2.1.1 Multicolor STED nanoscopy

In most cellular applications it is desirable not only to resolve a single structure at a time, but to highlight the relative sites and proximities of different molecules. In fluorescence microscopy this is usually realized by tagging the different molecules with different fluorescent labels, whose emission is then distinguished by its specific color, lifetime or potentially other fluorescence parameters. In a preferred implementation, the wavelength of the emitted light is chosen as a delimiter, and the fluorescence emission of the different labels is excited with lasers of different wavelength and detected on separate detectors monitoring different wavelength ranges. This principle is transferable to STED microscopy, however with the requirement of supplying a multitude of additional STED lasers, strictly speaking one for each label used. While this approach has rendered two-color STED imaging (Donnert et al. Reference Donnert, Keller, Wurm, Rizzoli, Westphal, Schoenle, Jahn, Jakobs, Eggeling and Hell2007b) possible, and revealed the co-localization of different proteins and structures on the nanometer-scale (Meyer et al. Reference Meyer, Wildanger, Medda, Punge, Rizzoli, Donnert and Hell2008), it entails a rather complex setup with the correct alignment of four lasers, two excitation and two STED beams. Furthermore, the simultaneous recording of the two colors proves itself somewhat difficult, since the more blue-shifted STED laser usually leads to a strong excitation and thus massive photobleaching of the more red-emitting dye. Therefore, initial two-color STED images were recorded sequentially. This limitation has been solved by straightforward optimizations of the choices of labels and wavelengths: (1) the combination of two labels with overlapping emission spectra and with a long (Stokes) shift between the excitation and emission spectrum of one of the labels allows the recording of two-color STED images with two excitation lasers but only one STED laser (serving both labels) (Clausen et al. Reference Clausen, Galiani, Bernardino De La Serna, Fritzsche, Chojnacki, Gehmlich, Lagerholm and Eggeling2013; Dean et al. Reference Dean, Liu, Staudt, Stahlberg, Vingill, Buckers, Kamin, Engelhardt, Jackson, Hell and Chapman2012; Friedemann et al. Reference Friedemann, Turshatov, Landfester and Crespy2011; Pellett et al. Reference Pellett, Sun, Gould, Rothman, Xu, Correa and Bewersdorf2011; Schmidt et al. Reference Schmidt, Wurm, Jakobs, Engelhardt, Egner and Hell2008). (Quasi-) simultaneous recording of both colors is possible in either a line-by-line or a pulse-interleaved excitation scheme, in both cases rapidly switching between the two excitation lasers. (2) Nanoscopic co-localization studies of various different proteins have been enabled by a carefully optimized choice of two conventional Stokes-shifted fluorophores, whose spectra differ by only about 60 nm, the use of two pairs of excitation/STED lasers timed in a pulsed interleaved excitation scheme, and with an elimination of the detection cross-talk by applying linear unmixing algorithms (Fig. 3c, d) (Blom et al. Reference Blom, Rönnlund, Scott, Spicarova, Rantanen, Widengren, Aperia and Brismar2012; Dean et al. Reference Dean, Liu, Staudt, Stahlberg, Vingill, Buckers, Kamin, Engelhardt, Jackson, Hell and Chapman2012; Neumann et al. Reference Neumann, Bückers, Kastrup, Hell and Jakobs2010; Opazo et al. Reference Opazo, Punge, Bückers, Hoopmann, Kastrup, Hell and Rizzoli2010; Reisinger et al. Reference Reisinger, Bresee, Neef, Nair, Reuter, Bulankina, Nouvian, Koch, Bückers, Kastrup, Roux, Petit, Hell, Brose, Rhee, Kügler, Brigande and Moser2011; Osseforth et al. Reference Osseforth, Moffitt, Schermelleh and Michaelis2013). In this scheme, the number of distinguishable labels could be increased to four by separating the emission based on emission wavelength and lifetime (Bückers et al. Reference Bückers, Wildanger, Vicidomini, Kastrup and Hell2011). (3) Recent two-color STED imaging has been realized using two excitation and a single STED laser only, e.g. 594 and 640nm excitation in combination with a 775nm STED laser (Göttfert et al. Reference Göttfert, Wurm, Mueller, Berning, Cordes, Honigmann and Hell2013). (4) Instituting rigorous linear unmixing with a single excitation and a single STED laser were sufficient to record two-color STED images of labels such as the yellow fluorescent protein (YFP) and green fluorescent protein (GFP), whose spectra are separated by about 20 nm (Tonnesen et al. Reference Tonnesen, Nadrigny, Willig, Wedlich-Soldner and Nägerl2011). Similarly, combining two reversibly photoswitchable fluorescent proteins (RSFPs) with opposite activation properties, two-color STED images were recorded with just one pair of excitation and STED lasers, without the necessity of applying linear unmixing, but with the addition of blue-light photoswitching (Willig et al. Reference Willig, Stiel, Brakemann, Jakobs and Hell2011). With the ongoing development of fluorescent labels and lasers, the number of fluorophores and matching excitation/STED laser pairs useful for multi-color STED imaging schemes will increase further.

2.1.2 3D STED nanoscopy

So far we have presented images recorded with STED nanoscopes which confine fluorescence emission and thus improve spatial resolution along the lateral direction only (using, e.g. the previously mentioned doughnut-like intensity distribution). The combination of this modality with an evanescent wave illumination (TIRF) scheme for excitation (Gould et al. Reference Gould, Myers and Bewersdorf2011; Leutenegger et al. Reference Leutenegger, Ringemann, Lasser, Hell and Eggeling2012) is sufficient for selectively imaging membranes of flat cells with penetration depths of <100 nm, and the combination with two-photon excitation realized imaging in ~800 nm thick sections with deep penetration depths (Bethge et al. Reference Bethge, Chereau, Avignone, Marsicano and Nägerl2013; Bianchini & Diaspro, Reference Bianchini and Diaspro2012; Ding et al. Reference Ding, Takasaki and Sabatini2009; Li et al. Reference Li, Wu and Chou2009b; Moneron & Hell, Reference Moneron and Hell2009; Takasaki et al. Reference Takasaki, Ding and Sabatini2013). However, the imaging of intra-cellular structures such as the Golgi apparatus or mitochondria often requires both a deeper penetration depth and an improvement of the spatial resolution along the axial z-direction as well. Four approaches have been applied so far: (1) The use of a phase plate that, upon interference, inhibits fluorescence along z and preferentially its combination with the doughnut-shaping plate creates the desired fluorescence restriction along all spatial directions (Fig. 3e) (Harke et al. Reference Harke, Keller, Ullal, Westphal, Schoenle and Hell2008a, Reference Harke, Ullal, Keller and Hellb; Klar et al. Reference Klar, Jakobs, Dyba, Egner and Hell2000; Wildanger et al. Reference Wildanger, Medda, Kastrup and Hell2009b; Osseforth et al. Reference Osseforth, Moffitt, Schermelleh and Michaelis2013). (2) The use of the doughnut-shaped STED focus in conjunction with illumination by two opposing objectives (as for 4Pi) has realized spatial resolution along the axial direction of down to 33 nm (Dyba & Hell, Reference Dyba and Hell2002; Dyba et al. Reference Dyba, Jakobs and Hell2003), or was used to create an effective isotropic observation volume with a spatial resolution of down to 30 nm along all spatial directions (Hell et al. Reference Hell, Schmidt and Egner2009; Schmidt et al. Reference Schmidt, Wurm, Jakobs, Engelhardt, Egner and Hell2008). Such an isoSTED microscope has given new insights into mitochrondrial structure, for example for the first time resolving the mitochondrial cristae with an optical microscope (Fig. 3f) (Schmidt et al. Reference Schmidt, Wurm, Punge, Egner, Jakobs and Hell2009). (3) Recently, a combination of STED with single plane illumination microscopy (SPIM) realized an almost two-fold improvement in axial resolution along with an 1·3-fold improvement along the lateral directions compared to conventional SPIM. Applying this illumination scheme, STED-SPIM should allow penetration depths of >100 μm and thus realize imaging inside zebra fish (Friedrich et al. Reference Friedrich, Gan, Ermolayev and Harms2011). (4) Adaptive optics allows the correction of aberrations occurring predominantly when imaging in 3D deep inside tissue (Gould et al. Reference Gould, Burke, Bewersdorf and Booth2012, Reference Gould, Kromann, Burke, Booth and Bewersdorf2013).

2.1.3 Photo-physical and -chemical considerations in STED nanoscopy

The signal strength and observation time of fluorescence experiments is limited by the population of metastable dark states and the photobleaching of the fluorophore label (e.g. Eggeling et al. Reference Eggeling, Widengren, Rigler and Seidel1998, Reference Eggeling, Widengren, Rigler, Seidel, Rettig, Strehmel, Schrader and Seifert1999; Tsien et al. Reference Tsien, Ernst, Waggoner and Pawley2006). Fluorescence emission follows the excitation of the fluorophore from its ground to its excited electronic state by, for example, laser light. The frequency and number of emitted photons depend on how fast and how many times one can cycle the fluorophore between the ground and excited state, respectively. In the excited state, the fluorophore becomes more fragile as reaction pathways to non-fluorescent species (such as ionization and/or breaking of double bonds) are opened up which can result in photobleaching, an irreversible loss of the ability to fluoresce. Furthermore, in the excited state the fluorophore may cross to metastable dark states of microsecond- to second-long lifetimes (such as the triplet state or radical states for usual organic dyes or fluorescent proteins), where the fluorophore is disengaged from the fluorescence cycling process, thus reducing the number of emitted photons. These long-lived dark states are much more prone to photobleaching than the first excited singlet state. For example, the triplet state efficiently interacts with molecular oxygen, generating highly reactive singlet oxygen (for a review see e.g. Eggeling et al. Reference Eggeling, Widengren, Rigler, Seidel, Rettig, Strehmel, Schrader and Seifert1999). On the other hand, high laser irradiances as for example used in scanning confocal microscopy open up new efficient photobleaching pathways by further exciting the already excited fluorophore to higher electronic states (e.g. Eggeling et al. Reference Eggeling, Widengren, Rigler and Seidel1998, Reference Eggeling, Volkmer and Seidel2005; Widengren & Rigler, Reference Widengren and Rigler1996). In aqueous environments, these excited species couple quite efficiently with ionic states and are thus highly reactive (e.g. Anbar & Hart, Reference Anbar and Hart1964; Reuther et al. Reference Reuther, Laubereau and Nikogosyan1996), usually characterized by more than ten-fold higher photobleaching probabilities than found from the first excited electronic states (e.g. Eggeling et al. Reference Eggeling, Widengren, Rigler and Seidel1998, Reference Eggeling, Volkmer and Seidel2005). Such non-linear photobleaching mechanisms are exceedingly efficient from the dark states due to their relatively long lifetime.

