Biomedical Engineering Reference
In-Depth Information
techniques are less easily optimized for more than two colors and
often work best under conditions of one color localization study.
It is possible to do two-color imaging, as shown by Figs.
5
and
7
.
Although STED systems can be used for two-color imaging, they
have been primarily used for studies utilizing just one photoactivat-
able or photoswitchable probe. Dye choice primarily limits multi-
color imaging for PALM/STORM and STED techniques.
6.2 3D Imaging
Three-dimensional data are a distinct advantage at the super-
resolution level. We illustrate this with a dSTORM example. As
shown in Fig.
5
, the interpretation of two-color STORM images is
clearer in 3D as illustrated by two observational angles. We note
here that both SIM and STED were designed from the beginning
to be 3D techniques in which vertical stacks of images are taken.
PALM/STORM can be a 3D approach as illustrated by implemen-
tations from various manufacturers such as Nikon, Vutara, and
Zeiss (listed in alphabetical order). The acquisition of 3D data is
particularly essential to demonstrating
Z
resolution and generally
comes at the cost of increased image collection time and process-
ing time. One striking example of successful 3D STORM is pro-
vided by the mapping of the mitochondrial network in whole cells
using dye pairs by Zhuang laboratory in [
33
].
Live cell imaging primarily requires the ability to maintain focus and
collect images at short exposures as quickly as possible without
shifts in temperature or movement. By necessity, it requires as little
photobleaching as possible and reduced background noise from
outside light sources. As has been mentioned, super-resolution
techniques falter with live cell imaging primarily due to speed and
the absolute requirement for stability. To date, variations on SIM
have been most successful. Recent developments have shown vast
improvements in utilizing super-resolution for three-dimensional
microscopy of living
Drosophila
cells in TIRF mode in which sam-
ples were fl attened against a pad of agarose gel [
15
]. Of particular
signifi cance in this application was the use of spatial light modulators
to generate phase shifts without mechanical movement. Camera
effi ciency is important in any imaging application but is particularly
imperative under live cell imaging considerations. Increased sensi-
tivity and speed and reduced specimen damage could be gained by
using either a high-effi ciency EMCCD camera or a scientifi c
CMOS camera. For live cell, multichannel 3D SIM approaches,
the use of individual cameras for each channel increases collection
speed with some cost in system complexity. STED as a laser scan-
ning technique has most of the advantages and disadvantages of
laser scanning confocal microscopy. STED has indeed been applied
to live cell imaging but is limited presently to the
XY
dimension
and to small fi elds, for which this strategy is most effi cient [
34
].
Perhaps parallel STED image collection can someday be implemented
6.3
Live Cell Imaging
