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1 Journal of Biotechnology 149 (2010) Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: Fluorescence microscopy beyond the diffraction limit Mike Heilemann Applied Laser Physics and Laser Spectroscopy, Physics Department, Bielefeld University, Universitätsstrasse 25, Bielefeld, Germany article info abstract Article history: Received 1 November 2009 Received in revised form 8 March 2010 Accepted 22 March 2010 Keywords: Fluorescence microscopy Subdiffraction resolution Super-resolution Stimulated-emission depletion Structured-illumination microscopy Photoswitching microscopy In the recent past, a variety of fluorescence microscopy methods emerged that proved to bypass a fundamental limit in light microscopy, the diffraction barrier. Among diverse methods that provide subdiffraction spatial resolution, far-field microscopic techniques are in particular important as they can be operated in complex biological samples such as cells or tissue. Valuable new insights into biomolecular structure, organization and even dynamic processes in living cells have been gained with these novel microscopic techniques. In the present review, the most important concepts of far-field microscopy with subdiffraction resolution are introduced. The underlying physical concepts are discussed, and practical considerations for the application of these methods are made Elsevier B.V. All rights reserved. 1. Introduction In the past decades, fluorescence microscopy has gained tremendous importance for biological and medical research. Fluorescence microscopy allows non-invasive and very specific imaging of single biomolecules, cells and tissue, in three dimensions, with multiple colours and even in living organisms (Lakowicz, 1983; Pawley, 2006). The variety of available fluorescent probes has largely increased and spans the whole visible part of the electromagnetic spectrum, including organic fluorophores, fluorescent proteins (Giepmans et al., 2006), semiconductor nanocrystals (Michalet et al., 2005) and new probes such as fluorescent nanodiamonds (Fu et al., 2007). At the same time, a multitude of strategies for specific labelling of biomolecules with fluorescent probes have been developed (Chen and Ting, 2005). As any lens-based light microscope, the spatial resolution of fluorescence microscopy is limited due to the wave nature of light and consequential diffraction (Abbe, 1873). As a result, an infinitesimally small object that emits light will be detected as a finite-sized spot, which is referred to as the point-spread function (PSF). The dimension of the PSF can be described by its full-widthhalf-maximum (FWHM), which has a dimension of typically half the wavelength or around nm in the focal plane. In axial direction (and along the optical axis), the FWHM of the PSF is substantially larger and reaches nm. The reason for the Tel.: ; fax: address: heileman@physik.uni-bielefeld.de. URL: elongated shape of the PSF along the optical axis lies in the nature of the non-symmetrical wavefront that emerges from a conventional objective lens. Compared to the biomolecular length scale which is in the range of a few nanometres, one can immediately figure out that structural details or the organization of biomolecular assemblies cannot be adequately resolved. It has been a motivation for researchers to overcome this limit imposed by the diffraction barrier and to find ways to resolve important details of biomolecular structures and interactions at the molecular level and inside the cell. This has led to the development of a number of far-field based microscopic techniques that surpass the diffraction limit (Hell, 2007, 2009; Hell et al., 2009; Huang et al., 2009; Huser, 2008; Ji et al., 2008). The purpose of this review is to summarize the efforts made in the past nearly two decades, to introduce the underlying physical concepts, to present current achievements and to discuss their practical aspects and limitations. 2. Concepts for super-resolution 2.1. Improving the axial resolution The first efforts to improve the resolution in far-field microscopy were aimed at improving the axial resolution in confocal microscopy, which is around nm and considerably worse than the lateral resolution. This fact severely hampers isotropic three-dimensional imaging of biological samples. A first strategy that successfully allowed for optical sectioning in axial direction was two-photon microscopy (Denk et al., 1990). Two-photon /$ see front matter 2010 Elsevier B.V. All rights reserved. doi: /j.jbiotec