The accommodation of a large number of continuous excitation/de-excitation cycles and thus the maximization of the fluorescence signal has been approached by choosing experimental conditions which minimize the reactivity of the excited states as well as the populations of dark and higher excited states. This can either be done by choosing an appropriate dye with low photobleaching probabilities, low absorption cross-sections of the excited states and low dark state populations (e.g. Dittrich & Schwille, Reference Dittrich and Schwille2001; Eggeling et al. Reference Eggeling, Widengren, Rigler, Seidel, Rettig, Strehmel, Schrader and Seifert1999, Reference Eggeling, Widengren, Brand, Schaffer, Felekyan and Seidel2006; Tsien et al. Reference Tsien, Ernst, Waggoner and Pawley2006), or by the addition of chemicals (such as radical quencher or mercapto-compounds) that result in a reduction of photobleaching probabilities (especially of the higher excited electronic states) or in a quenching of the dark states (e.g. Dave et al. Reference Dave, Terry, Munro and Blanchard2009; Dittrich & Schwille, Reference Dittrich and Schwille2001; Eggeling et al. Reference Eggeling, Widengren, Rigler, Seidel, Rettig, Strehmel, Schrader and Seifert1999, Reference Eggeling, Widengren, Brand, Schaffer, Felekyan and Seidel2006; Rasnik et al. Reference Rasnik, Mckinney and Ha2006; Vogelsang et al. Reference Vogelsang, Kasper, Steinhauer, Person, Heilemann, Sauer and Tinnefeld2008; Widengren et al. Reference Widengren, Chmyrov, Eggeling, Lofdahl and Seidel2007). Unfortunately, an appropriate fluorophore may not always exist, or the addition of (sometimes toxic) chemicals is often invasive and not live-cell compatible. The minimization of dark state populations and non-linear photobleaching processes therefrom was approached with a method termed dark- or triplet-state relaxation (D- or T-Rex) microscopy (Donnert et al. Reference Donnert, Keller, Medda, Andrei, Rizzoli, Lurmann, Jahn, Eggeling and Hell2006, Reference Donnert, Eggeling and Hell2007a). The D- or T-Rex principle makes use of low-repetition pulsed laser light (Donnert et al. Reference Donnert, Keller, Medda, Andrei, Rizzoli, Lurmann, Jahn, Eggeling and Hell2006, Reference Donnert, Eggeling and Hell2007a) or of ultra-fast scanners (Donnert et al. Reference Donnert, Eggeling and Hell2009) to allow for an efficient depopulation of any dark state population in-between excitation events, thereby minimizing dark-state build-up and non-linear photobleaching from these states. The use of fast beam-scanning microscopes, where D-/T-Rex is effectively implemented, is especially suitable for live-cell fluorescence imaging experiments (Borlinghaus, Reference Borlinghaus2006; Conchello & Lichtman, Reference Conchello and Lichtman2005; Tsien et al. Reference Tsien, Ernst, Waggoner and Pawley2006; Vukojevic et al. Reference Vukojevic, Heidkamp, Minga, Johansson, Tereniusa and Rigler2008; Webb et al. Reference Webb, Wells, Sandison, Strickler, Herman and Jacobson1990).

In STED, the fluorophores are forced to undergo numerous transitions between their ground and excited state from which photobleaching or a dark-state transition may occur (Fig. 4a) (Donnert et al. Reference Donnert, Keller, Medda, Andrei, Rizzoli, Lurmann, Jahn, Eggeling and Hell2006; Dyba & Hell, Reference Dyba and Hell2003). In addition, increasing the irradiance of the STED laser to increase the gain in spatial resolution requires an adaptation of the scanning imaging process, with smaller pixel sizes and thus a larger number of scanning steps, unfortunately increasing the number of cycles that a molecule has to undergo during the recording of an image. Therefore, a key to sub-diffraction STED (or, similarly, GSD and RESOLFT) imaging is to ensure that the marker is able to switch repeatedly between its ON and OFF states in the presence of both the switch-on and switch-off light.

Fig. 4.

STED nanoscopy: photophysics and bleaching. (a) Photobleaching pathways: excitation (Exc) elevates a fluorophore from its ground S ₀ to its first excited electronic state S ₁, from where it either returns to S ₀ by spontaneous fluorescence emission (Flu) or by STED, or it traverses with probability Φ _D to a dark state whose lifetime τ _D (time before return to S ₀) is much longer than that of S ₁. Photobleaching may occur from S ₁ and the dark state, and is most pronounced from higher excited electronic states after further absorption of excitation or STED light (higher-order photobleaching, dashed lines). Horizontal lines: electronic states (thick) and vibrational sub-states (thin). Curved lines: vibrational transitions. (b) Suppression of photobleaching rate by STED: stimulated emission shortens the lifetime of S ₁ and thus the probability of photobleaching as exemplified by the percentage of signal bleached after scanning a layer of the organic dye KK114 with and without the addition of STED light (excitation at 488 nm with 9 kW/cm² and STED at 760 nm with 0·8 GW/cm², repetition rate 76 MHz, scanning dwell time 10 ms). (c) Higher-order photobleaching from long-lived dark states, as exemplified by subsequent scanning STED images of 40-nm sized fluorescent beads, showing a significant loss of signal due to irreversible photobleaching (upper images). D-Rex illumination, i.e. increasing the time ΔT between subsequent pairs of excitation and STED pulses (i.e. decreasing the repetition rate 1/ΔT) above the dark states lifetime τ _D allows the dark states to relax before incidence of the next pulses, avoiding higher-order photobleaching (lower images). D-Rex illumination by fast beam scanning (lower panel): for a fluorophore the incidence of only a few excitation-STED pulse pairs of high repetition rate (e.g. 80 MHz) is followed by a long resting period ΔT until the scanned beam pair hits the same spot again. (d) Higher-order photobleaching from the first excited electronic state S ₁, as exemplified for the organic dyes pDI and pTDI and for eGFP. Absorption spectra of S ₀ (blue) and S ₁ (red) and fluorescence emission spectra (green) show that excitation to higher excited states from S ₁ by the STED light may be significant for pDI and for eGFP at >595 nm, but not for pTDI, and for eGFP at <595 nm, resulting in far less photobleaching for pTDI, exemplified in CW-STED images of single pDI and pTDI molecules (confocal images were taken prior to STED recordings, pTDI: STED (white circle) and confocal recording (outer region), scale bar 500 nm, adapted from (Hotta et al. Reference Hotta, Fron, Dedecker, Janssen, Li, Muellen, Harke, Bückers, Hell and Hofkens2010), and for eGFP the STED wavelength between 556 and 592 nm, exemplified in STED recordings of live Vero cells expressing eGFP in the endoplasmic reticulum (middle panel, lower left corner: confocal image, scale bar: 1 μm, adapted from (Rankin et al. Reference Rankin, Moneron, Wurm, Nelson, Walter, Schwarzer, Schroeder, Colon-Ramos and Hell2011)). (e) STED at nitrogen temperatures. (Left) The cross-section of stimulated emission is highest at wavelengths close to the fluorescence emission maximum as exemplified for the organic dye Atto532 (columns: cross-sections of stimulated emission at selected wavelengths, black line: fluorescence emission spectrum scaled to the cross-section value at 568 nm). (Middle) As a consequence, less intensity of STED light is required for 568 nm compared to 605 nm to increase the spatial resolution in STED images of 80 nm-large Atto532-labeled beads, as determined for different intensities of the STED laser. (Right) STED imaging at 568 nm is however only possible at nitrogen temperatures, since the relative fluorescence emission evoked by the STED light increases for wavelengths closer to the emission maximum, but this anti-Stokes fluorescence excitation is efficiently suppressed at nitrogen temperatures <100 K, as exemplified for the dye Atto532 in polyvinyl-alcohol (PVA) (adapted from Giske Reference Giske2007).

Stimulated emission reduces the lifetime of the excited state and therefore in principle increases the photostability of the fluorophore in comparison to action of the excitation laser only (Fig. 4b). However, unwanted excitation to the higher excited states usually antagonizes this process (Fig. 4a). As a consequence, it was the realization of D- or T-Rex (see above) STED imaging that demonstrated for the first time down to 20 nm macro-molecular spatial resolution in cells (Donnert et al. Reference Donnert, Keller, Medda, Andrei, Rizzoli, Lurmann, Jahn, Eggeling and Hell2006), and it is the implementation of fast beam-scanning that nowadays allows routine live-cell STED nanoscopy (compare Section 2.1.5) (Fig. 4c) (Moneron et al. Reference Moneron, Medda, Hein, Giske, Westphal and Hell2010). Additionally, STED nanoscopy becomes much more feasible by choosing fluorophores with a low absorption cross-section of the first excited state at the wavelength of the STED laser (Hotta et al. Reference Hotta, Fron, Dedecker, Janssen, Li, Muellen, Harke, Bückers, Hell and Hofkens2010), or by appropriately adapting the latter to minimize non-linear photobleaching (Fig. 4d) (Rankin et al. Reference Rankin, Moneron, Wurm, Nelson, Walter, Schwarzer, Schroeder, Colon-Ramos and Hell2011). This has recently allowed STED imaging in an intact living organism, namely Caenorhabditis elegans expressing eGFP (Rankin et al. Reference Rankin, Moneron, Wurm, Nelson, Walter, Schwarzer, Schroeder, Colon-Ramos and Hell2011). STED at nitrogen temperatures attempted to minimize photobleaching and to maximize the efficiency of stimulated emission (Fig. 4e) (Giske, Reference Giske2007), unfortunately making live-cell studies less feasible. The choice of other promising emitters such as the very photostable nitrogen vacancy (NV) centers in diamond has recently allowed STED imaging with a spatial resolution of down to 5 nm (see Section 2.3) (Han et al. Reference Han, Wildanger, Rittweger, Meijer, Pezzagna, Hell and Eggeling2012; Rittweger et al. Reference Rittweger, Han, Irvine, Eggeling and Hell2009a). Similarly, special quantum dots (QDs) could be used in a STED-like fashion (Irvine et al. Reference Irvine, Staudt, Rittweger, Engelhardt and Hell2008). Unfortunately, the use of most conventional QDs for STED is so far impeded by their low Stokes shift and narrow band of emission. Nevertheless, a large range of appropriate fluorophores for STED nanoscopy is now known. Still, the development of new bright and photostable dyes, often specialized for STED applications, has and continuously will enhance the applicability and flexibility of experiments using this super-resolution technique (Boyarskiy et al. Reference Boyarskiy, Belov, Medda, Hein, Bossi and Hell2008; Kolmakov et al. Reference Kolmakov, Belov, Bierwagen, Ringemann, Mueller, Eggeling and Hell2010a, Reference Kolmakov, Belov, Wurm, Harke, Leutenegger, Eggeling and Hell2010b, Reference Kolmakov, Wurm, Hennig, Rapp, Jakobs, Belov and Hell2012; Mitronova et al. Reference Mitronova, Belov, Bossi, Wurm, Meyer, Medda, Moneron, Bretschneider, Eggeling, Jakobs and Hell2010; Wurm et al. Reference Wurm, Kolmakov, Göttfert, Ta, Bossi, Schill, Berning, Jakobs, Donnert, Belov and Hell2012).

The number of cycles that a molecule has to undergo is severely increased when recording 3D images, where one plane after the other is scanned at different optical (z) axis positions. Here, an intelligent light exposure scheme was put into practice. Related to controlled light exposure microscopy – introduced for decreasing photobleaching and increasing the number of detected fluorescence photons in scanning microscopy (Hoebe et al. Reference Hoebe, Van Oven, Gadella, Dhonukshe, Van Noorden and Manders2007) – the reduction of state transition cycles (RESCue)-STED scheme uses an online feedback algorithm that rapidly switches off the excitation and STED light during a scanning step when no or only low signal is detected (Staudt et al. Reference Staudt, Engler, Rittweger, Harke, Engelhardt and Hell2011). Thereby, the cycling of nearby fluorophores is minimized and the total number of on–off cycles significantly reduced. For 3D scanning imaging, this reduces the probability of dark state transitions and photobleaching in the adjacent axial planes, consequently increasing the number of planes that can be successively recorded (Fig. 3f).

