Super-resolution Microscopy 1

microscopy
andanalysis
Super-resolution
microscopy
Essential
Knowledge
Briengs
2 Super-resolution Microscopy
Front cover image courtesy Professor Alberto Diaspro and Dr
Paolo Bianchini, of the Nanophysics Department, Istituto Italiano
di Tecnologia, Genoa
2013 John Wiley and Sons Ltd, The Atrium, Southern Gate,
Chichester, West Sussex PO19 8SQ, United Kingdom
Microscopy EKB Series Editor: Dr Julian Heath
Spectroscopy and Separations EKB Series Editor: Nick Taylor
Super-resolution Microscopy 3
CONTENTS
5 INTRODUCTION
8 HISTORY AND BACKGROUND
18 IN PRACTICE
27 PROBLEMS AND SOLUTIONS
31 WHATS NEXT?
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comprise a series of short guides to the latest techniques,
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4 Super-resolution Microscopy
Super-resolution Microscopy 5
INTRODUCTION
Imagine being able to see deep down inside a cell, to witness its
fine structure and all its internal workings and processes. Much as
you can see the internal workings of a watch, the click and whirr of
all its gears and levers, by simply taking off the back cover. To see
the complex mosaic of proteins in the cell membrane, the flexible
network of filaments making up the cytoskeleton, and the firing of
neurons. This is the enticing prospect held out by super-resolution
microscopy.
Until recently, the diffraction of light had placed a funda-
mental limit on how far biologists could peer into cells with optical
microscopes, preventing them from resolving features less than
250nm in size, missing critical structures within cells. Biologists
have still been able to view many of these features with non-opti-
cal microscopy techniques such as electron microscopy, but only
in freeze-frame, as these techniques require cellular samples to be
essentially frozen in place.
Over the past 20 years, however, scientists have developed
several ingenious techniques for lifting the veil caused by diffrac-
tion, allowing them to resolve features as small as 20nm. All these
techniques are based on fluorescence microscopy, but either
they finely control the fluorescence, such that only small groups
of fluorescent molecules or even individual molecules emit light at
any one time, or they apply specific patterns of illumination.
Commercial versions of all these techniques, produced by
companies such as Leica Microsystems, are now available and their
use is becoming increasingly widespread, allowing scientists to
study the structure and workings of cells in unprecedented detail.
Already, these techniques have been used to study the structure of
6 Super-resolution Microscopy
the inner mitochondrial membrane, the release of toxic granules
by immune system cells, and the cellular response to viral and bac-
terial infection.
But that is just the start: these techniques are poised to trans-
form our understanding of the internal workings of cells, as well as
of other processes that take place at the nanoscale. Furthermore, by
enhancing our understanding of how the immune system works
and how cells respond to viral and bacterial infection, they should
also lead to new treatments for major diseases.
This Essential Knowledge Briefing provides a general intro-
duction to the field of super-resolution microscopy, explaining
how the various techniques work and providing examples of what
they can do. It also outlines various practical issues related to the
techniques, describes potential problems that may arise and how
to solve them, and reveals forthcoming advances.
Super-resolution Microscopy 7
Principles of super-resolution microscopy
(A) In SIM the sample plane is excited by a nonuniform wide-field illumination. Laser
light passes through an optical grating, which generates a stripe-shaped sinusoidal
interference pattern. This combines with the sample information originating from
structures below the diffraction limit to generate moir fringes. The image detected by
the CCD camera thus contains high spatial frequency sample information shifted to a
lower spatial frequency band that is transmitted through the objective.
(B) In STED microscopy the focal plane is scanned with two overlapping laser beams,
typically being pulsed with a mutual time delay. While the first laser excites the
fluorophores, the second longer wavelength laser drives the fluorophores back to the
ground state by the process of stimulated emission.A phase plate in the light path of
the depletion laser generates a donut-shaped energy distribution, leaving only a small
volume from which light can be emitted that is then being detected. Thus, the PSF is
shaped to a volume smaller than the diffraction limit.
(C) Single molecule localization microscopy assures that only a relatively low number
of fluorophores are in the emitting (active) state. These molecules are detected on the
CCD camera as diffraction-limited spots, whose lateral position is determined with
very high accuracy by a fit. Courtesy L. Schermelleh, 2010
8 Super-resolution Microscopy
HISTORY AND BACKGROUND
Advances in optical microscopy over the years have increased
magnification and resolution, allowing scientists to glimpse ever
smaller objects at ever decreasing scales.
Originally, this increase was achieved with improved lenses,
but more recently it has required developing improved microscopy
techniques such as confocal microscopy. This is a form of fluo-
rescence microscopy that allows optical sectioning with subse-
quent three-dimensional reconstruction by scanning the sample
with focused laser light and using a pinhole to filter out fluorescent
signals from outside the focal plane.
But there is a fundamental limit, known as the diffraction
barrier, below which scientists were unable to glimpse. As its
name suggests, this limit is caused by the diffraction of light and
is defined as roughly half the wavelength of the light used to probe
the specimen. In practice, this means that objects smaller or closer
together than 250nm cannot be resolved.
