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(SIM) illuminates the entire field with a striped the PSF to reduce its effective diameter. They toactivation localization microscopy (PALM)
pattern of light (Gustafsson, 2000). When this surround a laser-scanning focal excitation PSF (Betzig et al., 2006) and fluorescent PALM
excitation pattern mixes with the spatial pattern with an annulus of longer-wavelength light that (fPALM) (Hess et al., 2006) use genetically
of the sample they produce an interference has an intensity high enough to saturate all expressed photoactivatable probes to achieve
pattern (called a moiré fringe) that is much fluorophores in the annulus to the ground state the on–off states of the fluorophore. An
coarser than either pattern alone and is (STED) (Hell and Wichmann, 1994) or the alternative approach, stochastic optical
detectable by the microscope. The excitation meta-stable dark state (GSD) (Bretschneider et reconstruction microscopy (STORM) (Rust et
pattern is translated and rotated to generate a al., 2007) to suppress their fluorescent emission. al., 2006), uses pairs of cyanine (Cy) dyes,
series of images with different moiré fringes. As Increasing the intensity of light in the annulus typically coupled to antibodies up to 15 nm in
the illumination pattern is known, it can be expands the zone of saturation to create an length, to act as reporter and activator pairs in
mathematically removed from the moiré to gain increasingly smaller excitation PSF that is order to cycle multiple times between the dark
access to the normally irresolvable higher smaller in diameter than the diffraction limit of and light states. In direct STORM (dSTORM)
resolution information in the sample. SIM 200 nm. The smaller PSF is then scanned over (Heilemann et al., 2008), several stand-alone
increases resolution to ~100 nm in the x-y the sample to generate the enhanced-resolution synthetic dyes, such as Alexa-Fluor dyes, can
direction and ~400 nm axially (Schermelleh et image. The best two-dimensional (2D) also be used in a blinking mode to attain super-
al., 2008). SIM is limited to this factor-of-two resolution in a biological context for STED or resolution images. Despite the technical
improvement because the periodicity of the GSD that has been achieved so far is 20–30 nm differences between these techniques, they all
illumination pattern is created by diffraction- full width at half maximum (FWHM) across the localize the position of the molecule of interest
limited optics and is, therefore, limited by the PSF (Westphal and Hell, 2005). The commercial to ~20 nm.
PSF of conventional microscopy (Gustafsson, implementation of STED currently has a
2000). resolution of ~70 nm laterally with no increase The resolution of super resolution
Other super-resolution imaging techniques in axial resolution above diffraction. Although The resolution of any image, conventional or
modulate the excitation light to exploit the the excitation volume is below the diffraction super resolution, depends on the number of
ability to saturate the emission of fluorophores limit, these techniques only make ensemble points that can be resolved on the structures
Journal of Cell Science
in order to break the diffraction barrier by a measurements because they do not distinguish of interest. According to the Nyquist–Shannon
greater amount. Saturation can be achieved by between individual molecules within an criteria (Shannon, 1949), a structural feature can
using intense illumination to produce a excitation volume. only be resolved when the distance between two
photophysical transition of the fluorophore to There are also good prospects for improving labels is less than half the feature size.
a transient dark state that can lead to either a the resolution of SIM. The non-linear Therefore, at least two points need to be
permanently dark state (bleaching) or the relationship between excitation and emission resolved within the minimum spatial feature size
emission of light on a microsecond or can be combined with the illumination pattern that is to be imaged. Localizing individual
millisecond time scale, which is much slower used in SIM. This technique – saturated SIM molecules with high precision does not create a
than the nanosecond time scale of fluorescence. (SSIM) (Gustafsson, 2005) – can be thought of super-resolution image when there are not
Alternatively, super-resolution techniques can as the inverse of STED, in which sharp dark enough labeled molecules within the PSF to
use light to induce photochemical reactions regions are created instead of sharp bright identify the spatial and temporal features of the
in photoswitchable or photoactivatable regions. The resolution of SSIM scales with the structure. Thus, when a single-molecule-based
fluorophores, and either transition them level of saturation, and 50 nm lateral resolution super-resolution image is reported as having a
between on and off states or change their color. has been demonstrated using fluorescent beads resolution of 20 nm, the number must refer to
As long as these transitions can be limited to a (Gustafsson, 2005). The general term for super- structural resolution and not simply indicate that
subset of fluorophores that are spatially resolution techniques that make use of the image is only displaying molecules whose
separated by the distance of the microscope PSF, the reversible non-linear switching of the centroids could be identified with ≤20 nm
the molecules can be located with precision fluorophore state is reversible saturable optical certainty.
approaching 5 nm. Super-resolution techniques fluorescence transitions (RESOLFT) (Hell,
can be separated into two categories depending 2003). Speed of acquisition
on whether these effects are exploited at the Many super-resolution techniques obtain
ensemble level or at the single-molecule level. Single-molecule techniques increased resolution at the cost of the speed of
Single-molecule-based techniques overcome image acquisition, simply because they use
Ensemble techniques the diffraction limit by using light to turn on only conventional microscope optics and hardware.