2 244 M. Heilemann / Journal of Biotechnology 149 (2010) Fig. 1. (A) Following absorption of a photon of appropriate energy, a fluorophore enters the first excited state, S 1. Following fast relaxation to the vibrational ground state of S 1, the emission of fluorescence can occur. The key principle of STED microscopy is that this excited state is locally depopulated by inducing stimulated emission. (B) A first laser that excites fluorophores into the S 1 state is overlaid with a depletion laser which has a doughnut-shaped intensity profile, where the area of zero-intensity scales with the irradiation intensity of the depletion beam. The resulting effective PSF represents the remaining area where fluorescence emission is still observed, and which is well below the diffraction limit. (C) Comparison of a confocal (top) and a STED image (bottom) of the endoplasmatic reticulum of a living mammalian cell labelled with the fluorescent protein Citrine (scale bar 1 m; reprinted with permission from Hein et al., 2008; Copyright (2008) National Academy of Sciences, U.S.A.). microscopy does not lead to a resolution enhancement per se: although the size of the excitation point-spread function (PSF) is reduced, the trade-off is that the wavelength has to be doubled to excite the fluorescent probes. However and more importantly, two-photon microscopy reduces out-of-focus light considerably and allows for better optical sectioning in the axial direction at lower irradiation intensities. At the same time, the imaging depth of two-photon microscopy is much larger because of the reduced scattering of infrared light that is used to excite the sample. Therefore, two-photon microscopy is an ideal tool to study deep tissue and even living animals (Helmchen and Denk, 2002). Two main concepts that were capable to effectively improve the axial resolution have been introduced independently, i.e. 4Pi microscopy (Hell and Stelzer, 1992) and I 5 M microscopy (Gustafsson et al., 1999). Both concepts use a set of opposing microscope lenses that sharpen the PSF along the optical axis through interference of the counter-propagating wavefronts. At the same time, the efficiency of collecting light is increased by the presence of two objectives. 4Pi microscopy is a spot-scanning method that achieved a four- to seven-fold increase in axial resolution using different experimental configurations (Gugel et al., 2004). On the other hand, I 5 M microscopy is a wide-field method and achieves a similar axial resolution of 100 nm (Gustafsson et al., 1999). Both 4Pi and I 5 M microscopy have mainly been used to study the structure of subcellular organelles in 3D (Egner et al., 2004; Gustafsson, 1999; Medda et al., 2006; Nagorni and Hell, 1998), but have also been combined with other microscopic techniques that improve the lateral resolution. A concise comparison of both methods can be found in (Bewersdorf et al., 2006) Stimulated-emission depletion (STED) The idea to use the fundamental physical process of stimulated emission to locally turn off the fluorescence emission of fluorophores (Fig. 1A) and hereby to bypass the diffraction limit was first introduced in 1994 and termed stimulated-emission depletion (STED) microscopy (Hell and Wichmann, 1994). In the actual experiment, a sample is spot-scanned with two lasers that are overlaid and synchronized: the first laser excites a fluorophore, and a second laser ( STED-beam ) that is red-shifted and has an intensity distribution of doughnut shape generated with a phase mask (Fig. 1B) depletes the fluorophores form their excited state. Provided that the irradiation intensity of the STED-beam is high enough, the particular intensity profile of the STED-beam leads to efficient quenching of fluorophores that reside in the excited state everywhere except close to the position of zero intensity inside the doughnut profile. The higher the irradiation intensity of the STED-beam, the smaller is the central area with zero intensity, such that the resolution enhancement in STED microscopy scales with the intensity of the depletion beam (Hell, 2007). With respect to the saturation intensity, I S, which represents an experimentally determined parameter for a particular fluorophore under particular conditions (i.e. its microenvironment), the achievable resolution r can be expressed as r =, 2n sin 1 + I STED /I S where is the wavelength, n is the refractive index, is the collection angle of the objective and I STED is the irradiation intensity of the STED-beam. In its early days, STED microscopy required the use of both high irradiation intensities and short pulses for the STED-beam, to ensure that the first excited state of the fluorophores was depleted efficiently enough to observe an increase in resolution. Routinely, irradiation intensities of >1 GW/cm 2 were necessary to achieve a moderate resolution enhancement (Klar et al., 2000). As a consequence, STED microscopy was in practice limited to only a small number of fluorophores that were photostable enough to pass a sufficient number of excitation cycles needed to produce a STED image. This has changed in the recent past for a number of reasons: (i) a palette of fluorophores with increased photostability has been developed, (ii) concepts that increase the photostability of fluorophores have been introduced (Heilemann et al., 2006; Vogelsang et al., 2008), and (iii) the photophysical processes that increase photobleaching from the excited state are better understood (Tinnefeld et al., 2004; Widengren et al., 2007). A further improvement that reduced the experimental complexity of STED microscopy is the use of continuous-wave laser excitation for the depletion beam (Willig et al., 2007). The fundamental concept of STED microscopy has been extended experimentally in many ways. Certainly, it has been a milestone to demonstrate STED microscopy in living cells. This was simplified by he fact that STED microscopy is compatible with the more photostable variants of fluorescent proteins, such that a lateral resolution of 50 nm inside a living cell was achieved (Fig. 1C) (Hein et al., 2008). Similarly, STED has been applied to study the dynamics of dendritic spines in live neurons with a lateral resolution of 70 nm (Nagerl et al., 2008). However, although lower