Simply put, an optical microscope working with visible light
is incapable of imaging a spot smaller than 200-250nm wide, mean-
ing that everything within that spot gets merged together. Tech-
niques such as confocal microscopy and its advanced derivatives
can get close to the diffraction limit, but it is impossible for them
to go below it.
This means that many interesting cellular components, such
as microtubules, vesicles and individual proteins, which are all
under 100nm in size, cannot be resolved with optical microscopes,
even when marked with fluorescent labels. Obviously, these cellu-
lar components can still be viewed with non-optical microscopy
technologies, such as electron microscopy and scanning tun-
Super-resolution Microscopy 9
nelling microscopy (STM). But such technologies are limited
because they require specimens to undergo complex sample prepa-
ration procedures and be exposed to extreme operating conditions,
preventing them from working with live cells.
Fortunately, human ingenuity can sometimes bypass fun-
damental physical laws, and over the past 20 years scientists have
developed quite a few methods for circumventing the diffraction
barrier, bringing the resolution of optical microscopy down to
beneath 50nm.
Collectively termed super-resolution microscopy, these meth-
ods adopt a number of different approaches, but all take advantage
of the latest advances in lasers, fluorescent labels and computer
processing.
The theoretical basis of such methods is that, if fluorescent
labels are switched on in turn while all the others remain off, then
each label can be distinguished and its location determined with
great accuracy.
All thats needed, then, is a way to switch on fluorescent labels
on a specimen individually, while leaving all the others switched
off, allowing each label to be distinguished even when closer
together than 250nm.
10 Super-resolution Microscopy
STIMULATED EMISSION DEPLETION
MICROSCOPY(STED)
One of the first super-resolution techniques was stimulated
emission depletion (STED) microscopy, originally developed in
the mid-1990s by Stefan Hell, now at the Max Planck Institute for
Biophysical Chemistry in Gttingen, Germany.
STED works on the principle that although an optical micro-
scope cannot distinguish two fluorescent labels that are closer
together than 250nm, thats only if both labels are switched on at
the same time. In that case, diffraction will cause the light from
both labels to merge with each other, preventing them from being
distinguished.
STED uses a laser beam to stimulate fluorescence in a small
area of a specimen. Because of diffraction, however, this beam
cannot be focused tightly enough to stimulate just a single fluores-
cent label, or even a small collection of labels. So a second laser beam
is applied to the specimen, consisting of light at an intensity that
depletes the label fluorescence, switching the labels off. This second
laser beam is doughnut-shaped, meaning there is a tiny gap, much
smaller than the diffraction limit, in the centre of the beam where
the fluorescence is not depleted.
With the fluorescence now restricted to a very narrow region,
only individual fluorescent labels, or a small collection of labels,
can emit light at any one time, while all the other nearby labels
are switched off by the surrounding doughnut-shaped beam. By
scanning this system of laser beams across a specimen, fluorescent
labels can be switched on in turn at specific locations, allowing an
image of the labelled specimen to be built up at a resolution as low
as 50nm.
Super-resolution Microscopy 11
This is an elegant proof that the sum can be more than its parts.
While the excitation and depletion lasers each come up against the
limitations imposed by the diffraction barrier, the overlay of both
generates a light-emitting spot with a diameter far below the dif-
fraction barrier.
Commercially STED is only available from Leica Microsys-
tems, and is being used to study cellular organelles. For example,
Jordan Orange at the University of Pennsylvania School of Med-
icine in Philadelphia, USA, used it to study how immune system
cells deploy toxic particles known as granules to destroy tumour
cells and cells infected by viruses. Meanwhile, Stefan Jakobs at the
University of Gttingen in Germany recently used it to study the
structure of the inner mitochondrial membrane.
Comparison of resolution STED vs. confocal
Colloidal crystal structure of fluorescent nano-
spheres labeled with ATTO 647N.
Layer of fluorescent nano-beads imaged under
optimal confocal conditions (bottom) and using
the same settings with the STED-depletion laser
turned on (top).
The scalebar indicates 2 m.
Courtesy of Max Planck Institute for Biophysical Chemistry,
Gttingen, Germany
12 Super-resolution Microscopy
STED MICROSCOPY
a) Simple diagram of a STED microscope. The excitation and STED beams are merged
by dichroic mirrors DM1 and DM2 and focused by the objective lens (OBJ) into a
common focus. A helical phase mask (PM) in the STED beam path creates a dough-
nut-shaoed STED focus in the sample. Fluorescence is collected by the objective,
separated from laser light by DM1 and DM2, bandpass-filtered (F), and focused onto a
detector (D).
b) Detailed view of a helical phase ramp used to produce a doughnut-shaped STED
focus.
c) Hypothetical excitation (dotted line) and emission spectra of a fluorophore showing
wavelengths used for excitation (green line), depletion (red line) and the spectral win-
dow for fluorescence detection (orange box).
From: Fluorescence Microscopy: From Principles to Biological Applications, First Edition. Edited by Ulrich Kubitscheck.
