super-resolution microscopy

Super-resolution Microscopy 1

microscopyandanalysis

Super-resolution microscopy

Essential Knowledge

Briefings

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 HeathSpectroscopy and Separations EKB Series Editor: Nick Taylor


CONTENTS5 INTRODUCTION8 HISTORY AND BACKGROUND18 IN PRACTICE27 PROBLEMS AND SOLUTIONS31 WHATS NEXT?

About Essential Knowledge Briefings Essential Knowledge Briefings, published by John Wiley and Sons, comprise a series of short guides to the latest techniques,applications and equipment used in analytical science. Reviewed and updated annually, EKBs are an essential resource for scientists working in both academia and industry looking to update their understanding of key developments within each specialty. Free to download in a range of electronic formats, the EKB range isavailable at www.essentialknowledgebriefings.com


INTRODUCTIONImagine 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


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.


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


HISTORY AND BACKGROUNDAdvances 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-


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.


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.


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. confocalColloidal 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


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.


LOCALIZATION MICROSCOPYA 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.


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


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


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


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.


IN PRACTICEAs 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 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 confocalmicroscope (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 togenerate 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.


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.


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).


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


THEORY INTO PRACTICE: CASE STUDIES

GSDIMUsing 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,


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 TECHNIQUESDaniel 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


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 STUDIESRalf 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


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).

STEDBiologists 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]


PROBLEMS AND SOLUTIONSResolution

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.


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 CellsAnother 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


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.


Additional Image ProcessingSIM 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.


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 MicroscopyPerhaps 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 DevelopmentAnother way to overcome the limitations is to improve the

tools that super-resolution microscopy has to work with. Although


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


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,


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


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.


FURTHER INFORMATIONSuper-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).

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