3 M. Heilemann / Journal of Biotechnology 149 (2010) Fig. 2. (A) Structured-illumination microscopy (SIM) illuminates an unknown structural feature of a sample with a known periodical pattern. The spatial frequencies of the original structure appear as a beat pattern with lower spatial frequencies and can thus become resolvable. (B) Total internal reflection fluorescence (TIRF) (left) and reconstructed SIM image (middle) from the microtubular network of a mammalian cell (scale bar 1 m; reprinted from Kner et al., 2009; reproduction with kind permission from Nature Publishing Group). irradiation intensities can nowadays be used in STED microscopy, one still cannot exclude photodamage of a cell and inevitably alter biomolecular processes, such that careful controls are necessary. Two-colour STED has been demonstrated and used to study the distribution of proteins in the mitochondrial membrane (Donnert et al., 2007) as well as of synaptic proteins in neurons (Meyer et al., 2008) with a lateral resolution of nm. However, the two-colour STED setup is experimentally demanding, as it requires the alignment of four laser beams, i.e. one excitation and one stimulated-emission beam for each fluorophore. Furthermore, as a scanning technique, STED microscopy is limited by the speed of the scanning system itself. A considerably faster acquisition rate of 28 Hz was achieved using fast beam scanners and applied to study the movement of synaptic vesicles in living neurons (Westphal et al., 2008). STED microscopy has been extended by implementing the 4Pi principle (STED-4Pi microscopy) and achieved an axial resolution of 50 nm (Dyba et al., 2003). A three-dimensional STED microscope has recently been realized by implementing a combination of two phase masks (Wildanger et al., 2009). Some very impressive and recent developments combined STED microscopy with other techniques, profiting from the fact that the obtained STED-PSF (or effective PSF) is drastically reduced in size compared to e.g. the confocal PSF. It was demonstrated that the position of fluorescent nitrogen vacancies in diamond can be determined with even sub-nanometre precision by localizing the centre of the STED-PSF of single emitters (Rittweger et al., 2009). This has been possible as a single fluorophore is localized with a precision that is determined by the number of photons emitted by the fluorophore as well as proportional to the full-width-half-maximum (FWHM) of a PSF (Thompson et al., 2002). In another approach, the reduced size of a STED-PSF was combined advantageously with fluorescence correlation spectroscopy (STED-FCS) and allowed the direct observation of nanoscale dynamics of membrane lipids in a living cell (Eggeling et al., 2009). The concept of STED can be generalized to a more general principle for super-resolution microscopy using reversible saturable optical fluorescence transitions (RESOLFT) between two distinguishable molecular states (Hell, 2003; Hofmann et al., 2005). For example, other transitions than stimulated emission can be used to achieve super-resolution, such as photoswitchable fluorophores in combination with phase mask imaging (Dedecker et al., 2007) or triplet shelving (Bretschneider et al., 2007) Structured-illumination microscopy Structured-illumination microscopy (SIM) is a concept that combines wide-field imaging and illumination of a sample with a known pattern of excitation light, and achieves a two-fold resolution improvement (Gustafsson, 2000). Experimentally, a periodic illumination pattern of parallel stripes of excitation light is projected onto the sample with the help of a fine grating (Fig. 2A). A series of images is recorded where the light pattern is moved along the sample laterally and rotated into different angles. Structural features with spatial frequencies that are higher than the frequency of the illumination pattern are modulated by the latter resulting in so-called Moiré fringes, and can be extracted mathematically. As such, a reconstructed image with increased spatial resolution can be obtained (Fig. 2B). As a pure physical approach, SIM does not depend on any particular fluorophore properties, such as high photostability or particular transitions between orthogonal states, and can therefore be generally applied. For example, multi-colour SIM has been used to study the nuclear periphery of mammalian cells (Schermelleh et al., 2008). Furthermore, the acquisition time of SIM as a parallelized imaging method is substantially shorter than spot-scanning methods. As such, SIM is a well suited method to study dynamic processes in living cells (Hirvonen et al., 2009; Kner et al., 2009). The principle of SIM has been extended to three dimensions by two different experimental configurations. In a first approach, three coherent beams were used to record an interference pattern varying both laterally and axially, yielding a two-fold enhancement in resolution in all three dimensions after image reconstruction (three-dimensional SIM (3D-SIM)) (Gustafsson et al., 2008). In an alternative approach, SIM was combined with the I 5 M concept by additionally introducing two opposing lenses (termed I 5 S). This optical arrangement increased the axial resolution substantially,