2013 Wiley-VCH Verlag GmbH & Co.
Super-resolution Microscopy 13
LOCALIZATION MICROSCOPY
A related super-resolution microscopy technique, sometimes
termed localization microscopy, also stimulates individual
fluorescent labels on a specimen, but it switches them on randomly
rather than sequentially.
There are several different versions of this technique, which
differ in terms of the labels they use and the mechanism by which
the labels randomly switch on and off.
These different versions include: photoactivated localiza-
tion microscopy (PALM), developed by Eric Betzig at Howard
Hughes Medical Institute in Virginia, USA; stochastic optical
reconstruction microscopy (STORM), developed by Xiaowei
Zhuang at Harvard University, Cambridge, USA; and ground
state depletion followed by individual molecule return
(GSDIM), developed by Hell.
The idea behind all these techniques is that only a fraction of
the labels on a specimen fluoresce at any one time, ensuring that no
labels located within 250nm of each other fluoresce simultaneously.
This is done by getting the labels to fluoresce randomly over an
extended period of time, such that eventually all the labels fluoresce.
As long as neighbouring labels dont fluoresce at the same
time, the location of each individual label can be pinpointed with
great accuracy. This is because, although diffraction will cause the
light emitted by the label to spread out, producing a broad fluores-
cent spot larger than 250nm across, the fluorescent molecule will
always be located at the centre of this spot.
By recording thousands of images of these randomly fluoresc-
ing spots and calculating where their centres are, an image of the
specimen can be built up with a resolution as low as 20nm.
14 Super-resolution Microscopy
Its like standing some distance from a large structure such
as the Eiffel Tower in Paris when outlined with external lights at
night. With all the lights turned on, you will just see the outline of
the structure, rather than the individual lights. But if the lights are
set to a blinking mode, where they turn on and off randomly, it is
much easier to distinguish the individual lights. If you then cap-
ture enough images to ensure that each lamp has blinked at least
once, and merge them together, youll produce a single image of the
structure in which all the individual lights can be distinguished.
The trick is to find labels that fluoresce at random times, with
different versions of localization microscopy employing different
types of labels. PALM employs genetically-expressed photoacti-
vatable fluorescent proteins, such as photoactivatable versions of
green fluorescent protein (GFP), while STORM employs pairs of
organic dyes such as cyanine coupled to antibodies. In both cases,
the labels are activated by exposing them to a low intensity light
that triggers fluorescence via a comparatively rare mechanism
that only happens occasionally, ensuring that the labels switch on
randomly. After a certain period of fluorescence, the labels switch
back off again, either automatically or by deliberately quenching
them.
GSDIM also employs organic fluorescent dyes attached to
antibodies, but in this case the labels are initially illuminated
with high-intensity light, transferring them into a temporary off
state, termed dark state. After a random interval, they come out
of this dark state and start to fluoresce, switching themselves on
one at a time before switching back off. The advantage of GSDIM
(and a similar technique known as direct STORM) is that it can
work with a much greater variety of fluorescent labels than PALM
Super-resolution Microscopy 15
or STORM, but it does require that the specimen is immersed in
a medium that can maintain the majority of fluorescent labels in
the dark state.

Schematic representation of the GSDIM method based on a simplified Jablonski dia-
gram. Delocalized electrons in fluorophores can be, for instance, in the ground state
S0, in the excited state S1 (both so-called ON states) or in a triplet or radical dark state
(both OFF states). When fluorescent light is emitted, electrons circulate between the
ground and the excited state. Unlike these ON states, fluorophores in the OFF state are
not able to emit light. These OFF states are usually of long lifetime, but they are diffi-
cult to attain, as an inter system crossing is required. By setting the right ambient con-
ditions in the embedding medium and through the clever choice of standard fluoro-
phores for immunofluorescence, it is possible to reversibly switch off fluorophores by
exciting them with an extreme light intensity. When enough molecules are in the OFF
state, it is possible to detect individual molecules in the sample. Courtesy Leica Science Lab
16 Super-resolution Microscopy
Commercial versions of PALM, STORM and GSDIM are avail-
able, and are also being widely used to study cellular organelles. For
example, Jeri Timlin at Sandia National Laboratories in California,
USA, has used STORM to study cell membranes as they try to fend
off bacterial pathogens, while Jrg Wiedenmann at the University
of Southampton, UK, has used PALM to study the actin filaments
making up the cells cytoskeleton.
Principles of STORM
Fluorophores
too close to
resolve

Stochastic activation and
localization of individual
molecules

Super-resolution image
reconstructed from
localizations
Super-resolution Microscopy 17
STRUCTURED ILLUMINATION
MICROSCOPY (SIM)
The third and final super-resolution microscopy technique
works in a completely different way to STED and localization
microscopy, relying much more on computer processing. Known
as structured illumination microscopy (SIM), it was developed
in the late 1990s by Mats Gustafsson at the University of California,
San Francisco, USA, and takes advantage of an interference pattern
known as a moir fringe. This is produced when two grids of parallel
lines are overlaid at an angle, creating distinct patterns of fuzzy lines
running across the parallel lines. Such moir fringes are seen when
someone wearing a striped shirt appears on television, because the
stripes interact with the scanned lines that produce the picture.