2.1.4 Cluster analysis in STED nanoscopy

Clustering of molecules is key in a lot of cellular processes. Unfortunately, these clusters can often not be characterized accurately, especially in the living cell, mainly because their sizes are usually below the diffraction barrier and thus cannot be determined with conventional light microscopy. The use of a STED nanoscope is an obvious way to overcome this limitation. STED was applied to image various molecular assemblies in different cells, tissue or membranes. To name a few, STED imaging was used to study the clustering to ~70-nm large spots of a synaptic vesicle protein after exocytosis (Fig. 3a) (Willig et al. Reference Willig, Rizzoli, Westphal, Jahn and Hell2006c), the anatomy of supra-molecular membrane protein clusters of approximately 50–60 nm in diameter (Sieber et al. Reference Sieber, Willig, Kutzner, Gerding-Reimers, Harke, Donnert, Rammner, Eggeling, Hell, Grubmuller and Lang2007), the dynamics of approximately 80-nm large synaptic vesicle movements (Westphal et al. Reference Westphal, Rizzoli, Lauterbach, Kamin, Jahn and Hell2008), or the formation of domains in phase separated membrane bilayers of down to below 40 nm in diameter (Honigmann et al. Reference Honigmann, Mueller, Hell and Eggeling2013b, Reference Lau, Lee, Sahl, Stearns and Moerner2012). These examples have in common that previously blurred structures are now revealed as a multitude of single isolated clusters. Besides the cluster size, STED imaging also gives access to further parameters such as cluster brightness (and thus an estimate of the number of molecules per cluster) and the cluster density. An advantage of the STED approach is that it gives a direct image, i.e. cluster parameters such as cluster size can directly be inferred from the recorded image without having to introduce considerable image processing. In the following we will give an example of such a cluster analysis using STED nanoscopy.

We had previously shown that syntaxin 1A, a protein of the soluable N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) family of receptors which are involved in exocytosis, forms clusters of about 50–60 nm in diameter in the plasma membrane of PC12 cells (Sieber et al. Reference Sieber, Willig, Heintzmann, Hell and Lang2006, Reference Sieber, Willig, Kutzner, Gerding-Reimers, Harke, Donnert, Rammner, Eggeling, Hell, Grubmuller and Lang2007). An important question remains how these clusters change with increasing expression levels of syntaxin. For this, we created membrane sheets of PC12 cells, after fixation added labelled antibodies against syntaxin (HPC-1 monoclonal antibody and a secondary antibody decorated with the green-emitting dye Atto 532) and imaged their distribution using a custom-built STED nanoscope, as described previously (Sieber et al. Reference Sieber, Willig, Heintzmann, Hell and Lang2006). To test the performance of our STED cluster analysis, we artificially created syntaxin clusters with a broad range of sizes and densities, as follows: (1) direct fixed: the cells were fixed directly after the membrane sheet preparation (i.e. after removing the upper part of the cell). (2) Patched 1: the sheets were incubated for 1 h at 37 °C prior to fixation (in 200 μl/CS sonication buffer with 1% BSA). (3) Patched 2: preparation as for patched 1 but with a polyclonal antibody during the 1 h incubation. For cases (2) and (3) we expect an enhanced (artificial) clustering of syntaxin, which should result in larger and brighter but less dense protein assemblies. Figure 5a shows representative STED images together with conventional confocal counterparts (circles) of these three different preparations. Clearly, the clusters can be much more accurately resolved with STED, and their density behaves as expected.

Fig. 5.

STED nanoscopy: Cluster analysis. (a) ICS, FIDA and SCA analysis of syntaxin clusters recorded by STED. Upper panels: STED and confocal (circled areas) images of membrane sheets of PC12 cells immunolabeled for syntaxin for three different preparations (directly fixed (left) and patched 1 (middle) and 2 (right)). For the latter two, clustering is reinforced and cluster density decreased, dashed rectangles: analysed area, scale bar: 500 nm. Lower panels: values of cluster density (left), brightness (middle) and size (right) determined for different cells of the different preparations (dots: direct fixed, triangles: patched 1, diamonds: patched 2); left panel: 3D data (black) and projections to different value pairs (blue, green, red) of cluster density by ICS, FIDA and SCA, showing agreeing results by the different analysis techniques. (b) Syntaxin cluster morphology is independent of its expression level. Left panel: STED and confocal (left part) image of three representative membrane sheets generated from PC12 cells expressing different levels of immunolabeled myc syntaxin 1A (cell 1 with a low (probably endogenous) level of syntaxin, and cells 2 and 3 with different overexpression levels, adapted from (Sieber et al. Reference Sieber, Willig, Heintzmann, Hell and Lang2006)). Plot of cluster brightness (upper middle), size (lower middle) and density (right) against the expression level (~average pixel brightness) determined by ICS (black) and FIDA (red) from the STED images of different cells, revealing no variation of brightness and size with expression level, and a linear increase of density with expression level.

We applied three different statistical approaches to gain accurate values of cluster size, brightness and density. (1) Single cluster analysis (SCA): using an image analysis algorithm for identifying single isolated spots, clusters were separated and their brightness, diameter and the number of clusters per area determined (Willig et al. Reference Willig, Rizzoli, Westphal, Jahn and Hell2006c). (2) Image correlation analysis (ICS) (Petersen et al. Reference Petersen, Hoddelius, Wiseman, Seger and Magnusson1986): the calculation and analysis of the autocorrelation function of the number of counts in the image pixels over space allows the determination of cluster size and density. (3) Fluorescence intensity distribution analysis (FIDA) or photon-counting-histogram (PCH) image analysis (Chen et al. Reference Chen, Muller, So and Gratton1999; Kask et al. Reference Kask, Palo, Ullmann and Gall1999): The assembly and analysis of the histogram of the number of counts in the image pixels establishes relative values of cluster brightness and density (compare Digman et al. Reference Digman, Dalal, Horwitz and Gratton2008; Sergeev et al. Reference Sergeev, Costantino and Wiseman2006). Applied to the STED images of the three different syntaxin preparations (direct fixed, patched 1 and patched 2), the three different analysis approaches (SCA, ICS and FIDA) congruently confirmed larger cluster brightness and sizes and lower densities for the patched compared to the direct fixed preparations, as expected (Fig. 5a). The advantage of ICS and FIDA over SCA is that these two analysis methods may still be applied to images with higher cluster densities, where SCA starts to fail at clearly separating single isolated spots, similar to advantages of fluorescence correlation spectroscopy (FCS) and FIDA over single fluorescence burst analysis in the study of diffusing molecules (e.g. Eggeling et al. Reference Eggeling, Schaffer, Volkmer, Seidel, Brand, Jaeger and Gall2001b).

With the cluster analysis of the STED images tested, we now studied the formation of syntaxin clusters under different levels of syntaxin expression. Figure 5b depicts a representative STED image and its confocal counterpart of the syntaxin clusters of the membrane sheets of three PC12 cells, each expressing different levels of syntaxin. The preparation and labelling of these sheets was performed as for the direct fixed case. Again, clusters can be much better resolved with STED, and their sizes, brightness and densities accurately determined from subsets of the STED images with ICS and FIDA, even for the highly expressing cell. We used the average number of counts per pixel as a relative measure of the expression level in each image subset. As a result we could recognize that the expression level only determined the density but not the size and brightness (i.e. number of molecules) of the clusters. This is an important insight into the characteristics of the protein clusters (Sieber et al. Reference Sieber, Willig, Heintzmann, Hell and Lang2006, Reference Sieber, Willig, Kutzner, Gerding-Reimers, Harke, Donnert, Rammner, Eggeling, Hell, Grubmuller and Lang2007): increasing the concentration of the protein more than ten-fold seems to increase only the number of clusters formed, but not their composition.

2.1.5 Live-cell and in-vivo STED nanoscopy

Due to the aforementioned potential phototoxic effects of the image recording schemes, STED imaging was for long believed to be non-compatible with the study of living cells. However, first images of YFP-labeled endoplasmatic reticulum (ER) and microtubular networks in PtK2 cells proved the opposite (Hein et al. Reference Hein, Willig and Hell2008). Especially the use of fast scanning units nowadays allows a straightforward use of STED nanoscopy for the study of the living cell using genetically encoded markers such as fluorescent proteins (Bethge et al. Reference Bethge, Chereau, Avignone, Marsicano and Nägerl2013; Eggeling et al. Reference Eggeling, Ringemann, Medda, Schwarzmann, Sandhoff, Polyakova, Belov, Hein, Von Middendorff, Schönle and Hell2009; Hein et al. Reference Hein, Willig and Hell2008; Li et al. Reference Li, Wu and Chou2009b; Moneron & Hell, Reference Moneron and Hell2009; Morozova et al. Reference Morozova, Piatkevich, Gould, Zhang, Bewersdorf and Verkhusha2010; Nägerl et al. Reference Nägerl, Willig, Hein, Hell and Bonhoeffer2008; Rankin et al. Reference Rankin, Moneron, Wurm, Nelson, Walter, Schwarzer, Schroeder, Colon-Ramos and Hell2011; Tonnesen et al. Reference Tonnesen, Nadrigny, Willig, Wedlich-Soldner and Nägerl2011; Urban et al. Reference Urban, Willig, Hell and Nägerl2011; Willig et al. Reference Willig, Kellner, Medda, Hein, Jakobs and Hell2006b), or tagging proteins such as SNAP-, HALO- or CLIP-tags for proteins (Eggeling et al. Reference Eggeling, Ringemann, Medda, Schwarzmann, Sandhoff, Polyakova, Belov, Hein, Von Middendorff, Schönle and Hell2009; Hein et al. Reference Hein, Willig, Wurm, Westphal, Jakobs and Hell2010; Lukinavicius et al. Reference Lukinavicius, Umezawa, Olivier, Honigmann, Yang, Plass, Mueller, Reymond, Correa, Luo, Schultz, Lemke, Heppenstall, Eggeling and Johnsson2013; Pellett et al. Reference Pellett, Sun, Gould, Rothman, Xu, Correa and Bewersdorf2011; Schröder et al. Reference Schröder, Benink, Dyba and Los2008) or specifically for actin or microtubule-networks (Lukinavicius et al. Reference Lukinavicius, Reymond, D'Este, Masharina, Göttfert, Ta, Güther, Fournier, Rizzo, Waldmann, Blaukopf, Sommer, Gerlich, Arndt, Hell and Johnsson2014), or fluorogen-activating tags (Fitzpatrick et al. Reference Fitzpatrick, Yan, Sieber, Dyba, Schwarz, Szent-Gyorgyi, Woolford, Berget, Waggoner and Bruchez2009) that covalently bind functionalized and membrane-permeable organic dyes. Ranging from the study of the nanoscale dynamics of different cellular molecules, molecular assemblies and structures such as the cytoskeleton, ER, mitochondria, peroxisomes, caveolae, membrane lipids and proteins or vesicles (e.g. Eggeling et al. Reference Eggeling, Ringemann, Medda, Schwarzmann, Sandhoff, Polyakova, Belov, Hein, Von Middendorff, Schönle and Hell2009; Hein et al. Reference Hein, Willig and Hell2008, Reference Hein, Willig, Wurm, Westphal, Jakobs and Hell2010; Moneron & Hell, Reference Moneron and Hell2009; Morozova et al. Reference Morozova, Piatkevich, Gould, Zhang, Bewersdorf and Verkhusha2010; Muller et al. Reference Muller, Schumann and Kraegeloh2012; Pellett et al. Reference Pellett, Sun, Gould, Rothman, Xu, Correa and Bewersdorf2011; Rankin et al. Reference Rankin, Moneron, Wurm, Nelson, Walter, Schwarzer, Schroeder, Colon-Ramos and Hell2011; Tonnesen et al. Reference Tonnesen, Nadrigny, Willig, Wedlich-Soldner and Nägerl2011; Westphal et al. Reference Westphal, Rizzoli, Lauterbach, Kamin, Jahn and Hell2008; Willig et al. Reference Willig, Stiel, Brakemann, Jakobs and Hell2011), live-cell STED nanoscopy experiments have also been realized with multi-color detection (Bethge et al. Reference Bethge, Chereau, Avignone, Marsicano and Nägerl2013; Pellett et al. Reference Pellett, Sun, Gould, Rothman, Xu, Correa and Bewersdorf2011; Tonnesen et al. Reference Tonnesen, Nadrigny, Willig, Wedlich-Soldner and Nägerl2011; Willig et al. Reference Willig, Stiel, Brakemann, Jakobs and Hell2011), and video-rate STED imaging has been pushed to a time resolution of up to 80–200 frames per second. The STED method is therefore the fastest reported super-resolution imaging mode to date (Lauterbach et al. Reference Lauterbach, Keller, Schönle, Kamin, Westphal, Rizzoli and Hell2010a, Reference Lauterbach, Ullal, Westphal and Hellb; Westphal et al. Reference Westphal, Lauterbach, Di Nicola and Hell2007, Reference Westphal, Rizzoli, Lauterbach, Kamin, Jahn and Hell2008).