4 246 M. Heilemann / Journal of Biotechnology 149 (2010) such that I 5 S yields 100 nm optical resolution in all three dimensions (Shao et al., 2008). An interesting extension of SIM that provides theoretically unlimited resolution has been reported recently. Employing a nonlinear structural illumination scheme, saturated SIM (SSIM) experimentally demonstrated a lateral resolution of 50 nm and is essentially unlimited (Gustafsson, 2005). The key feature that is exploited in SSIM is the nonlinear response of the fluorescence intensity with respect to the excitation intensity, a feature that has been proposed earlier for enhanced optical resolution (Heintzmann et al., 2002). However, a practical limit is set by high irradiation intensities that are necessary to observe such nonlinear effects, which on the one hand requires very photostable fluorophores and at the same time careful considerations in live-cell experiments Single-molecule based localization microscopy with subdiffraction resolution A large set of fluorescence microscopy methods that achieve subdiffraction resolution rely on the localization of single fluorophores, exploiting the fact that the position of a single emitter can be determined with a precision of a few nanometres. The localization precision depends on the number of photons that are collected and can reach a few nanometres (Thompson et al., 2002), and fluorescence imaging with one nanometre accuracy (FIONA) has been used to study individual steps of motor proteins along filaments (Yildiz et al., 2003; Yildiz and Selvin, 2005). First attempts that successfully resolved multiple fluorophores within a diffraction-limited area combined single-molecule localization with photobleaching. Nanometre-localized multiple single-molecule (NALMS) fluorescence microscopy isolated single emitters by sequential photobleaching and reconstructing the individual PSF of each fluorophore by subtracting subsequent emission profiles (Qu et al., 2004). Very recently, this concept has been combined with defocused imaging and extended to SPIDER microscopy (subtracting patterns in defocused imaging to enhance the resolution), providing both positional precision of a few nanometres as well as accurate information on the molecular orientation (Dedecker et al., 2009). Other approaches have used a reversible docking process of a fluorophore or a fluorophore-labelled probe to a target structure in combination with single-molecule localization, e.g. in points accumulation for imaging in nanoscale topography (PAINT) (Sharonov and Hochstrasser, 2006). Histogrammed PAINT (H-PAINT) images have revealed binding hotspots of enzymes on lipid bilayers (Rocha et al., 2009). Employing photoactivatable or photoswitchable fluorescent probes in combination with nanometre-precise localization of single emitters offers an alternative route to localization-based super-resolution microscopy. Today, a wealth of fluorescent probes that exhibit the necessary photoactivation or photoswitching properties are available (Fernandez-Suarez and Ting, 2008; Heilemann et al., 2009a), including conventional organic fluorophores (Bates et al., 2005; Heilemann et al., 2005, 2008; Vogelsang et al., 2009), caged fluorophores (Belov et al., 2009), photochromic compounds (Irie, 2000; Irie et al., 2002; Seefeldt et al., 2010) and a large variety of fluorescent proteins (Andresen et al., 2008; Schönle and Hell, 2007; Stiel et al., 2008). All photoswitchable fluorophores have in common that they exist in at least two different states that are distinguishable, e.g. in their fluorescence emission properties (Fig. 3A). Photoswitchable fluorophores populate a fluorescent on - and a non-fluorescent off -state (dark state), and the interconversion between these states is reversible and can be controlled by light or the chemical nanoenvironment. Photoactivatable fluorophores are initially found in a dark state and require activation to become fluorescent, typically achieved by irradiation with light. Photoconvertible fluorophores exist in two different fluorescent states and can be interconverted upon irradiation with light. All methods that use photoswitchable fluorophores for subdiffraction fluorescence microscopy employ a temporal confinement of the fluorescence signal. The starting point in these experiments is that all fluorophores in a sample are either in their off -state already (i.e. if photoactivatable fluorophores are used) or turned into this off -state (i.e. if photoswitchable fluorophores are used). In a next step, only a small subset of fluorophores is transferred into a fluorescent on -state. Here one has to make sure that less than one fluorophore is present in a diffraction-limited region, such that it can be detected as individual emitter. The fluorescence emission signal of such a single fluorophore is read