The idea behind SIM is to illuminate a specimen with a striped
pattern of light, such that the grid of parallel lines of light interacts
with the pattern of fluorescent labels on the surface of the specimen
to produce a moir fringe. This is repeated up to 15 times with the
striped pattern of light at different angles and offsets, producing
lots of moir fringes. Because moir fringes essentially present high
resolution information at lower resolutions, they can be processed
by a computer to reveal the hidden high resolution information
about the distribution of fluorescent labels, allowing the specimen
to be studied at resolutions as high as 100nm.
This resolution is not as good as can be achieved with STED
and localization microscopy, but it has still proved very effective
at producing images of organelles and processes in live cells. For
example, Gustafsson has used it to visualize tubulin and kinesin
dynamics in live cells, while Rainer Heintzmann at Kings College
London, UK, has used it to visualize mitochondria in live cells.
18 Super-resolution Microscopy
IN PRACTICE
As with any form of fluorescence microscopy, the first step in
super-resolution microscopy is making sure you have a good qual-
ity specimen. The results obtained with any super-resolution tech-
nique are only as good as the specimen being studied.
The next step is to cover the specimen in fluorescent labels,
and there are two basic ways of doing this, both of which are also
employed in conventional fluorescence microscopy.
Fluorescent proteins such as GFP can be attached to speci-
mens via genetic engineering. This involves modifying the cells
genome such that the fluorescent protein is always expressed in
conjunction with a cellular protein of interest.
Alternatively, organic dyes can be physically attached to the
specimen. Conventionally, this is done by linking the dye to a pro-
tein or antibody that naturally binds with a protein of interest.
Another option is to link the dye to a secondary antibody, which
binds with the first antibody bound to the protein of interest.
A major advantage of this approach is that more than one sec-
ondary antibody can bind with the first antibody, meaning that
several dyes can be linked to the protein of interest, increasing the
amount of light given off by each label, which is particularly useful
in localization microscopy.
Both approaches have their advantages and disadvantages for
super-resolution microscopy. Genetically-expressed fluorescent
proteins are less invasive than organic dyes and so are appropriate
for live imaging, but they can be unstable and alter the structure of
the co-expressed protein.
Organic dyes tend to be brighter and more stable than fluores-
cent proteins, but they are not always appropriate for live imaging
Super-resolution Microscopy 19
Super-resolution microscopy of
biological samples. (A) Conventional
wide-field image (left) and 3D-SIM
image of a mouse C2C12 prometa-
phase cell stained with primary
antibodies against lamin B and
tubulin, and secondary antibodies
conjugated to Alexa 488 (green) and
Alexa 594 (red), respectively. Nuclear
chromatin was stained with DAPI
(blue). 3D image stacks were acquired
with a DeltaVision OMX prototype
system (Applied Precision). The
bottom panel shows the respective
orthogonal cross sections. (B) HeLa
cell stained with primary antibodies
against the nuclear pore complex
protein Nup153 and secondary
antibodies conjugated with
ATTO647N. The image was acquired
with a TCS STED confocal
microscope (Leica). (C) TdEosFP-pax-
illin expressed in a Hep G2 cell to
label adhesion complexes at the lower
surface. Single molecule positional
information was projected from
10,000 frames recorded at 30 frames
per second. On the left, signals were
summed up to
generate a TIRF image with conven-
tional wide-field lateral resolution.
Bars: 5 m (insets, 0.5 m). Courtesy L
Schermellah, 2010
and their use with antibodies means they are prone to binding with
various other proteins on the specimen rather than just the protein
of interest.
Fluorescent proteins also benefit from being located right
next to the protein of interest, ensuring that they accurately reflect
its position in the cell. In contrast, the intervening presence of one
or more antibodies, which can each be more than 10nm in size,
means that organic dyes can actually be some distance away from
the protein of interest.
20 Super-resolution Microscopy
This distance doesnt matter in normal fluorescent micros-
copy, because it is too small to show up, but it does matter in
super-resolution microscopy because of potential inaccuracies in
the resultant image. For example, the cellular structural compo-
nents called microtubules are known to be 25nm wide, but when
labelled with organic dyes attached to antibodies they can appear
to be 60nm wide.
The labelling approach adopted will also depend on the spe-
cific super-resolution technique being employed, because different
techniques demand fluorescent labels with different properties.
SIM can work with the same fluorescent labels as conventional
fluorescence microscopy, while STED requires a careful combina-
tion of fluorescent dyes and depletion laser wavelengths.
Localization microscopy techniques such as PALM and
STORM require labels that only fluoresce for a short time, mini-
mizing the possibility that two neighbouring labels will fluoresce
simultaneously, but produce a lot of light when they do fluoresce,
allowing high precision localization.
In all cases, however, the labels need to be stable and robust
enough to withstand repeated excitation with high intensity light
from a powerful laser, otherwise they can suffer from photobleach-
ing, where the labels switch off for good.