Ensemble-based techniques increase resolution a sparse subset of the fluorescent molecules of It takes longer to scan the smaller PSF of the
by shaping the excitation light, and the interest. Even though the visualized molecules STED excitation beam across a specimen than it
resolution of the images obtained by these that are turned on appear to have the size of a would take to scan the larger PSF of a
techniques is determined by the size of the PSF in a conventional microscope, if they are conventional spot-scanning microscope. The
super-resolution PSF. By contrast, single- separated from each other by at least 200 nm, temporal resolution of STED microscopy has
molecule techniques rely on localization then the concept of single-molecule localization been reported as 35 mseconds per image with a
precision, which is the uncertainty in the can be used to determine their centroids with 1.8 m ´ 2.5 m field of view, at a 2D
identification of the center of a molecule’s PSF nanometer-level precision (Gelles et al., 1988; resolution of 62 nm (Westphal et al., 2008).
and depends on photon output (Thompson et al., Yildiz et al., 2003). This process is repeated over However, with a larger field of view, imaging
2002). Importantly, localization precision is not many cycles, with new molecules turning on and speed slows down dramatically. Acquisition
the resolution of the image. Stimulated emission other molecules turning off in a stochastic times of 10 seconds per image over a 2.5 m ´
depletion (STED) microscopy and ground-state manner. Super-resolution images are created by 10 m field of view for a 50 nm resolution have
depletion (GSD) microscopy optically modify assembling all of the localized points. Both pho- been reported for the very bright fluorescence-
Journal of Cell Science 124 (10) 1609
filled dendritic spines in living cells (Nagerl et reversibly or irreversibly switchable between a excitation and the STED beam (Wildanger et al.,
al., 2008). Similarly, if image acquisition light and a dark state, or they need to change 2008). Multi-color STORM requires either
requires moving a grid and collecting as many as from one wavelength to another wavelength. pairing the same activator Cy dye to one of three
15 images to generate one SIM super-resolution The probes should be as bright as possible and spectrally different reporter Cy dyes or pairing
image, then super-resolution images will be should have a high contrast ratio between the spectrally distinct activators to the same
obtained at a slower rate than conventional two states. Of course, different techniques have reporter. In the first scenario, distinct emission
images, i.e. at ~30 seconds per image. However, different criteria for what makes a good probe. spectra are the multicolor read out and, in the
a recent implementation of SIM using a For STED, dyes need to be easily driven into second scenario, distinct activation spectra are
ferroelectric liquid crystal on a silicon spatial stimulated emission, have no excitation at the used to temporally separate the constant
light modulator to produce the patterns has depletion wavelength, and have photostability emission (Bates et al., 2007). Finally, multi-
proven to be much faster. Image fields of 32 m to withstand high intensities at both the color PALM schemes originally imaged
´ 32 m and 8 m ´ 8 m have been depletion and the excitation wavelengths. ATTO multiple colors sequentially, by photoconverting
successfully imaged at 3.7 Hz and 11 Hz, dyes were originally used for STED; however, one fluorophore from a lower-wavelength to a
respectively (Kner et al., 2009). Therefore, it is an extensive list of additional dyes, their higher-wavelength color and then reversibly
important to consider whether dynamic resolution, parameters used for depletion and switching a lower-wavelength fluorophore
biological structures change on a time scale that excitation, and references can be found online at (Shroff et al., 2007). More recently, two-color
is slower than the time required to collect an the website of the Department of imaging has been done by using spectrally
image when choosing a super-resolution NanoBiophotonics at the Max Planck Institute separated dark-to-light probes (Subach et al.,
method. for Biophysical Chemistry, Göttingen, Germany 2010). However, there is still a need for
The trade-off between speed and resolution is (http://www.mpibpc.mpg.de/groups/hell/STED additional probe development, especially
typically more pronounced with single- _Dyes.html). for green fluorophores, where low photon
molecule techniques. Two molecules cannot be All single-molecule super-resolution outputs and the inability to identify transfected
turned on within the same PSF at any given time, techniques rely on localization precision, which cells prior to activation have slowed down
which limits the speed of the molecular read- is approximated as s/冑 (N), where s is the simultaneous dual-color super-resolution
Journal of Cell Science
out. Although the concept of turning on a sparse standard deviation of the PSF, and N is imaging efforts. The continued development of
subset of labeled molecules has enabled high- the number of photons detected (Thompson et technologies such as dSTORM, which rely on
speed single-molecule tracking with high al., 2002). Therefore, the approximately tenfold manipulating the photophysics of dyes used in
molecular density (Hess et al., 2007; Manley et higher number of photons detected with an conventional light microscopy, will increase the
al., 2008), the spatial constraint on molecular inorganic dye compared with that when using a number of color choices.