out, and the position of the fluorophore is determined by approximating the PSF with a Gaussian function (Fig. 3B). The precision of this singlemolecule localization depends mainly on the number of emitted photons (Thompson et al., 2002) and can reach nanometre accuracy (Yildiz et al., 2003; Yildiz and Selvin, 2005). This procedure is repeated many times, and the ensemble of coordinates collected from localizing single fluorophores is used to generate ( reconstruct ) an artificial image that provides subdiffraction resolution information (Fig. 3C). Prominent examples of concepts that rely on photoswitchable fluorophores are photoactivated localization microscopy (PALM) (Betzig et al., 2006), fluorescence photoactivation localization microscopy (FPALM) (Hess et al., 2006), stochastic optical reconstruction microscopy (STORM) (Rust et al., 2006), PALM with independently running acquisition (PALMIRA) (Egner et al., 2007) and direct STORM (dstorm) (Heilemann et al., 2008). A faster variant of PALM has been reported using a stroboscobic illumination scheme (Flors et al., 2007). A related method is groundstate depletion followed by individual molecule return (GSDIM), which switches off fluorophores by populating the non-fluorescent triplet state out of which spontaneous return to the ground state occurs (Folling et al., 2008). Photoswitching-based methods have been extended in many ways. Multi-colour imaging with 20 nm lateral resolution has been demonstrated (Andresen et al., 2008; Bates et al., 2007; Bock et al., 2007; Schönle and Hell, 2007; Shroff et al., 2007; van de Linde et al., 2009a), setting the prerequisite to study biomolecular interactions at the molecular level. A variety of concepts that allow 3D-imaging have been developed, either by introducing astigmatism (Huang et al., 2008) or a helical shape (Pavani et al., 2009) into the beam path, or by recording two imaging planes simultaneously and approximating the PSF to a three-dimensional model function (Juette et al., 2008), or by using an interferometric arrangement (Shtengel et al., 2009). Photochromic rhodamine derivatives have been used in combination with two-photon activation, such that selective activation of fluorophores can be achieved by optical sectioning and three-dimensional images can be reconstructed (Folling et al., 2007). A challenge for all methods employing photoswitchable fluorophores is compatibility with live-cell imaging. Photoswitchable fluorescent proteins have the advantage that they can easily be implemented via genetic labelling, and that they can be operated as photoswitchable units inside a living cell (Hess et al., 2007; Shroff et al., 2008). Organic fluorophores require special labelling procedures, and photoswitching typically requires very special buffer conditions (Bates et al., 2005; Heilemann et al., 2005). However, a refined understanding of the impact of redox reactivity on photoswitching has paved the way to use organic fluorophores even in living cells (Heilemann et al., 2009b; van de Linde et al., 2008a). A major drawback for the use of localization-based super-resolution methods is that a large set of individual images have to be recorded, typically several thousands, which drastically reduces the temporal resolution.

5 M. Heilemann / Journal of Biotechnology 149 (2010) Fig. 3. (A) Molecular optical switches that can be photoactivated or reversibly photoconverted between two states ( on or fluorescent and off or non-fluorescent) are the key of single-molecule based super-resolution methods. (B) The PSF of a single emitter imaged on a wide-field microscope can be approximated to a Gaussian function, which allows determining the molecule s position with few nanometre precision. (C) By selectively observing only a small subset of all fluorophores in a sample, single emitters can be identified and localized. The localization coordinates determined over many imaging cycles are used to reconstruct an image with superior resolution. (D) Microtubular filaments of a COS-7 cell imaged in both TIRF mode (lower left of the image) and according to the dstorm concept. Beyond imaging the cellular cytoskeleton or other biomolecular structures, photoswitching microscopy with subdiffraction resolution has successfully been used to study protein organization. The self-organization of the chemotaxis network in E. coli was studied with PALM (Greenfield et al., 2009), and the organization of respiratory chain proteins organized in the inner mitochondrial membrane was investigated with dstorm (van de Linde et al., 2008b). Another target of substantial biological importance which is packed in such a dense fashion that it is unresolvable with conventional microscopy is DNA. Unsymmetric dimeric cyanine dyes, such as YOYO-1 and others are known to intercalate into double stranded DNA. It has been shown that under similar experimental conditions as used for classic carbocyanine switching (Bates et al., 2005; Heilemann et al., 2005), these intercalating fluorophores can be operated as photoswitches and used to image DNA with a resolution better than 40 nm (Flors et al., 2009) Other super-resolution methods There are a number of experimental techniques that have demonstrated fluorescence