If photobleaching occurs too quickly, it will be impossible to
produce a detailed image of the specimen. Photobleaching can be a
particular risk for SIM, because it involves repeatedly exciting the
same fluorescent molecules.
In practice, SIM and STED can use most types of fluorescent
label. PALM employs fluorescent proteins, while STORM employs
pairs of organic dyes attached to antibodies.
Super-resolution Microscopy 21
GSDIM was deliberately developed to be more flexible in its
labelling requirements than PALM or STORM, and can work with
a wide variety of organic fluorescent dyes but tends to have stricter
requirements regarding the medium surrounding the specimen
than PALM or STORM, with this medium needing to contain
anti-oxidants and oxygen scavengers to keep the majority of the
labels in a dark state.
Many of these proteins and dyes are available in various
different colours, allowing biologists to visualise cellular processes
or interactions between organelles by labelling two or three groups
of proteins with different colour labels.
In order to provide a detailed image, however, the labels do
need to be attached at sufficiently high densities. This is more of
a challenge with super-resolution microscopy than conventional
fluorescence microscopy, because the higher resolution means
the labels need to be located much closer to each other to provide
a detailed image. Otherwise, all you see is a seemingly random col-
lection of spots.
Despite these inherent difficulties and challenges, scientists
have already used super-resolution microscopy to image cellular
organelles and structures that have never been viewed before with
optical microscopy, expanding our knowledge about the inner
workings of cells (see Case studies).
22 Super-resolution Microscopy
Resolvable volumes obtained with current commercial super-resolution microscopes.
A schematic 3D representation of focal volumes is shown for the indicated emission
maxima. The approximate lateral (x,y) and axial (z) resolution and resolvable volumes
are listed. Note that STED/CW-STED and 3D-SIM can reach up to 20 m into the
sample, whereas PALM/STORM is usually confined to the evanescent wave field near
the sample bottom. It should be noted that deconvolution approaches can further
improve STED resolution. For comparison the focal volume for PALM/STORM was
estimated based on the localization precision in combination with the z-range of TIRF.
These indications do not necessarily constitute actual resolution as many other effects
(e.g., fluorophore orientation, local refractive index variations, flatfield quality of the
camera, local aberrations, and statistical selection bias) influence image quality and
final resolution. Courtesy L Schermelleh, 2010
Super-resolution Microscopy 23
THEORY INTO PRACTICE: CASE STUDIES
GSDIM
Using a GSDIM-based microscope, German scientists have
been able to determine the position of fluorescent labels with an
accuracy of below 1nm. This has allowed them to investigate the
organization of the 30 or so proteins making up the pores in the cells
nuclear membrane, in which rings of proteins surround a central
channel through which transport occurs.
Using an iterative procedure we could align several thou-
sand images of single nuclear pores and generate an average image,
explains team member Anna Szymborska from the European
Molecular Biology Laboratory in Heidelberg. From the profile of
the average image we could determine the average distance of the
fluorescent label from the centre of the pore with a very high precision
and accuracy.
As a consequence, the scientists were able to determine for the
first time how one of the protein subcomplexes forming the scaf-
fold of the nuclear pore is oriented. Super-resolution microscopy
was particularly useful here because it combines high resolution
and information about the molecular identity of the protein, says
Szymborska. In principle electron microscopy with immunogold
labelling provides similar information, but in practice such experi-
ments are much harder to perform.
This was Szymborskas first experience with super-resolution
microscopy, but she predicts it will soon be routinely used in biology
laboratories. Already at the moment super-resolution is useful to see
how different proteins are organized in a whole cell, or, as in our case,
24 Super-resolution Microscopy
in a single protein complex, she says. I expect that many new pos-
sibilities will open up once super-resolution methods become easily
applicable for imaging live cells at physiological conditions. This kind
of technology will make it possible to visualize dynamic changes in
cells at the level of single molecules.
Science, 2013 (DOI: 10.1126/science.1240672)
MULTIPLE TECHNIQUES
Daniel Davis, director of research at the Manchester Collabo-
rative Centre for Inflammation Research, UK, employs a variety of
different super-resolution microscopy techniques in his work, includ-
ing SIM, STED, PALM and GSDIM. For example, in 2011, he
led a team that used SIM to image the meshwork of actin filaments
that underlie all cell membranes, discovering that this meshwork
opens up in immune cells known as natural killer cells when primed
to kill diseased cells.
Most recently, he used PALM and GSDIM to image proteins
on the surface of natural killer cells. This revealed that natural killer
cells alter the organization of these surface proteins when activated
by a type of protein found on tumour cells and on cells infected by a
virus.
We have shown that immune cell proteins are not evenly dis-
tributed as once thought, but instead they are grouped in very small
clumps a bit like if you were an astronomer looking at clusters of
stars in the Universe and you would notice that they were grouped in
clusters, explains Davis. We studied how these clusters or proteins
Super-resolution Microscopy 25
change when the immune cells are switched on to kill diseased cells.
