Biomedical Engineering Reference
In-Depth Information
provides a resolving power beyond that of the diffraction limit to
be super-resolution microscopy.
In reality, the development of fl uorescence microscopy can be
compared to the process of peeling an onion in which one layer
after another is revealed. In practice, the fi rst limit was not diffrac-
tion but brightness. In the early 1970s, the introduction of epi-
fl uorescence microscopy revolutionized the fi eld by overcoming
much of the brightness limitations at high magnifi cations (40-100×),
making possible localization studies at the full diffraction-limited
resolution of the light microscope. In this confi guration, the objec-
tive serves both as the condenser and objective, and the light
source was placed 90° to the objective. Light was refl ected onto
the specimen using a dichroic mirror. This arrangement minimized
background contributions from the excitation light, and more
importantly, the intensity of the collected fl uorescence light now
increased with the objective's numerical aperture. In contrast, for
previous optical confi gurations in which the condenser and objec-
tive were separate optical elements, fl uorescence intensity failed to
increase with numerical aperture of the objective, and brightness
decreased with magnifi cation [
3
]. With these advances, routine
studies of protein co-localization became possible with a resolution
approaching 200 nm in nearly fl at, two-dimensional space, condi-
tions found most commonly at the periphery of well-spread tissue
culture cells.
Cells, though, are not two-dimensional objects. Even tissue
culture fi broblasts are thicker toward the cell center where the
nucleus is located. Cell types such as epithelial cells lining an organ
(e.g., the intestine) are columnar in three-dimensional space. The
overall planar resolution of the epifl uorescence microscope, often
simply referred to as the wide-fi eld microscope, is limited in prac-
tice by sample thickness. Light from both above and below the
plane of focus is collected. As shown in Fig.
1a
, this affects image
acuity even for small cells such as human platelets that are discoid
in shape with a thickness of about 1.5
ʼ
m and a diameter of about
3
-granule pro-
teins, von Willebrand factor and fi brinogen, in an isolated platelet
appears hazy making the extent of co-localization diffi cult to assess.
Deconvolution algorithms in which the light distribution is cor-
rected on the basis of the point spread function allow for contrast
improvement as shown in Fig.
1b
, but not a gain in resolution [
4
].
In this review, we compare developing super-resolution tech-
niques including structured illumination microscopy (SIM), stim-
ulated emission depletion microscopy (STED), and photoactivated
localization microscopy/stochastic optical reconstruction micros-
copy (PALM/STORM) with conventional wide-fi eld and confocal
microscopy. We briefl y explain the basic principles of each tech-
nique and consider advantages and challenges in confronting these
new possibilities in microscopy. Microscope setup and probe use
ʼ
m. Hence, an image co-localizing two different
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