imaging below the diffraction limit but do not fit in the aforementioned categories. However, some of these recent approaches have huge potential to contribute to the field of super-resolution imaging, and hence shall be mentioned briefly. One recently introduced approach employs multi-photon absorption of semiconductor quantum dots and the subsequent generation of multiple excitonic states (Ben-Haim and Oron, 2008; Hennig et al., 2009). In contrast to two- or multi-photon microscopy, the generation of multi-excitonic states in quantum dots does not require the use of infrared light, such that a 2-fold resolution enhancement can be achieved experimentally. As a pure physical process, this approach operates under any experimental condition and in particular in living cells (Hennig et al., 2009). Another concept that has been recently introduced exploits the nonlinear response of the fluorescence signal with respect to the excitation intensity (similar to SSIM), generating a high-resolution image from higher order contributions and providing theoretically unlimited resolution (Fujita et al., 2007; Humpolickova et al., 2009; Yamanaka et al., 2008). Another technique, super-resolution optical fluctuation imaging (SOFI), exploits the temporal information of fluorescence fluctuations and mathematically extracts highresolution spatial information (Dertinger et al., 2009). The general concept of using temporal information of transient fluorescence signals to improve spatial resolution had been theoretically introduced earlier as dynamic saturation optical microscopy (DSOM) (Enderlein, 2005). Super-resolution techniques have very recently entered the research area of nanoscale materials sciences. A super-resolved map of reactivity of nanostructured catalyst particles has been generated with pro-fluorescent substrates (Roeffaers et al., 2009). Spectrally selective imaging of single fluorophores embedded in a matrix and sequentially recorded at low temperature using tunable laser frequencies has also demonstrated to achieve subdiffraction resolution (Naumov et al., 2009). 3. Limitations and requirements 3.1. Fluorophores All of the above presented methods have successfully demonstrated super-resolution. However, many methods require fluorophores with specific properties, such as high photostability or the existence of two molecular states that can be distinguished and addressed. Methods such as 4Pi and I 5 M do not have any special requirements for fluorophores, as they simply extend a microscope by introducing a second lens and increase the collection angle. Linear SIM can be realized with almost any fluorophore that exhibits a minimum of photostability, ensuring that the very few necessary images can be recorded without substantial loss of signal because of photobleaching. Photostability is more an issue for nonlinear approaches such as SSIM, where saturation of less photostable fluorophores may not be reached and thus limits the attainable resolution enhancement. STED microscopy is far more demanding: to achieve a maximum in resolution enhancement, irradiation intensities may reach dimensions of GW/cm 2 in the focal spot, and as a consequence, the number of suitable fluorophores is still limited. However, specific buffers that enhance photostability might be used in experiments with fixed cells (Vogelsang et al., 2008). Despite the fact that fluorescent proteins are not as stable as organic fluorophores, novel mutants have become available and can be used for STED microscopy with nearly comparable resolution enhancement (Hein et al., 2008). Approaches that rely on photoswitching of fluorophores and subsequent single-molecule localization require that the photophysical or photochemical properties of the fluorophores are compatible with the nano-environment of the experiment. For example, photoactivatable or photoswitchable fluorescent proteins

6 248 M. Heilemann / Journal of Biotechnology 149 (2010) work fine in living cells and can be introduced genetically (Hess et al., 2007; Shroff et al., 2008). Compared to organic fluorophores, the photon yield of fluorescent protein is lower, which affects the localization accuracy (Thompson et al., 2002) and hence the attainable resolution. On the other hand, organic fluorophores have widely been used to image cellular structures (Bates et al., 2007; Belov et al., 2009; Flors et al., 2009; Folling et al., 2008; Heilemann et al., 2008; Huang et al., 2008; Steinhauer et al., 2008; van de Linde et al., 2008a, 2009a; Vogelsang et al., 2009) or the organization of proteins in fixed cells (van de Linde et al., 2008b), and first attempts of live-cell imaging have been published targeting mrna in mammalian nuclei (Heilemann et al., 2009b). In summary, the choice of the best-suited fluorescent probe for photoswitching microscopy methods should be made with respect to the actual experimental conditions (Fernandez-Suarez and Ting, 2008; Heilemann et al., 2009a) Labelling strategies An important aspect for biomedical applications of fluorescence microscopy in general is the availability of labelling strategies and their suitability for different types of biomolecules. Fluorescent proteins can be co-expressed genetically to target proteins, and