Looking at our cells in this much detail gives us a greater understand-
ing about how the immune system works and could provide useful
clues for developing drugs to target disease in the future.
According to Davis, the advantage of using multiple super-res-
olution techniques is that they tend to be complementary, with the
abilities of one technique making up for weaknesses in another. Take
STED and GSDIM: Both techniques have their pros and cons, he
says, GSDIM can offer higher resolution, whereas STED is better
for imaging live cells. But he adds that the technology is improving so
fast that these differences in ability may soon be a thing of the past.
Science Signaling, 2013, 6, ra62 (DOI: 10.1126/scisig-
nal.2003947).
CELLULAR STUDIES
Ralf Jacob at Philipps University of Marburg in Germany
regularly employs GSDIM in his cellular studies. For example, he
has used it to visualize the allocation of post-translationally-modified
tubulin on microtubules and to study the distribution of the protein
galectin-3 within membrane-bound organelles known as endosomes.
He was also part of a team that used GSDIM to probe how cells
respond to viral infection, discovering that a cellular enzyme known
as RNA helicase recognizes a viral invader by interacting with a
panhandle structure on the outer coat of the virus.
These studies could also have been conducted with electron
microscopy, says Jacob, but super-resolution techniques such as
26 Super-resolution Microscopy
GSDIM have certain important advantages. Electron microscopy
is certainly an alternative to super-resolution, he says. Neverthe-
less, sample preparation and labelling is much easier with fluorescent
techniques.
As such, he thinks that super-resolution microscopy will be an
increasingly attractive option for those exploring the intricacies of
cellular structure and processes. Super-resolution will help us to get
a more detailed view of subcellular structures, he asserts. This may
lead to a deeper understanding of molecular interaction partners and
their interplay within cellular compartments.
Cell Host & Microbe, 2013, 13, 336346 (DOI: 10.1016/j.
chom.2013.01.012).
STED
Biologists led by Jordan Orange at the University of Pennsyl-
vania School of Medicine in Philadelphia, US, used a Leica TCS
STED microscope to produce unprecedented images of the immune
system in action. Specifically, they used the microscope to study how
natural killer cells deploy toxic particles known as granules to destroy
tumour cells and cells infected by viruses. They were able to witness
the granules travelling through a dense network of protein filaments
produced by the natural killer cells, which delivered the granules into
the diseased cells. [PLoS Biology, 2011, 9, e1001151]
Super-resolution Microscopy 27
PROBLEMS AND SOLUTIONS
Resolution
With the current generation of super-resolution microscopy
techniques able to circumvent the diffraction barrier, achieving res-
olutions as high as 20nm, its no surprise that commercial versions
have been readily adopted by the biological community and are now
in widespread use. Despite this success, however, the current genera-
tion of techniques still have certain constraints.
One of the main limitations is now starting to be overcome
for certain super-resolution microscopy techniques. Initially, these
techniques could only achieve super-resolution laterally, over the
flat surface of the specimen. None of the super-resolution micros-
copy techniques could achieve the same level of resolution axially, in
the up-down direction.
This means that these techniques were basically confined to
producing high-resolution images in just two dimensions. Extend-
ing into the third dimension requires super-resolution techniques
that can achieve similar resolutions in the axial direction as in lateral
directions, and these have begun to appear over the past few years.
Xiaowei Zhuang and her colleagues came up with a three-
dimensional version of STORM, known as 3D STORM, in 2008. This
uses a cylindrical lens to alter the shape of the spot produced by each
fluorescent label, with the precise shape of the spot depending on
whether the label is positioned above or below the focal plane of the
lens. In this way, they can localize the label in both lateral and axial
directions, achieving an axial resolution as high as 50nm.
Also in 2008, Mats Gustafsson came up with a three-
dimensional version of SIM, by utilizing an illumination pattern
that varies both laterally and axially.
28 Super-resolution Microscopy
To produce a three-dimensional version of STED, Stefan
Hell used a system of opposing lenses to replace the flat dough-
nut-shaped spot of laser light with a hollow sphere.
Termed isotropic STED, this technique is able to achieve an
axial resolution of 30nm. A similar approach was taken by Jennifer
Lippincott-Schwartz at the US National Institute of Child Health
and Human Development in Bethesda, USA, to produce iPALM,
which combines opposing lenses with PALM to achieve an axial
resolution of just 10nm.
GSDIM, STED, SIM and STORM systems capable of three-
dimensional super-resolution are already commercially available,
and others are surely set to follow.
Light-optical section through two mouse
cell nuclei in prophase, recorded with 3D
Structured Illumination Microscopy
(3D-SIM-microscopy). condensed
chromosomes are red, the nuclear
envelope blue and microtubuli, which
belong to the cytoskeleton, are green.
Scale bar is 5 m
Courtesy Lothar Schermelleh, 2010
Acquisition and Challenges with Living Cells
Another limitation is that high resolution comes at the
expense of speed. It takes time to scan a laser beam across a specimen,
as required for STED, or to produce numerous separate images of a
specimen, as required for SIM and localization microscopy. This
means it can take 1030 seconds to produce a single STED or SIM
Super-resolution Microscopy 29
image, and up to 60 seconds to produce a single localization micros-
copy image, which can be made up of thousands of separate images.