proximity has made live-cell super-resolution photoactivatable fluorophore suggest that
imaging challenging. However, large fields inorganic dyes provide a far greater localization Other dimensions
(28 m ´ 28 m) with a single-molecule precision – approaching 5 nm FWHM (Shtengel Super-resolution microscopy began by
localization precision of 20 nm can be obtained et al., 2009). However, in biological improving resolution in the x and y dimensions,
every 25–60 seconds, but the images have a applications, inorganic dyes are often coupled but biological structures are 3D. Although the
spatial resolution of ~60 nm (Shroff et al., 2008). to antibodies whose size (>10 nm) is two- to simplest way to generate 3D super-resolution
Spatial resolution is normally less than threefold larger than the 3–4 nm of genetically images is to combine the serial sectioning of
localization precision, not only because of the expressed fluorescent proteins. The length of the tissue with standard lateral super-resolution
number of fluorophores localized along a given antibody adds uncertainty to the location of techniques (Punge et al., 2008), many super-
feature, but also because the structures in living the centroid and decreases the higher precision resolution techniques have now been extended
cells translocate, gain molecules and lose that is inherent to inorganic dyes. However, new to 3D. In 3D STORM a cylindrical lens is simply
molecules during image acquisition. Hence, labeling strategies are continuously emerging added in the light path to create astigmatic
live-cell single-molecule super-resolution with the use of Halo-, SNAP- and CLIP-tag imaging so that the ellipticity of the PSF
requires that sampling is fast enough – both protein labeling systems that enable specific, becomes a sensitive measure of its distance from
temporally and spatially – to avoid blurring and covalent attachment of virtually any molecule the focal plane, and yields a resolution of 20–
to quantify dynamics. It also requires that to a protein of interest and have already been 30 nm laterally and 50–60 nm axially (Huang et
control experiments that estimate the number of used in PALM (Lee et al., 2010), STED (Hein et al., 2008). Isotropic STED (isoSTED) is a more
molecules in a structure are used as a guide for al., 2010) and STORM (Dellagiacoma et al., complicated hardware implementation of STED
parsing the correct number of single-molecule 2010). These new technologies promise the that uses opposing objectives to create a hollow
frames into single-molecule super-resolution possibility of continually increasing localization sphere of light, which replaces the more-
time series images. precision. cylindrical doughnut-shaped STED, yielding
As super resolution frequently relies on 30 nm isotropic resolution (Schmidt et al.,
Super-resolution probes photophysics, the task of selecting multiple 2009). To date, the greatest axial improvement
Like all fluorescent techniques, the key to the labels requires more consideration than simply has been achieved by using opposing objectives
super-resolution techniques lies in the choice of choosing spectral separation. For example, to combine axial interferometry with 2D PALM
probes. SIM is the closest to traditional multi-color SIM is best achieved with two dyes in a technique termed iPALM (Shtengel et al.,
fluorescence microscopy, requiring no special that have similar excitation spectra but a large 2009). iPALM demonstrates 20 nm lateral
probes but, as multiple intermediate images are enough shift between their emission maxima to resolution and 10 nm axial resolution (Shtengel
collected, photobleaching must be considered. spectrally separate the dyes. Similarly, to avoid et al., 2009). Previously, structures of this size
The other super-resolution techniques require using pairs of either excitation or depletion could only be accurately visualized by electron
fluorescent probes whose state can be lasers, a pulsed-supercontinuum laser was microscopy (EM), but now the EM resolution
controlled. The probes need to be either recently used with two-color STED for both the can be combined with the molecular specificity
1610 Journal of Cell Science 124 (10)
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Cell Science at a Glance on the Web
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Journal of Cell Science
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795. Multilayer three-dimensional super resolution imaging of