labelling techniques for mrna using proteins that target specifically introduced structural motifs have been developed (Shav-Tal et al., 2004). However, fluorescent proteins are relatively large, with a typical size of 27 kda for most monomeric derivatives of the green fluorescent protein (GFP) (Giepmans et al., 2006), and thus might interfere with the functionality of a target protein. In contrast, organic fluorophores are much smaller, yet require a chemical labelling procedure. One of the most common procedures to label cellular targets in fixed cells is immunofluorescence, and fluorophore-labelled antibodies are widely available. In particular, a large selection of commercially available antibodies already carries an organic fluorophore that can be operated as reversible photoswitch (Heilemann et al., 2009b). Alternatively, organic fluorophores can be conjugated to small peptides that specifically bind to cellular structures, for example the actin-binding peptides phalloidin and LifeAct (Riedl et al., 2008). Oligonucleotide sequences can be chemically modified with fluorophores at nearly any position and can probe for specific mrna. Furthermore, labelled oligonucleotides can be transfected into living cells and used for live-cell microscopy. Other labelling techniques that allow high-specific labelling of biomolecules in living cells include specific tag-proteins, e.g. SNAP-Tag (Keppler et al., 2003) or TMP-Tag (Calloway et al., 2007). Recently, a dual-colour approach for protein tagging employing both the SNAP- and the CLIP-Tag has been demonstrated, allowing for specific labelling with organic fluorophores in living cells (Gautier et al., 2008) Resolution versus labelling density A fundamental limit for super-resolution microscopy is the achievable labelling density with respect to the required resolution. According to the Nyquist Shannon criterion, it is necessary to label a structure with at least twice the spatial frequency as that of the desired resolution (Shannon, 1949). In other words, to obtain an optical resolution of 20 nm in one dimension, fluorophores have to be positioned at least every 10 nm. To super-resolve a two-dimensional structure with the same resolution, one needs a labelling density of about 10 4 fluorophores/ m 2, and about 10 6 fluorophores/ m 3 for a similarly resolved three-dimensional structure. To put these numbers into some context, one has only to remind typical high values of biomolecular concentrations that can occur in mammalian cells. As an example: a concentration of about 10 M corresponds to only about molecules/ m 3. It is intuitively easy to understand that this degree of labelling can hardly be achieved with the necessary specificity, and that these large numbers of fluorescent probes will certainly alter the function of e.g. a living cell. For approaches that temporally separate the fluorescence emission by employing photoswitchable fluorophores, the ratio of the on- and off-switching kinetics is an additional experimental parameter that has to be adjusted to the labelling density accordingly. If we consider again a labelling density of 10 4 fluorophores/ m 2 to achieve a resolution of 20 nm (two-dimensional case), this translates to about 600 fluorophores that have to be placed within the lateral projection of the PSF. However, localization-based super-resolution only works if not more than one molecule per diffraction-limited area is in its fluorescent state at a time, in order to ensure unambiguous single-molecule localization with high precision. As a consequence, this requires that the average time the fluorophore spends in its non-fluorescent (dark) state is at least 600 times longer than the average time it stays in its fluorescent state. This issue is further addressed in an article by van de Linde et al., also published in this issue (van de Linde et al., 2010). Finally, all the considerations on labelling density discussed in this section refer to an immobile structure. As soon as dynamics of structural elements are observed, the achievable spatial resolution will be reduced. A comprehensive discussion of this fact has been published recently (Shroff et al., 2008) Temporal resolution All the above introduced super-resolution techniques have successfully demonstrated their ability to resolve objects which are smaller than the diffraction limit. However, most published work so far only demonstrated imaging of cellular structures in fixed cells. Biological processes are dynamic, and it is therefore desirable to make these methods applicable to study structural changes and biomolecular interactions of different kinetics in living cells or tissue. As a direct imaging approach, the temporal resolution in STED microscopy is only limited by the scanning process. The use of fast resonant scanners make STED microscopy at video rate possible, which is fast enough to observe the dynamics of synaptic vesicles inside the axons of cultured neurons (Westphal et al., 2008). However, these fast imaging rates limit the observable area to a rather small region of 1.8 m 2.5 m. Furthermore, the fast scanning goes at the cost of the achievable resolution, which was determined to about 62 nm. In contrast to sequential