In an application to neurobiology it was shown that it is possible to image dendritic spines of YFP-positive or organic-dye-filled hippocampal neurons in organotypic slices (Fig. 6a) (Ding et al. Reference Ding, Takasaki and Sabatini2009; Nägerl et al. Reference Nägerl, Willig, Hein, Hell and Bonhoeffer2008; Urban et al. Reference Urban, Willig, Hell and Nägerl2011). Spines are the dendritic processes that form the postsynaptic part of most excitatory synapses in the mammalian brain. In neurobiology, confocal and two-photon microscopy are widely used to study activity-dependent changes in synaptic morphology by recording time lapse images. However, the diffraction-limited resolution of light microscopy is often inadequate, forcing researchers to complement the live-cell imaging strategy by electron microscopy. Time-lapse STED nanoscopy outperforms confocal microscopy in revealing important structural details and can be used for quantification of morphological parameters, such as the neck width and curvature of the heads of spines, which play critical roles for the function and plasticity of synaptic connections (Urban et al. Reference Urban, Willig, Hell and Nägerl2011), and can be correlated to synaptic signaling (Tonnesen et al. Reference Tonnesen, Katona, Rozsa and Nägerl2014). Here, the use of aberration-reducing optics has realized STED imaging deep inside scattering biological tissue with penetration depth of up to 120 μm (Urban et al. Reference Urban, Willig, Hell and Nägerl2011).

Fig. 6.

Live-cell STED nanoscopy. (a) Time-lapse STED imaging of dendritic processes in a living hippocampal slice culture labeled with the fluorescent protein YFP (1 frame every 40 s, adapted from (Nägerl et al. Reference Nägerl, Willig, Hein, Hell and Bonhoeffer2008)). Arrows indicate a change in shape over time of a cup-like spine head. Scale bar: 1 μm. (b) In-vivo STED nanoscopy of a YFP-labeled neuron in the molecular layer of the somatosensory cortex of a mouse (left, inset depicts imaging setup). A maximum intensity projection of dendritic and axonal structures proves a spatial resolution of <70 nm (upper right). Temporal dynamics of spine morphology (lower right). Scale bar: 1 μm. Adapted from (Berning et al. Reference Berning, Willig, Steffens, Dibaj and Hell2012). (c) Two-color in-vivo STED imaging of astrocytes and neurons offers the possibility to study the influence of the astrocyte on synaptic transmission in vivo. Confocal overview (left, 100 × 100 μm, scale bar: 10 μm) and STED close-up image (right, scale bar: 500 nm) of the somatosensory cortex in a double transgenic mouse expressing cytosolic EYFP in neurons (TgN(Thy1-EYFP)) and cytosolic GFP in astrocytes (TgN(GFAP-EGFP)GFEC).

With STED nanoscopy available to study the living cell it will be exciting to see where it can unravel fundamental details. The imaging of spines is a good example because synaptic function is related to its shape. Super-resolution is necessary because the size of the spine neck is below the diffraction barrier. However, the main function of neurons is information processing by forming connections with their neighbours. This can only be studied where they are embedded in their natural environment and, if possible, in the living animal. Therefore, STED imaging was adapted to resolve neurons and their subtle dynamics in the cerebral cortex of a living mouse with so far unachieved spatial resolution. An upright STED nanoscope was constructed and adapted to the spectroscopic properties of YFP. The somatosensory cortex of the anaesthetised mouse was exposed through a glass-sealed hole in the skull (Berning et al. Reference Berning, Willig, Steffens, Dibaj and Hell2012). Figure 6b shows an image of the setup as well as of dendritic processes within the molecular layer of a TgN (Thy1-YFP) mouse taken by STED. The line profile shows that the smallest structures are <70 nm in diameter, indicating that the resolution is at least of that order. Recording images over 30 min in the living organism revealed that the dendritic spine can undergo morphologic changes and movements on the time scale of minutes. Furthermore, multi-colour STED recordings of the somatosensory cortex in double transgenic mice have the potential to study the influence of astrocytes on synaptic transmission in vivo (Fig. 6c). This shows that STED nanoscopy can be a tool to study brain function or the origin of brain diseases which are related to a structural change.

2.1.6 Lasers for STED

The development of the STED method has significantly benefited from improvements in laser technologies. STED nanoscopy was first realized with pulsed and high-repetition (~80 MHz) Ti:Sa laser systems for stimulated emission and laser diodes for excitation (Klar et al. Reference Klar, Jakobs, Dyba, Egner and Hell2000). This configuration requires the exact timing of both lasers. This is usually accomplished by triggering the laser diodes using custom-built delay electronics, and stretching of the Ti:Sa pulses to 50–300 ps by, for example, glass rods and fibers. The triggering and pulse stretching optimize the timing and efficiency of the stimulated emission relative to the excitation laser pulses, and minimize bias due to polarization effects, jitters in the timing of the excitation and the STED pulses, multi-photon excitation processes, non-linear photobleaching via higher excited electronic states and direct excitation by the STED light (e.g. Dyba & Hell, Reference Dyba and Hell2003). While the Ti:Sa laser with wavelengths typically around 700–800 nm may directly be used for STED imaging of dyes emitting in the red spectrum (>600 nm), fluorescent labels such as GFP, YFP, Alexa 488, TMR and similar emit at around 500–590 nm and require STED wavelengths in the range of 590–650 nm. Pulsed laser light in this wavelength range and with average powers of 100–150 mW is delivered by an optical parametric oscillator (OPO) pumped by a Ti:Sa laser (Willig et al. Reference Willig, Kellner, Medda, Hein, Jakobs and Hell2006b, Reference Willig, Rizzoli, Westphal, Jahn and Hellc). Such Ti:Sa arrangements are usually rather complex and costly. Nevertheless, the use of Ti:Sa lasers is still one of the preferred options for STED nanoscopy (e.g. Auksorius et al. Reference Auksorius, Boruah, Dunsby, Lanigan, Kennedy, Neil and French2008; Berning et al. Reference Berning, Willig, Steffens, Dibaj and Hell2012; Eggeling et al. Reference Eggeling, Ringemann, Medda, Schwarzmann, Sandhoff, Polyakova, Belov, Hein, Von Middendorff, Schönle and Hell2009; Gould et al. Reference Gould, Myers and Bewersdorf2011; Lau et al. Reference Lau, Lee, Sahl, Stearns and Moerner2012; Leutenegger et al. Reference Leutenegger, Ringemann, Lasser, Hell and Eggeling2012; Nägerl et al. Reference Nägerl, Willig, Hein, Hell and Bonhoeffer2008; Pellett et al. Reference Pellett, Sun, Gould, Rothman, Xu, Correa and Bewersdorf2011; Sieber et al. Reference Sieber, Willig, Heintzmann, Hell and Lang2006, Reference Sieber, Willig, Kutzner, Gerding-Reimers, Harke, Donnert, Rammner, Eggeling, Hell, Grubmuller and Lang2007; Westphal et al. Reference Westphal, Rizzoli, Lauterbach, Kamin, Jahn and Hell2008; Willig et al. Reference Willig, Kellner, Medda, Hein, Jakobs and Hell2006b, Reference Willig, Rizzoli, Westphal, Jahn and Hell2006c). Ti:Sa-based laser systems were employed in the first demonstration of two-color STED imaging (Donnert et al. Reference Donnert, Keller, Wurm, Rizzoli, Westphal, Schoenle, Jahn, Jakobs, Eggeling and Hell2007b; Meyer et al. Reference Meyer, Wildanger, Medda, Punge, Rizzoli, Donnert and Hell2008), have been integrated in a commercial system (Clausen et al. Reference Clausen, Galiani, Bernardino De La Serna, Fritzsche, Chojnacki, Gehmlich, Lagerholm and Eggeling2013; Fitzpatrick et al. Reference Fitzpatrick, Yan, Sieber, Dyba, Schwarz, Szent-Gyorgyi, Woolford, Berget, Waggoner and Bruchez2009; Morozova et al. Reference Morozova, Piatkevich, Gould, Zhang, Bewersdorf and Verkhusha2010; Schröder et al. Reference Schröder, Benink, Dyba and Los2008) and have facilitated the D- or T-Rex modality (using a regenerative amplified (Rega) mode-locked Ti:sapphire oscillator) (Donnert et al. Reference Donnert, Keller, Medda, Andrei, Rizzoli, Lurmann, Jahn, Eggeling and Hell2006).

Several options have been presented to reduce complexity and cost of the laser setup. Giving up the wavelength tunability, the use of single-wavelength pico- or nanosecond laser modules allows reducing costs (Westphal et al. Reference Westphal, Blanca, Dyba, Kastrup and Hell2003), and their high pulse peak powers have facilitated imaging with spatial resolutions of <10–20 nm (Göttfert et al. Reference Göttfert, Wurm, Mueller, Berning, Cordes, Honigmann and Hell2013; Rittweger et al. Reference Rittweger, Han, Irvine, Eggeling and Hell2009a). The lowest-cost STED nanoscope to date has been realized by pulsing a 660 nm DVD-diode, i.e. a compact off-the-shelf laser diode (Schrof et al. Reference Schrof, Staudt, Rittweger, Wittenmayer, Dresbach, Engelhardt and Hell2011). Strong and compact single-wavelength lasers are therefore increasingly applied for STED, especially in new commercial STED nanoscopes. Multiple laser lines for STED and excitation can simultaneously be chosen from a pulsed white light or super-continuum laser, which offers highest flexibility for wavelength optimization, and disburdens from the necessity of synchronizing several lasers (Auksorius et al. Reference Auksorius, Boruah, Dunsby, Lanigan, Kennedy, Neil and French2008; Blom et al. Reference Blom, Rönnlund, Scott, Spicarova, Widengren, Bondar, Aperia and Brismar2011; Wildanger et al. Reference Wildanger, Rittweger, Kastrup and Hell2008, Reference Wildanger, Medda, Kastrup and Hell2009b). The advent of white light lasers alleviated the setup of multi-color STED instruments (Blom et al. Reference Blom, Rönnlund, Scott, Spicarova, Rantanen, Widengren, Aperia and Brismar2012; Bückers et al. Reference Bückers, Wildanger, Vicidomini, Kastrup and Hell2011; Neumann et al. Reference Neumann, Bückers, Kastrup, Hell and Jakobs2010). In another development, a ~530 nm pico- or nanosecond microchip or fiber-amplified, frequency doubled laser was coupled into a standard single-mode fiber to produce a tunable spectrum of discrete peaks between 530 and 620 nm via stimulated Raman scattering (SRS) (Rankin & Hell, Reference Rankin and Hell2009; Rankin et al. Reference Rankin, Kellner and Hell2008). This again allowed a flexible choice of STED laser wavelengths, demonstrating STED imaging with spatial resolution down to 20–30 nm (Rankin & Hell, Reference Rankin and Hell2009; Rankin et al. Reference Rankin, Kellner and Hell2008, Reference Rankin, Moneron, Wurm, Nelson, Walter, Schwarzer, Schroeder, Colon-Ramos and Hell2011).