One of the consequences of this lack of imaging speed is that
these techniques are simply not fast enough to visualise short-lived
processes taking place in live cells and certain features on an image
of a live cell produced by these super-resolution microscopy tech-
niques will appear blurred if they are moving on a timescale that is
shorter than the imaging time.
Indeed, although these super-resolution microscopy tech-
niques dont require the kind of chemical treatment of specimens
and the extreme imaging conditions employed by EM and STM,
they are still not always ideal for imaging live cells. As well as the
long imaging times, the strong laser light needed to stimulate
emission by the fluorescent labels can damage live cells. Another
limitation is that the laser light is scattered as its passes through
biological tissue, as is the light emitted by the fluorescent labels,
preventing these microscopy techniques from probing too far
beneath the surface of specimens.
Another issue to bear in mind when conducting super-
resolution microscopy is that the resultant image is, to a greater or
lesser extent, manufactured. Most of the techniques use computer
processing and various algorithms to construct the images from
the optical data; unlike with conventional fluorescent micros-
copy, you are not seeing a direct image of the specimen. As such,
there is a risk that these constructed images may contain artefacts
produced by the algorithms, rather than being actual features of
the specimen.
30 Super-resolution Microscopy
Additional Image Processing
SIM is the technique that relies most heavily on algorithms,
as it involves extracting high resolution data hidden in low resolu-
tion data. The localization microscopy techniques are also reliant
on algorithms to identify the centres of the fluorescent spots and to
build up a single image from thousands of separate images of those
spots. STED is the only technique that doesnt rely on algorithms,
although deconvolution can be carried out to further enhance the
resultant image.
One way to ensure that the image produced by localization
microscopy techniques is an accurate representation of the spec-
imen and doesnt contain any artefacts is to try producing the
same image with various different algorithms. Any features that
appear on the images produced by some algorithms but not others
could well be artefacts. To this end, biologists would like access to a
greater range of algorithms for producing super-resolution images
from optical data.
As with conventional fluorescence microscopy, a range of
other factors, including optical aberrations and changes in envi-
ronmental conditions such as temperature, can also cause artefacts
to appear in the image. Indeed, factors such as thermal drift can be
even more of a problem for super-resolution microscopy, because
their effects are magnified at high resolutions.
Fortunately, a few simple measures can help to reduce the
chance of artefacts occurring. These include performing exper-
iments in a controlled environment with a stable temperature,
ensuring the microscope is mechanically isolated and conducting
an initial check of the microscope with a known reference sample.
Super-resolution Microscopy 31
WHATS NEXT?
The current generation of super-resolution microscopy tech-
niques may have certain limitations, but scientists are hard at
work trying to overcome them. In the process, they are developing
the next generation of techniques, which will be faster, more flexi-
ble and able to penetrate deeper into tissue. They will also push the
resolution even higher.
Multi-modal Microscopy
Perhaps the most straight-forward way to overcome some
of the limitations of the current generation of super-resolution
microscopy is to combine it with other forms of microscopy. For
example, scientists are combining super-resolution microscopy
with EM, overlaying images of the same specimen produced by
each technique.
Combining fluorescence microscopy and EM in this way is
known as correlative light and electron microscopy (CLEM)
and allows the structures imaged by fluorescence microscopy to
be placed within the landscape revealed by EM. Super-resolution
microscopy enhances CLEM because its resolution is much closer
to that of EM, allowing the fluorescently-labelled structures to be
located within the cellular landscape much more accurately. Scien-
tists are also combining techniques such as PALM and STED with
multiphoton microscopy, which uses wavelengths of light that can
penetrate deeper into tissue.
Fluorescent Dye Development
Another way to overcome the limitations is to improve the
tools that super-resolution microscopy has to work with. Although
32 Super-resolution Microscopy
there are already a large number of fluorescent dyes that can be
used with super-resolution microscopy, scientists are always on
the look-out for varieties that are both more effective and have a
broader range of properties.
At first, scientists discovered appropriate labels for PALM
and STORM more or less by trial and error. Indeed, STORM actu-
ally came about as a result of the chance discovery by Mark Bates,
a doctoral researcher in Xiaowei Zhuangs group at Harvard Uni-
versity, that the red emission of a fluorescent cyanine molecule
known as Cy5 could be switched on and off with pulses of light.
Now scientists have more experience with these super-resolu-
tion techniques and the kind of properties required in the fluores-
cent labels, the search has become more directed. In 2011, Zhuang,
Bates and their colleagues conducted the first systematic assess-
ment of a selection of fluorescent labels for their ability to be used
in STORM.
This involved characterizing 26 organic dyes that can revers-
ibly switch between on and off states, based on the number of pho-
tons they produce, the length of their duty cycle (how quickly they
switch between on and off states), and their stability. The work
uncovered several high quality labels for STORM that could fluo-
resce at different colours, allowing Zhuang and his team to conduct
four-colour STORM.