spot-scanning in STED microscopy, SIM as a parallelized imaging approach allows fast imaging of larger areas of a size that is essentially determined by the imaging optics and the camera chip. SIM has been used to study tubulin and kinesin dynamics in living cells with a lateral resolution of 100 nm and a frame rate of up to 11 Hz (Kner et al., 2009). Here, the constraint is the resolution limit of about 120 nm that is inherent to linear SIM, and the number of images that have to be recorded at different experimental settings. Very different constraints have to be considered in singlemolecule based super-resolution approaches that rely on precise localization of photoactivatable or photoswitchable fluorophores. These approaches do not directly generate a subdiffraction image, but rather rely on the generation of an artificial image that is reconstructed from individual molecules localizations that were determined from thousands of individual imaging frames. In other words, the temporal resolution is, at first order, determined by the number of imaging frames that is required to obtain a satisfactorily reconstructed image with subdiffraction resolution. Typically, thousands of images are required, recorded in an experimental time of tens of seconds to minutes. Other constraints lie in the nature of the photoswitchable fluorescent probes themselves. On

7 M. Heilemann / Journal of Biotechnology 149 (2010) one hand, the fluorescent probes need to exhibit photoswitching under the particular experimental conditions, e.g. in living cells, and on the other hand, the kinetics of the photoswitching process determines the temporal resolution. Fluorescent proteins can readily be handled in living cells, but exhibit slow photoswitching kinetics. Live-cell imaging with subdiffraction resolution using fluorescent proteins has therefore only been demonstrated for relatively slow processes, such as the dynamics of adhesion complexes (Shroff et al., 2008). Organic fluorophores are brighter than fluorescent proteins and at the same time less prone to photobleaching, but their photoswitching requires specific chemical conditions (Bates et al., 2005; Heilemann et al., 2005; Heilemann et al., 2008, 2009b; Rust et al., 2006; Steinhauer et al., 2008; van de Linde et al., 2008a; Vogelsang et al., 2009). However, a more refined understanding of photophysical and photochemical processes that drive the transition of a fluorophore between a fluorescent and a dark state allows for identifying suitable candidates for live-cell imaging. On the basis of these considerations, specific mrna sequences have been targeted with fluorophore-labelled oligonucleotides in living cells (Heilemann et al., 2009b). An important advantage of organic fluorophores is that very fast photoswitching cycles can be achieved. Taking the example of carbocyanine fluorophores, e.g. the commercial derivatives Cy5 and Alexa Fluor 647, both the on- and the off-switching of the fluorophores is controlled by the irradiation intensity of a green and a red laser, respectively (Heilemann et al., 2008). Using these carbocyanine fluorophores, rapid photoswitching with an imaging frame rate up to 1 khz and a lateral resolution of 30 nm has been demonstrated using the dstorm principle (Wolter et al., 2009). As such, switching cycles of 1 ms can be realized with organic fluorophores that are about one hundred times faster than those reported for live-cell PALM (Shroff et al., 2008) or FPALM (Hess et al., 2007) experiments. Fast photoswitching of organic fluorophores has recently been used to study the dynamics of actin filaments moving across a myosin-coated surface (Endesfelder et al., 2010). However, the implementation of this concept into living cells remains to be demonstrated. An important aspect that has to be considered here is that high irradiation intensities can lead to photodamage, and hence alter biomolecular interactions inside a cell. A careful balance of irradiation intensity and switching kinetics is therefore necessary. 4. Outlook From the successful experimental realization of fluorescence microscopy below the diffraction limit and its first meaningful applications, we can anticipate an important contribution to various research areas in the future. However, it is important to keep in mind how these novel techniques can contribute to address a certain question, and which technique is best suited. In other words, super-resolution techniques will certainly be used in e.g. biomedical research if they (i) are simple to use for non-experts, (ii) do not require any expensive equipment (or even more ideally can be implemented on existing microscopic systems), (iii) are compatible with a simple and straightforward strategy for sample labelling with fluorescent probes or other labels, and (iv) allow for easy analysis and correct data interpretation. Beyond these issues, an important question to consider is where new experimental concepts should aim at. Certainly, it would be desirable if superresolution could operate in multi-colour mode, three-dimensional, and in living cells with a temporal resolution that allows for following cellular processes at fast time scales. 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