Further simplification of the setup was achieved by realizing STED nanoscopy with continuous-wave (CW) lasers, since no laser-pulse preparation is required (Willig et al. Reference Willig, Harke, Medda and Hell2007). CW-STED imaging was first realized with strong Argon–Krypton lasers (Willig et al. Reference Willig, Harke, Medda and Hell2007) or Ti:Sa lasers running in CW mode (Ding et al. Reference Ding, Takasaki and Sabatini2009; Harke, Reference Harke2008), but since then it has been shown that it is possible to utilize compact fibre lasers (Bianchini & Diaspro, Reference Bianchini and Diaspro2012; Moneron & Hell, Reference Moneron and Hell2009; Moneron et al. Reference Moneron, Medda, Hein, Giske, Westphal and Hell2010), diode-pumped solid-state (DPSS) lasers (Honigmann et al. Reference Honigmann, Eggeling, Schulze and Lepert2012; Mueller et al. Reference Mueller, Eggeling, Karlsson and Von Gegerfelt2012) or amplified diode lasers (Honigmann et al. Reference Honigmann, Mueller, Fernando, Eggeling and Sperling2013a) at wavelengths between 560 and 765 nm, also on commercial systems (Clausen et al. Reference Clausen, Galiani, Bernardino De La Serna, Fritzsche, Chojnacki, Gehmlich, Lagerholm and Eggeling2013; Friedemann et al. Reference Friedemann, Turshatov, Landfester and Crespy2011). However, CW-STED comes with two drawbacks compared to the pulsed STED modality. Firstly, much higher average laser powers have to be applied (Harke, Reference Harke2008; Willig et al. Reference Willig, Harke, Medda and Hell2007). This follows from the fact that tightly synchronized trains of excitation and STED pulses, as realized in the pulsed modality, yield an optimized efficiency of stimulated emission: the pulses of the STED beam reach the focal plane virtually simultaneously with or a few picoseconds after the excitation pulses so as to instantly inhibit fluorescence emission from excited molecules. In contrast, in the CW-STED beam implementation the STED intensity and thereby the probability of stimulated de-excitation is lower. As a consequence, to achieve the same fluorescence inhibition efficiency by stimulated emission, (ln(2) f τ)⁻¹-fold larger time-averaged powers of the CW than of a pulsed STED laser have to be supplied (with fluorescence lifetime τ of the label and repetition rate f of the pulsed STED modality) (Harke, Reference Harke2008; Willig et al. Reference Willig, Harke, Medda and Hell2007). For usual parameters f = 80 MHz and τ = 3·5 ns this amounts to about five-fold larger time-averaged CW powers, for example 800 mW in the CW compared to 160 mW in the pulsed case. Secondly, a further consequence of the CW STED modality is that a non-negligible fraction of the molecules emit fluorescence before having been exposed to much of the STED light, and thus residual fluorescence outside the zero-intensity point of the STED light leads to a pedestal in the effective observation spot, resulting in somewhat lower-contrast images (Leutenegger et al. Reference Leutenegger, Eggeling and Hell2010).

Besides the aforementioned simplifications of the laser arrangements (Kastrup et al. Reference Kastrup, Wildanger, Rankin, Hell and Diaspro2010), compact and more robust STED setups have been created by using optimized phase plates for realizing the focal intensity distribution of the STED laser (Reuss et al. Reference Reuss, Engelhardt and Hell2010; Wildanger et al. Reference Wildanger, Bückers, Westphal, Hell and Kastrup2009a). Based on diffractive optical elements, a single-beam-path STED nanoscope has been constructed, which includes a phase plate that selectively modulates the STED beam but leaves the excitation beam unchanged. In this configuration the beams are aligned by design and the alignment is hence insensitive to mechanical drift (easySTED) (Wildanger et al. Reference Wildanger, Bückers, Westphal, Hell and Kastrup2009a). Similarly, a birefringent device has been instituted which produces the doughnut-shaped focal spot with suitable polarization for the STED laser while leaving the excitation spot virtually intact, and which in addition can be adapted to reveal, through the resulting fluorescence image, the orientation of fluorophores in the sample, thus directly providing sub-diffraction resolution images of molecular orientation (Reuss et al. Reference Reuss, Engelhardt and Hell2010). Using Wollaston prisms, a common beam-shaping device realized a parallelized STED nanoscope featuring four pairs of scanning excitation and STED beams. This arrangement provides four-fold increased imaging speed of a given sample area, while maintaining the advantages of a single-beam easy STED instrument (Bingen et al. Reference Bingen, Reuss, Engelhardt and Hell2011). Further increases in image acquisition speed are achieved by the use of even more parallelized scanning excitation and STED beams, as recently introduced by camera-based detection and wide-field excitation together with well-designed optical patterns for STED (Yang et al. Reference Yang, Przybilla, Mestre, Trebbia and Lounis2014; Bergermann et al. Reference Bergermann, Alber, Sahl, Engelhardt and Hell2015). Spatial light modulators have been applied to auto-align a STED nanoscope (Gould et al. Reference Gould, Kromann, Burke, Booth and Bewersdorf2013).

Overall, it is to be expected that further developments in laser and optical technology will create more and more compact and less costly systems, and thus will further facilitate the use of STED imaging in day-to-day biophysical and medical research.

2.1.7 Gated CW-STED nanoscopy

The problem of the pedestal inherent to the CW-STED modality can be solved by implementing a pulsed-laser excitation in combination with the CW-STED laser and a time-gated detection scheme (Moffitt et al. Reference Moffitt, Osseforth and Michaelis2011; Vicidomini et al. Reference Vicidomini, Moneron, Han, Westphal, Ta, Reuss, Engelhardt, Eggeling and Hell2011). Time-gated detection is often used in fluorescence microscopy for suppressing background (e.g. Eggeling et al. Reference Eggeling, Berger, Brand, Fries, Schaffer, Volkmer and Seidel2001a; Shera et al. Reference Shera, Seitzinger, Davis, Keller and Soper1990), and, in a pulsed STED scheme, it is well known that photons should be detected after the STED pulse has left (Schrader et al. Reference Schrader, Meinecke, Bahlmann, Kroug, Cremer, Soini and Hell1995; Westphal & Hell, Reference Westphal and Hell2005), as shown in a recent experiment using time-correlated single photon counting (Auksorius et al. Reference Auksorius, Boruah, Dunsby, Lanigan, Kennedy, Neil and French2008). This is due to the fact that scattered laser light or residual fluorescence signal only occurs during the laser pulses. Time-gated detection also improves the contrast of CW-STED images by selectively suppressing image contents (or spatial frequencies) of low spatial resolution, i.e. the aforementioned pedestal or blurring (Fig. 7) (Moffitt et al. Reference Moffitt, Osseforth and Michaelis2011; Vicidomini et al. Reference Vicidomini, Moneron, Han, Westphal, Ta, Reuss, Engelhardt, Eggeling and Hell2011, Reference Vicidomini, Schoenle, Ta, Han, Moneron, Eggeling and Hell2013). This follows from the fact that fluorescence inhibition is lowest during the duration of the excitation pulse (usually <150 ps), while right afterwards only the CW-STED beam is acting and inhibiting fluorescence emission: the longer it lasts, the more likely it becomes that a fluorophore is switched off, i.e. the spatial resolution not only depends on the intensity of the STED light but also on the time span of the STED beam action (Hell et al. Reference Hell, Jakobs and Kastrup2003; Vicidomini et al. Reference Vicidomini, Schoenle, Ta, Han, Moneron, Eggeling and Hell2013). This can also be rationalized in the sense that STED reduces the fluorescence lifetime τ of the excited fluorescent state. Ensuring that photons are collected only for delays significantly after the excitation pulse largely suppresses signal from strongly inhibited molecules: fluorescence light is recorded mainly from fluorophores from the zero-intensity doughnut center, where the STED beam is inherently weak (and thus the fluorescence lifetime rather long). Along with the improved image contrast, the selection of high spatial frequencies by the gated detection scheme also allows to apply lower CW-STED powers compared to the non-gated CW-STED scheme to be able to similarly discern alike features (Vicidomini et al. Reference Vicidomini, Moneron, Han, Westphal, Ta, Reuss, Engelhardt, Eggeling and Hell2011). This, and the fact that CW in comparison to pulsed beams may be less prone to induce phototoxic multiphoton processes, reduces light-induced stress on the sample. It is worth noting that these improvements always come with a reduction in signal, since the gating suppresses valuable signal as well (Vicidomini et al. Reference Vicidomini, Schoenle, Ta, Han, Moneron, Eggeling and Hell2013). Gated-STED (gSTED) nanoscopy has been realized for different fluorophores and in living cells, and has now been incorporated into a commercial system, allowing the recording of live-cell images with sub-diffraction spatial resolution at moderate CW STED powers <80 mW (Clausen et al. Reference Clausen, Galiani, Bernardino De La Serna, Fritzsche, Chojnacki, Gehmlich, Lagerholm and Eggeling2013; Vicidomini et al. Reference Vicidomini, Moneron, Han, Westphal, Ta, Reuss, Engelhardt, Eggeling and Hell2011). Data acquisition for gSTED has been demonstrated by offline processing of time-correlated single-photon counting data, or in real-time using a fast electronic gate. As for CW-STED, gSTED nanoscopy can be realized with compact CW lasers, which are nowadays available at various wavelengths (Honigmann et al. Reference Honigmann, Eggeling, Schulze and Lepert2012, Reference Honigmann, Mueller, Fernando, Eggeling and Sperling2013a; Mueller, Reference Mueller2012) and show improved performance with reduced noise levels (Hernandez et al. Reference Hernandez, d'Amora, Diaspro and Vicidomini2014a). Of all current STED modalities, gSTED provides the sharpest STED images at the lowest peak laser powers.

Fig. 7.

gSTED nanoscopy. (a) Principle: the fluorescence lifetime of a fluorophore decreases with increasing STED power as depicted for representative fluorescence lifetime decays for different STED powers (left): using pulsed excitation (blue, Exc) and CW-STED (red) in conjunction with gated detection (detection within the time period ΔT (green) with a time lag T _g (grey) relative to the exciting pulse) favors signal from points of low STED power, i.e. from areas at or close to the intensity zero. (b) Scanning fluorescence intensity (left and upper right) and lifetime (lower right) images of a single fluorophore for confocal diffraction-limited (upper left), CW-STED (intensity: lower left, lifetime: lower right) and gSTED (upper right) recordings (scale bar: 200 nm), and (right panel) intensity line profiles through the middle images, indicating the removal of the pedestal of the CW-STED recordings (black) by the gated detection (gSTED, red). (c) Confocal and gSTED images (right: magnification of central area marked by the dashed white box) of keratin fused to the fluorescent protein citrine in a living PtK2 cell with low CW STED laser power. Scale bar: 1 μm. Adapted from (Vicidomini et al. Reference Vicidomini, Moneron, Han, Westphal, Ta, Reuss, Engelhardt, Eggeling and Hell2011).

Besides the aforementioned improvement in image contrast, gated detection also allows to specifically suppress background signal. On one hand, discarding signal during the excitation pulse (as done for gSTED) reduces scattering signal from the excitation laser (e.g. Eggeling et al. Reference Eggeling, Berger, Brand, Fries, Schaffer, Volkmer and Seidel2001a; Shera et al. Reference Shera, Seitzinger, Davis, Keller and Soper1990). On the other hand, the rejection of signal for time delays after the excitation pulse much longer than the fluorescence lifetime τ of the fluorophore specifically suppresses continuous background signal such as fluorescence light excited by the CW-STED laser or detector noise. This allows using wavelengths of the STED laser closer to the fluorescence emission maximum, which reduces the required laser powers (Vicidomini et al. Reference Vicidomini, Moneron, Eggeling, Rittweger and Hell2012; Hernandez et al. Reference Hernandez, Peres, Zanacchi, d'Amora, Christodoulou, Bianchini, Diaspro and Vicidomini2014b). Consequently, gated detection introduces high flexibility, especially if the acquired data is post-processed by software. This will not only allow an optimization of the image contrast, but also the investigation of the very same data for different image contrasts, i.e. for different gating positions (Vicidomini et al. Reference Vicidomini, Moneron, Han, Westphal, Ta, Reuss, Engelhardt, Eggeling and Hell2011).