Other researchers are investigating brand new types of flu-
orescent label for super-resolution microscopy, such as quantum
dots. These are semiconducting nanoparticles that fluoresce in var-
ious different colours according to their size.
Scientists are also exploring new ways to attach fluorescent
labels to the specimen, with the aim of locating the label much
Super-resolution Microscopy 33
closer to the protein of interest than is possible with antibodies.
Several new techniques for covalently attaching almost any mole-
cule to a protein of interest are now available, including Halo-tags
and SNAP-tags, and have already been applied to super-resolution
microscopy. For example, a team led by W. E. Moerner at Stanford
University, California, USA, used Halo-tags, which are based on a
combination of a special enzyme and substrate, to link fluorescent
labels to microtubules for imaging by PALM.
Still, even with fluorescent labels that emit lots of light and
are closely linked to the proteins of interest, current super-resolu-
tion techniques have a maximum resolution of around 20nm, even
though theoretically they should be able to achieve much higher
resolutions.
The reason they cant is due to a combination of background
noise, unavoidable vibration in the measurement equipment
and imperfections in the optics. In 2010, though, a team led by
Steven Chu from Stanford University, and a former US Secretary
of Energy, developed a feedback-based mathematical processing
system that could take account of this noise and unavoidable var-
iation. By monitoring exactly where photons released by individ-
ual fluorescent labels hit a charge-coupled device, Chu and his team
were able to resolve labels separated by just 0.5nm.
Mathematical processing can also help deal with the other
major limitation of super-resolution microscopy: speed. Locali-
zation microscopy is clearly the slowest of the super-resolution
microscopy techniques, but its speed can be increased by simply
increasing the number of fluorescent labels emitting light in each
image, as well as by using faster cameras and stronger lasers. More
labels switched on means fewer separate images need to be taken,
34 Super-resolution Microscopy
which means less time. Obviously, you then run the risk that
neighbouring labels will fluoresce at the same time, with the light
from each merging together, but this can be dealt with by mathe-
matical processing.
In 2012, a team led by Bo Huang at the University of Califor-
nia, San Francisco, USA, reported using a mathematical technique
known as compressed sensing to resolve overlapping fluorescent
labels in STORM. This meant they could produce images with a
much higher density of switched-on labels than in conventional
STORM, allowing them to produce images of microtubules in
living cells in just three seconds.
A different approach was taken by a team led by Jim Zhang
at the Johns Hopkins University in Baltimore, USA. Known as
photochromic stochastic optical fluctuation imaging (pcSOFI),
their approach involves taking images of a specimen labelled with
fluorescent proteins that repeatedly flash on and off like beacons.
Each image therefore contains a different pattern of light-emitting
labels.
Using statistical analysis, Zhang and his team were able to
combine the different patterns in these images into a single high
resolution image of the specimen. Crucially, this can be achieved
with a much lower number of images than with conventional
localization microscopy, in the region of hundreds rather than
thousands. As a consequence, Zhang and his team were able pro-
duce images of a cell at a resolution of 100nm in just five seconds.
Eventually, the fluorescent labels may no longer even be
required. In 2013, a team led by Ji-Xin Cheng at Purdue Univer-
sity in Indiana, USA, reported developing a version of STED that
doesnt require fluorescent labels. Instead, the scientists combine
Super-resolution Microscopy 35
STED with pump-probe spectroscopy, in which a pump laser
beam disturbs the density of charge-carriers such as electrons in a
small region of a specimen while a second probe beam detects this
disturbance. The scientists combined this two-beam system with
the doughnut-shaped quenching beam used in STED, restricting
the pump-probe beams to a spot around 200nm wide. Using this
approach, they were able to produce images of graphite nanoplate-
lets.
After circumventing the diffraction barrier, super-resolution
microscopy looks set to continue its downward trajectory, reveal-
ing the secrets of life at ever smaller scales.
36 Super-resolution Microscopy
FURTHER INFORMATION
Super-resolution microscopy section of Leica website (http://www.
leica-microsystems.com/science-lab/topics/super-resolution)
Hell SW, 2007. Far-field optical nanoscopy. Science 316: 11531158
(http://dx.doi.org/10.1126/science.1137395).
Schermelleh L, Heintzmann R and Leonhardt H, 2010. A guide to
super-resolution fluorescence microscopy. Journal of Cell Biology
190: 165175 (http://dx.doi.org/10.1083/jcb.201002018).
Vogelsang J, Steinhauer C, Forthmann C, Stein IH, Person-Skegro
B, Cordes T et al., 2010. Make them blink: Probes for super-res-
olution microscopy. ChemPhysChem 11: 24752490 (http://dx.doi.
org/10.1002/cphc.201000189).
Galbraith CG and Galbraith JA, 2011. Super-resolution micros-
copy at a glance. Journal of Cell Science 124: 16071611 (http://dx.doi.
org/10.1242/jcs.080085).
Super-resolution Microscopy 37
38 Super-resolution Microscopy
Super-resolution Microscopy 39
microscopy
andanalysis
Essential
Knowledge
Briengs