application of Super-Resolution Techniques: Specifi city Is Key
The resolution and contrast-enhancing ability of super-resolution
microscopy approaches make them attractive solutions for life sci-
entists. However, one super-resolution technique may be more
appropriate than another depending on the application and experi-
mental questions. Specifi city and knowledge of how to best answer
one's scientifi c question are key in selecting a super-resolution sys-
tem. We highlight three of the major applications in biology and
address context-specifi c application of super-resolution techniques
below. A brief summary is provided in Table
2
.
In our experience, SIM lends itself most easily to multicolor imag-
ing, as shown in Fig.
4
. Implementation of SIM is immediately
accessible for most life scientists due to the conventional nature of
dyes and fl uorescent proteins. By comparison, PALM/STORM
6.1 Multicolor
Imaging
Table 2
Applications of super resolution
Technique
Application
SIM
PALM/STORM
STED
Typical resolution (
XY
)
100 nm
20-50 nm
25-80 nm
Typical resolution (
Z
)
250 nm
50 nm
150-600 nm
Conventional probes available
All
Some
Some
Multicolor imaging
4-Color
2-Color ideally
2-Color ideally
Live cell imaging
X
Limited by sampling approach
Inherently diffi cult
3D imaging
X
X
X