2.1.8 STED-FCS: nanoscale single-molecule dynamics

The ongoing quest to study molecular dynamics not in the ensemble but at the molecular level calls for measurements both of molecular numbers and, crucially, with access to molecular spatial scales. Compared to ensemble measurements, studies at the single-molecule level reach a much increased sensitivity as they may reveal heterogeneities hidden in the ensemble and disclose deviations from the ergodic theorem (i.e. a time-averaged ensemble measurement may not be the same as the ensemble average of a large number of single-molecule measurements). Fluorescence microscopy has reached single-molecule detection sensitivity (Moerner & Kador, Reference Moerner and Kador1989; Orrit & Bernard, Reference Orrit and Bernard1990; Shera et al. Reference Shera, Seitzinger, Davis, Keller and Soper1990; Weiss, Reference Weiss1999; Zander et al. Reference Zander, Enderlein and Keller2002), and studies of single-molecule dynamics have given many new, detailed insights into physical, chemical and biological problems (e.g. Lord et al. Reference Lord, Lee and Moerner2010; Moerner, Reference Moerner2007). In this context, statistical analysis tools such as Fluorescence Correlation Spectroscopy (FCS) (Ehrenberg & Rigler, Reference Ehrenberg and Rigler1974; Haustein & Schwille, Reference Haustein and Schwille2003; Magde et al. Reference Magde, Webb and Elson1972) or photon-counting histogram analysis (PCH) (Chen et al. Reference Chen, Muller, So and Gratton1999) and Fluorescence Intensity Distribution Analysis (FIDA) (Kask et al. Reference Kask, Palo, Ullmann and Gall1999) have been extremely helpful. However, further insights into a lot of (single-molecule) dynamical processes have been again impeded by the limited spatial resolution of common diffraction-limited fluorescence microscopy. 1) The concentration of fluorescently labeled molecules has to be very low (<nM) to reach the single-molecule level with conventional confocal observation volumes – a concentration which is often far below that of endogenous (biological) conditions. In contrast to performing measurements in zero-mode waveguides (Leutenegger et al. Reference Leutenegger, Goesch, Perentes, Hoffmann, Martin and Lasser2006; Levene et al. Reference Levene, Korlach, Turner, Foquet, Craighead and Webb2003; Wenger et al. Reference Wenger, Conchonaud, Dintinger, Wawrezinieck, Ebbesen, Rigneault, Marguet and Lenne2007), or to photobleach (Moertelmaier et al. Reference Moertelmaier, Brameshuber, Linimeier, Schutz and Stockinger2005) or switch off (Eggeling et al. Reference Eggeling, Hilbert, Bock, Ringemann, Hofmann, Stiel, Andresen, Jakobs, Egner, Schönle and Hell2007) large parts of the ensemble, the most obvious way to handle larger, endogenous concentrations would be to lower the observation spot's length scale (its size) (Blom et al. Reference Blom, Kastrup and Eggeling2006; Kastrup et al. Reference Kastrup, Blom, Eggeling and Hell2005; Weiss, Reference Weiss2000). 2) The conventional confocal observation spot usually averages over details of nanoscale molecular dynamics. For example, strongly localized trapping cannot directly be distinguished from slow but regular molecular diffusion (Eggeling et al. Reference Eggeling, Ringemann, Medda, Schwarzmann, Sandhoff, Polyakova, Belov, Hein, Von Middendorff, Schönle and Hell2009). In the case of confocal FCS observations of molecular membrane dynamics, such discrimination has indirectly been realized, for example, by searching for anomalies in diffusion (Schwille et al. Reference Schwille, Korlach and Webb1999), by extrapolating to the nanoscopic case (Wawrezinieck et al. Reference Wawrezinieck, Rigneault, Marguet and Lenne2005), or by probing in the near-field on nanostructures or -holes (Leutenegger et al. Reference Leutenegger, Goesch, Perentes, Hoffmann, Martin and Lasser2006; Manzo et al. Reference Manzo, Van Zanten and Garcia-Parajo2011; Wenger et al. Reference Wenger, Conchonaud, Dintinger, Wawrezinieck, Ebbesen, Rigneault, Marguet and Lenne2007). However, very direct measurements at the length scale of interest (i.e. observing diffusion dynamics through sub-diffraction-sized far-field observation spots) deliver much more reliable and model-independent results about nanoscopic details of molecular diffusion and interactions and, at the same time, allow indirect methods to access even smaller length scales.

In an attempt to access smaller length scales, non-invasive far-field microscopy was thus combined with single-particle tracking (SPT), utilizing the high spatial localization precision of down to the 1-nanometer level for bright marker particles (Geerts et al. Reference Geerts, Debrabander, Nuydens, Geuens, Moeremans, Demey and Hollenbeck1987; Kusumi et al. Reference Kusumi, Nakada, Ritchie, Murase, Suzuki, Murakoshi, Kasai, Kondo and Fujiwara2005; Schutz et al. Reference Schutz, Schindler and Schmidt1997; Sheetz et al. Reference Sheetz, Turney, Qian and Elson1989; Yildiz et al. Reference Yildiz, Forkey, Mckinney, Ha, Goldman and Selvin2003). Yet this introduces other restrictions: to reach the desired spatial localization precision, SPT often applies bright but rather large signal markers, which potentially influence the system under study (Clausen & Lagerholm, Reference Clausen and Lagerholm2011). When using conventional fluorophore labeling the temporal resolution is lower. And while important insights can be gained in carefully optimized SPT experiments, for example if the full spatiotemporal resolution afforded by the fluorophore photon budget is harnessed in fast molecular tracking schemes (Sahl et al. Reference Sahl, Leutenegger, Hilbert, Hell and Eggeling2010, Reference Sahl, Leutenegger, Hell and Eggeling2014), the amount of data to be gathered for an accurate statement on an average molecular behavior is higher for SPT compared to FCS. In addition to the concentration issue, the stochastic sampling concomitant with SPT yields low statistics and random coverage from a single measurement only.

A remedy for these limitations is the combination of FCS with the STED principle (Eggeling et al. Reference Eggeling, Ringemann, Medda, Schwarzmann, Sandhoff, Polyakova, Belov, Hein, Von Middendorff, Schönle and Hell2009; Kastrup et al. Reference Kastrup, Blom, Eggeling and Hell2005; Ringemann et al. Reference Ringemann, Harke, Middendorff, Medda, Honigmann, Wagner, Leutenegger, Schoenle, Hell and Eggeling2009). STED-FCS delivers high temporal and spatial resolution together with a high degree of statistical averaging. The most straightforward parameters measured with FCS are the average transit time τ _D of the fluorescent molecules through the observation area/volume and the average number N of fluorescing molecules in the observation volume. One expects both τ _D and N to decrease with the confinement of the observation spot, i.e. with the STED power. Figure 8a shows exemplarily FCS data for fluorophores in solution exhibiting 3D free diffusion. While the expected decrease of τ _D is observed, N does not decrease as strongly, and even increases for high STED powers due to a reduced signal-to-background ratio (SBR) (Ringemann et al. Reference Ringemann, Harke, Middendorff, Medda, Honigmann, Wagner, Leutenegger, Schoenle, Hell and Eggeling2009). The low SBR is caused by non-inhibited, low-brightness fluorescence signals from out-of-plane volume shells. Contributions of the latter to the overall signal grow relative to the declining fluorescence signal from the more and more confined observation spot (Fig. 8a) (Ringemann et al. Reference Ringemann, Harke, Middendorff, Medda, Honigmann, Wagner, Leutenegger, Schoenle, Hell and Eggeling2009). This degradation in SBR has on the one hand been observed for confinement along x/y only, but also for confinement along z or along all three spatial directions (Kastrup et al. Reference Kastrup, Blom, Eggeling and Hell2005; Ringemann et al. Reference Ringemann, Harke, Middendorff, Medda, Honigmann, Wagner, Leutenegger, Schoenle, Hell and Eggeling2009), even when using two-photon excitation (Moneron & Hell, Reference Moneron and Hell2009) or two opposing objectives in an iso-STED arrangement (Schmidt et al. Reference Schmidt, Wurm, Jakobs, Engelhardt, Egner and Hell2008). The biased values of N can straightforwardly be corrected for by, for example, a combined or global FCS-FIDA analysis (Fig. 8a, right) (Ringemann et al. Reference Ringemann, Harke, Middendorff, Medda, Honigmann, Wagner, Leutenegger, Schoenle, Hell and Eggeling2009). Therefore, while such reduction effects on the SBR might limit the sensitivity of STED-FCS experiments for 3D diffusion, it does not necessarily preclude such measurements, such as assessments of nanoscale molecular diffusion dynamics inside living cells.

Fig. 8.

STED-FCS. (a) STED-FCS analysis of free 3D-diffusion of the organic dye Atto647N in aqueous solution. Representative correlation data (left panels, left: normalized amplitudes, right: original data) for confocal (black) and STED recordings (open dots) and (right panel) relative values of transit time τ _D (black), particle number N (original open dots, background-corrected grey triangles), and signal fraction of apparent background (grey crosses) determined from FCS and FIDA data recorded for increasing power of the doughnut-shaped (inset left) STED laser. The size reduction of the observation spot by increasing the STED power shortens the dye's transit time, but also introduces an increasing contribution of apparent un-depleted low-brightness background, which introduces noise and dampens the FCS data's amplitude and thus results in an apparent increase of N, which can be corrected for by a global FCS-FIDA analysis. (b) Relative apparent background for the STED-FCS recordings for two different pinhole sizes (decrease from left to right), and for a fluorescent lipid analog diffusing in a multi-lamellar membrane. Reduction of the pinhole size reduces un-depleted out-of-plane low-brightness fluorescence signal (apparent background), which diminishes further when measuring two-dimensional diffusion in membranes, where out-of-plane signal is absent. (c) STED-FCS analysis of free 2D-diffusion of a fluorescent lipid analog in a multi-lamellar membrane. Representative correlation data (left panels, left: normalized amplitudes, right: original data) for confocal (black) and STED recordings (open dots) and (right panel) relative values of transit time τ _D (black) and particle number N (original open dots, background-corrected grey triangles) determined from FCS and FIDA data recorded for increasing power of the doughnut-shaped STED laser. The confinement of the observation spot by increasing the STED power reduces both τ _D and N, without an influence by out-of-plane signal contributions. (d) STED allows single-molecule observations at high concentrations. Fluorescence signal over time for the same concentration of a fluorescent lipid analog diffusing in a multi-lamellar membrane indicates diffusion of single molecules only for the STED (right) but not for the confocal (left) recordings. Adapted from (Ringemann et al. Reference Ringemann, Harke, Middendorff, Medda, Honigmann, Wagner, Leutenegger, Schoenle, Hell and Eggeling2009).

The above out-of-plane background vanishes for 2D diffusion measurements such as within membranes, where no out-of-plane signal is present (Fig. 8b). Here, both τ _D and N decrease with increasing STED power, i.e. with the confinement of the observation area, as expected (Fig. 8c) (Ringemann et al. Reference Ringemann, Harke, Middendorff, Medda, Honigmann, Wagner, Leutenegger, Schoenle, Hell and Eggeling2009). Recently, STED-FCS has also been combined with evanescent (TIRF) illumination to reject out-of-plane background (Leutenegger et al. Reference Leutenegger, Ringemann, Lasser, Hell and Eggeling2012). However, the maximum achievable resolution of the TIRF-STED-FCS setup was only 50–60 nm due to side lobes inherent to the confocal TIRF illumination scheme. An important observation is that the STED mode reduces the transit times but hardly the detected fluorescence brightness (if the intensity zero of the STED focus is properly aligned, i.e. it really is close to zero). In Fig. 8d, the detected photon count-rate of a typical molecular trace of fluorescent lipids in multi-lamellar membrane layers peaks at 300–400 kHz which enables the analysis of single-molecule STED data at a good signal-to-noise ratio. Most importantly, signatures of single-molecule transits are only observable for small, sub-diffraction sized spots as created by STED, while the concentration is too high for conventional confocal recordings (Fig. 8d). Therefore, STED allows accurate single-molecule measurements on the cellular plasma membrane of living cells, and thus is a powerful tool for shedding new light on long-standing biological questions.

2.1.9 STED-FCS: live-cell membrane dynamics

Many membrane-associated processes such as signaling events are considered to be closely related to cholesterol-mediated interactions of some lipids such as sphingolipids (Brown & London, Reference Brown and London2000; Fielding, Reference Fielding2006; Hanzal-Bayer & Hancock, Reference Hanzal-Bayer and Hancock2007; Jacobson et al. Reference Jacobson, Mouritsen and Anderson2007; Simons & Ikonen, Reference Simons and Ikonen1997). In contrast to phosphoglycerolipids, these lipids are assumed to form molecular complexes or integrate, assisted by cholesterol, into <200 nm sized lipid nanodomains, usually denoted ‘rafts’ (Simons & Ikonen, Reference Simons and Ikonen1997). These interactions may disturb the diffusion of lipids and proteins in the plasma membrane, and on the other hand may compartmentalize cellular signaling (Pike, Reference Pike2006). Although several experiments indicate their existence, such complexes and/or lipid ‘rafts’ remained controversial due to the lack of suitable techniques for detecting these small-sized objects in living cells (Hancock, Reference Hancock2006; Jacobson et al. Reference Jacobson, Mouritsen and Anderson2007; Lommerse et al. Reference Lommerse, Spaink and Schmidt2004; Munro, Reference Munro2003; Shaw, Reference Shaw2006). A common problem is once again that the <200 nm sized domains cannot be resolved by conventional confocal microscopes, and thus common fluorescence recovery after photobleaching (Feder et al. Reference Feder, Brust-Mascher, Slattery, Baird and Webb1996; Yechiel & Edidin, Reference Yechiel and Edidin1987) or confocal FCS measurements (Fahey et al. Reference Fahey, Koppel, Barak, Wolf, Elson and Webb1977; Schwille et al. Reference Schwille, Korlach and Webb1999; Wawrezinieck et al. Reference Wawrezinieck, Rigneault, Marguet and Lenne2005) average over nanoscale details of molecular diffusion. STED nanoscopy opens up a new avenue to elucidate lipid diffusion.

Figure 9 shows FCS analysis of lipid diffusion in the plasma membrane of living mammalian cells recorded with a STED nanoscope. The unique feature of the STED method to continuously downscale the size of the observation area with laser intensity and/or gated detection allows the determination of the average transit time τ _D for different diameters d of the observation, revealing different modes of diffusion (Fig. 9d) (Eggeling et al. Reference Eggeling, Ringemann, Medda, Schwarzmann, Sandhoff, Polyakova, Belov, Hein, Von Middendorff, Schönle and Hell2009; He & Marguet, Reference He and Marguet2011; Ruprecht et al. Reference Ruprecht, Wieser, Marguet and Schuetz2011; Wawrezinieck et al. Reference Wawrezinieck, Rigneault, Marguet and Lenne2005). Unlike the confocal recordings, the STED-FCS data reveal distinct differences between fluorescent phosphoglycerolipid (PE) and sphingolipid (SM) analogs. Most importantly, it is revealed that while the PE lipids diffuse freely, the SM lipids diffuse heterogeneously on small spatial scales, being transiently (~10–20 ms) trapped in cholesterol-mediated molecular complexes with relatively slow-moving or immobilized binding partners. Such previously inaccessible molecular details depict how the non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells. Recent work could not detect any correlation between the nanoscale trapping as observed by STED-FCS in living cells and partitioning characteristics of the labeled lipids into liquid ordered domains of phase separated model membranes (which is often assumed as a physical model of lipid ‘rafts’) (Fig. 9f) (Eggeling, Reference Eggeling, Mely and Duportail2012; Mueller et al. Reference Mueller, Ringemann, Honigmann, Schwarzmann, Medda, Leutenegger, Polyakova, Belov, Hell and Eggeling2011, Reference Mueller, Honigmann, Ringemann, Medda, Schwarzmann, Eggeling and Tetin2013; Sezgin et al. Reference Sezgin, Levental, Grzybek, Schwarzmann, Mueller, Honigmann, Belov, Eggeling, Coskun, Simons and Schwille2012; Honigmann et al. Reference Honigmann, Mueller, Ta, Schoenle, Sezgin, Hell and Eggeling2014). Rather, the trapping characteristics and the dependence on cholesterol are highly dependent on the molecular structure of the lipid (however not on the dye label or the labeling position), identifying the ceramide or sphingosine group close to the water–lipid interface as well as the lipid's headgroup as the driving forces for molecular interactions (Fig. 9g) (Mueller et al. Reference Mueller, Ringemann, Honigmann, Schwarzmann, Medda, Leutenegger, Polyakova, Belov, Hell and Eggeling2011). Therefore, we may recall our previous statement (Eggeling, Reference Eggeling, Mely and Duportail2012; Mueller et al. Reference Mueller, Ringemann, Honigmann, Schwarzmann, Medda, Leutenegger, Polyakova, Belov, Hell and Eggeling2011, Reference Mueller, Honigmann, Ringemann, Medda, Schwarzmann, Eggeling and Tetin2013), ‘that the observed transient trapping with its rather strong binding to other membrane constituents follows a different molecular mechanism than that of the weak interactions responsible for the formation of ordered phases in model membranes. […] If one pictures the STED-FCS observations in the context of lipid ‘rafts’, one may support a current view that (sphingo)lipid rafts may establish fluctuating nanoscale assemblies of sphingolipid, cholesterol and proteins that can be stabilized to coalesce, forming platforms that function in membrane signaling and trafficking (Lingwood & Simons, Reference Lingwood and Simons2010). Here, the STED-FCS experiments may highlight the fluctuating nanoscale assemblies, which then seem to be highly diverse and strongly depending on the lipid structure. It remains to be shown whether these fluctuating nanoscale assemblies may be stabilized to coalesce to maybe more tightly packed domains (Joly, Reference Joly2004; Lingwood & Simons, Reference Lingwood and Simons2010). A fluorescent lipid analog that partitions into the liquid ordered phase of model membranes (like its natural counterpart) may be able to report on this coalescence.’ Recently, such a lipid analog has successfully been tested in STED experiments (Honigmann et al. Reference Honigmann, Mueller, Hell and Eggeling2013b), and initial experiments in live-cell membranes indicated no difference to the liquid-disordered partitioning of fluorescent lipid analogs (Sezgin et al. Reference Sezgin, Levental, Grzybek, Schwarzmann, Mueller, Honigmann, Belov, Eggeling, Coskun, Simons and Schwille2012; Honigmann et al. Reference Honigmann, Mueller, Ta, Schoenle, Sezgin, Hell and Eggeling2014). Caution has to be taken when designating any lipid-based interaction or heterogeneous organization as a ‘raft’, since the basis of these may be highly diverse (Eggeling, Reference Eggeling, Mely and Duportail2012; Mueller et al. Reference Mueller, Ringemann, Honigmann, Schwarzmann, Medda, Leutenegger, Polyakova, Belov, Hell and Eggeling2011, Reference Mueller, Honigmann, Ringemann, Medda, Schwarzmann, Eggeling and Tetin2013; Sezgin et al. Reference Sezgin, Levental, Grzybek, Schwarzmann, Mueller, Honigmann, Belov, Eggeling, Coskun, Simons and Schwille2012).

Fig. 9.

STED-FCS analysis of lipid plasma membrane diffusion. (a) Lipids and proteins are heterogeneously distributed in the cellular plasma membrane, stemming from often cholesterol-assisted lipid–protein interactions (which may be the basis for the coalescence of transient signaling platforms, denoted membrane domains or lipid ‘rafts’, i.e. spatially confined molecular assemblies of different lipids and proteins which are essential for a cellular signaling event), an asymmetric molecular distribution to the inner and outer leaflet of the bilayer, the underlying cytoskeleton (which is membrane-anchored via proteins), and from membrane curvature and pits. Adapted from (Lingwood & Simons, Reference Lingwood and Simons2010). (b) Structures of the fluorescent lipid analogs phosphoethanolamine (PE) and sphingomyelin (SM), both tagged with the organic dye Atto647N. Grey shaded area: ceramide or sphingosine group of the SM lipid. (c) Representative confocal and STED (observation diameter d = 40 nm) FCS data of PE (red), SM (black) and SM after cholesterol depletion by Cholesterol Oxidase (grey, SM+COase). The SM STED-FCS data can only be described by anomalous diffusion, revealing cholesterol-assisted hindered diffusion of the SM lipid analog. (d) The dependence of the transit time τ _D for different sub-diffraction sized observation areas ~d ² (as tuned by the STED laser power) shows an almost free diffusion (linear dependence, dark grey line diffusion coefficient 0·5 μm²/s) for PE (red squares) and SM after cholesterol depletion (open circles), and a hindered diffusion (non-linear dependence) for SM (grey circles). The minimal change of τ _D for very small observation areas (grey horizontal line) and Monte-Carlo simulations indicate that the hindrance in diffusion is caused by transient complexes with either relatively slow-moving or immobilized membrane molecules (red dotted line) and not by incorporation into ⩾20 nm large domains, where diffusion is slowed down (green dotted line). The direct observation of these transient interactions is impossible with the large diffraction-limited confocal observation area (grey shaded area). (e) Schematic drawing of normal free (red) and hindered SM diffusion (blue, dots: points of interactions or complexes). (f) Comparison of live-cell and model membrane data. Phase separation into liquid-disordered (Ld) and liquid-ordered (Lo) domains of a model membrane bilayer composed of a ternary mixture: both the fluorescent PE and SM lipid analogs hardly enter the Lo phase (upper panel: confocal scanning fluorescence image, black: low signal, white: high signal, adapted from (Mueller et al. Reference Mueller, Ringemann, Honigmann, Schwarzmann, Medda, Leutenegger, Polyakova, Belov, Hell and Eggeling2011)). Partitioning in model membranes and trapping characteristics observed by STED-FCS on live-cell plasma membranes are not correlated (lower panel): Trapping time (red, left axis, live-cell) and fraction of signal in Lo phase (grey, right axis, model membranes) of PE and SM (adapted from Sezgin et al. Reference Sezgin, Levental, Grzybek, Schwarzmann, Mueller, Honigmann, Belov, Eggeling, Coskun, Simons and Schwille2012). (g) STED-FCS analysis of the plasma membrane diffusion of different fluorescent lipid analogs, revealing lipid-specific interactions and independence on dye and label position. Average transit time τ _D for confocal (d ≈ 250 nm, upper panel) and STED (d ≈ 40 nm, lower panel) recordings of the Atto647N- or Atto532-labeled phosphoethanolamine (PE: head group and PE1: acyl-chain labeled), sphingomyelin (SM: acyl-chain and CPE: head-group labeled), ganglioside GM1 (GM1: acyl-chain, GM1^#: head-group and GM1^##: chain addition), and of an Atto647N-tagged transfected GPI-anchor. The meshed bars in the lower panel indicate the values of τ _D determined after cholesterol depletion by COase treatment. Error bars result from averaging over more than thirty measurements. No dependence of the STED-FCS data on the dye-label position has been observed, apart from using the dye Atto532 on the lipid's acyl chain, which accelerates diffusion and lowers trapping probability. Adapted from (Eggeling et al. Reference Eggeling, Ringemann, Medda, Schwarzmann, Sandhoff, Polyakova, Belov, Hein, Von Middendorff, Schönle and Hell2009).

2.2.1 STED: stimulated emission

STED requires rather large laser intensities in order to induce de-excitation from S ₁ more efficiently than the spontaneous decay (usually around 2–4 ns). Therefore, the threshold or saturation intensities required to inhibit half of the fluorescence are in the range of I _S = 1–10 MW/cm² (Fig. 10a). As mentioned in Section 2.1.6, a preferred implementation of STED is the use of pulsed lasers supplying high peak intensities. This STED modality provides an exponential, i.e. a very steep, dependence of the fluorescence inhibition on the STED laser, realizing a very sharp confinement of fluorescence emission (Harke et al. Reference Harke, Keller, Ullal, Westphal, Schoenle and Hell2008a; Hell, Reference Hell2009b). Inhibition of fluorescence by stimulated emission can be realized with basically any fluorophore. The spatial resolution of the STED concept is in principle limited only by the size of the quantum system to be imaged, i.e. by the molecule. (Section 2.3).

2.2.2 GSD: transient dark state shelving

GSD imaging was the second far-field nanoscopy concept concretely laid out (Hell & Kroug, Reference Hell and Kroug1995). In GSD, inhibition of fluorescence emission is realized by transiently shelving the fluorophore in a metastable dark state such as the triplet (Kasha, Reference Kasha1950) or other dark (redox) states populated therefrom (e.g. Vogelsang et al. Reference Vogelsang, Kasper, Steinhauer, Person, Heilemann, Sauer and Tinnefeld2008; Zondervan et al. Reference Zondervan, Kulzer, Orlinskii and Orrit2003). Since the lifetime of the involved dark states is usually much longer (μs to s) than that of the S ₁ (ns), GSD allows using much lower intensities than STED, in the range of 100–300 kW/cm² (Fig. 10b) (Bretschneider et al. Reference Bretschneider, Eggeling and Hell2007; Hell & Kroug, Reference Hell and Kroug1995). Since both dark state shelving and fluorescence excitation occur via S ₁, i.e. most efficiently by the same laser line, a pump-probe scheme has been introduced in the experimental realization of GSD nanoscopy (Bretschneider et al. Reference Bretschneider, Eggeling and Hell2007). Here, the pump beam is arranged with one or several zero intensity points (such as the doughnut-shaped intensity distribution) and essentially prepares the observation area by restricting molecules that are left in the bright S ₀–S ₁ system to sub-diffraction sized volumes. A subsequent conventional, diffraction-limited fluorescence excitation beam probes these residual bright molecules. Super-resolution images are again realized by scanning the resulting nanoscopic observation spot over the sample, as depicted for immunolabeled SNAP-25 protein clusters on a cell membrane in Fig. 10b. The silenced fluorophores have to return to the S ₀–S ₁ system before each scanning step, which puts a lower limit on the image acquisition time (e.g. pixel dwell times of 60 ms in Fig. 10b). The implementation of the GSD concept is further challenged by the increased involvement of the dark states in photobleaching. Therefore, the experimental realization of GSD had to discover conditions for which (1) the dark state lifetime is within a reasonable range (1–100 ms) to minimize laser intensity for optical pumping but also to reduce pixel dwell and thus image acquisition times, and (2) photobleaching is low enough to allow a reasonable number of pump-probe cycles per fluorophore (Bretschneider et al. Reference Bretschneider, Eggeling and Hell2007). This has, for example, been realized using special mounting media (Bretschneider et al. Reference Bretschneider, Eggeling and Hell2007) or nitrogen temperatures (Schwentker, Reference Schwentker2007) increasing both the dark state lifetime as well as the photostability. On the other hand, it was shown that the addition of UV or IR light after probing may shorten the return time to the S ₀–S ₁ system by reverse dark state/intersystem crossing (Giske, Reference Giske2007; Ringemann et al. Reference Ringemann, Schönle, Giske, Von Middendorff, Hell and Eggeling2008), introducing an additional degree of freedom for the optimization of GSD data acquisition (Schwentker, Reference Schwentker2007). A large list of fluorophores was introduced as potential labels for GSD nanoscopy (Bretschneider et al. Reference Bretschneider, Eggeling and Hell2007). Recently, GSD was realized with NV centers in diamond, featuring down to 12 nm spatial resolution (compare Fig. 11b) (Han et al. Reference Han, Kim, Eggeling and Hell2010). GSD nanoscopy will benefit from the exploration of fluorophores and mounting or buffer conditions which realize large and controllable dark-state populations (Kolmakov et al. Reference Kolmakov, Belov, Bierwagen, Ringemann, Mueller, Eggeling and Hell2010a; Vogelsang et al. Reference Vogelsang, Kasper, Steinhauer, Person, Heilemann, Sauer and Tinnefeld2008). For example, special redox buffers have allowed the controlled and efficient photoswitching of conventional organic dyes by promoting the population of their dark states as well as their photostability (Vogelsang et al. Reference Vogelsang, Kasper, Steinhauer, Person, Heilemann, Sauer and Tinnefeld2008). Figure 10b on the other hand depicts GSD imaging in aqueous environment, where fluorophores usually exhibit dark state lifetimes in the range of μs. The image has been recorded with an organic dye which is characterized by a high intersystem crossing rate, i.e. a high triplet state yield (Chmyrov et al. Reference Chmyrov, Arden-Jacob, Zilles, Drexhage and Widengren2008) and using somewhat larger laser intensities (Bretschneider et al. Reference Bretschneider, Eggeling and Hell2007). The use of dark states that are normally involved in bleaching pathways has however so far hampered the practicability of GSD for (live-) cell nanoscopy.

Fig. 11.

The resolution of coordinate-targeted nanoscopy is in principle limited only by the size of the quantum system (molecule or emitter) to be distinguished: imaging nitrogen-vacancy (NV) centers in diamond. (a) STED imaging of NV centers. (Upper left) NV centers (inset: molecular structure) are a nearly perfect switch: fluorescence inhibition versus STED laser intensity. (Upper middle) STED images of single isolated NV centers in bulk diamond (inset: scanning image, with confocal counterpart in the upper left corner) reveal down to 6 nm spatial resolution (or diameters of the observation area) with increasing STED laser intensity. Scale bar: 100 nm. (Upper right) Scanning image of a single isolated NV center (fast axis along y) with STED laser switched on only in the marked area, exemplifying the vast increase in spatial resolution from confocal 225 nm down to 8 nm. Scale bar: 100 nm. (Lower left) Repetitive STED images of single isolated NV centers in diamond (frame number in the upper right corner), showing the ultimate stability of these emitters. Scale bar: 200 nm. (Lower right) Confocal (left) and STED (right) images of 35 nm large NV-containing nanodiamonds. Scale bar: 100 nm. Adapted from (Han et al. Reference Han, Willig, Rittweger, Jelezko, Eggeling and Hell2009; Rittweger et al. Reference Rittweger, Han, Irvine, Eggeling and Hell2009a). (b, c) GSD nanoscopy of NV centers. (b) GSD by reversible laser-driven transitions into a long-lived dark state. (Upper panel) Energy level diagram of an NV center with ground ³A, excited ³E and dark states, transitions driven by red (excitation Exc and OFF-switching) and blue light (ON-switching), spontaneous fluorescence emission (Flu) and dark state return (dashed line, >100 s lifetime). (Lower panel) Spatial resolution versus power of the red laser driving the OFF transition, as determined by scanning images of isolated NV centers and respective intensity line profiles (insets, scale bar is 100 nm). Adapted from (Han et al. Reference Han, Kim, Eggeling and Hell2010). (c) GSD by saturated excitation. (Upper left panel) Energy level diagram of an NV center with ³A (OFF) and ³E (ON) states, and excitation by green light (Exc) and spontaneous fluorescence emission (Flu). (Upper right panel) Saturation of fluorescence signal: dependence of fluorescence signal of a single NV center on the power of the excitation laser. (Middle panel) Experimental setup with green 532 nm excitation laser, phase plate (PM) generating the doughnut-shaped intensity distribution (inset), dichroic mirror (DC), fluorescence signal (orange), and detector (Det). (Lower panels) Scanning images of a single NV center for increasing intensity of the excitation laser (left to right), depicting the confinement of the area in which no fluorescence is elicited. Adapted from (Rittweger et al. Reference Rittweger, Wildanger and Hell2009b). Scale bar: 50 nm.

2.2.3 SPEM/SSIM: GSD via saturated excitation

Saturated Patterned Excitation Microscopy (SPEM) (Heintzmann et al. Reference Heintzmann, Jovin and Cremer2002) or Saturated Structured Illumination Microscopy (SSIM) (Gustafsson, Reference Gustafsson2005) also depletes the ground state, as in GSD. It differs from GSD (or STED) in that it confines the dark state S ₀ rather than the emitting state S ₁, thus creating sub-diffraction sized dark regions that are surrounded by bright areas. Scanning of such spots consequently produces ‘negative data’ and the final ‘positive’ images have to be reconstructed computationally (Gustafsson, Reference Gustafsson2005). The intensities necessary to create dark regions of sub-diffraction sized extent, i.e. to efficiently deplete S ₀ and saturate the population of S ₁ are of similar magnitude as in the STED concept, because both rely on the same states. Introduced theoretically a decade ago and also promising in principle molecular resolution (Heintzmann et al. Reference Heintzmann, Jovin and Cremer2002), SPEM/SSIM has so far experimentally been realized once with fluorescent beads, displaying a lateral resolution of 50 nm (Gustafsson, Reference Gustafsson2005). The limitations of this technique result from the use of very large excitation intensities, which cause enhanced photobleaching from higher excited (dark) states, as well as an optically induced depopulation of dark states, which prevents saturation of the S ₁ (Giske, Reference Giske2007; Ringemann et al. Reference Ringemann, Schönle, Giske, Von Middendorff, Hell and Eggeling2008; Schwentker, Reference Schwentker2007). The aforementioned SPEM/SSIM experiment therefore implemented a D-/T-Rex-like illumination scheme, avoiding significant populations of long-lived dark states using a 5 kHz laser for excitation (Gustafsson, Reference Gustafsson2005). In other work, saturated excitation of ultra-stable NV centers in diamond yielded single-spot scanning GSD images with a spatial resolution of <10 nm (compare Fig. 11c) (Han et al. Reference Han, Wildanger, Rittweger, Meijer, Pezzagna, Hell and Eggeling2012; Rittweger et al. Reference Rittweger, Wildanger and Hell2009b).

2.2.4 RESOLFT: reversible photoswitchable labels

All of the above approaches may be summarized under a general name, RESOLFT nanoscopy (Hell, Reference Hell2004; Hell et al. Reference Hell, Jakobs and Kastrup2003, Reference Hell, Dyba and Jakobs2004). The realization of STED or GSD led to the consideration of molecular switches between states of very long lifetimes (even longer than the metastable dark states introduced in the GSD concept). The advantage is obvious: the utilization of states with very long lifetimes τ (or even the elimination of spontaneous transitions) would allow applying very low laser intensities for driving a fluorophore to a certain state (Hell et al. Reference Hell, Jakobs and Kastrup2003). Reversible photoswitching between states with very long τ can be realized through changes in molecular conformations. A prominent example is photoinduced cis–trans isomerization involving fluorescent and dark (or non-detectable) isomeric counterparts, which can be switched back and forth by light of different wavelengths. Cis–trans isomerization is for example known for cyanine dyes. The lifetime of these states for cyanines is however τ < μs in aqueous solution (Widengren & Schwille, Reference Widengren and Schwille2000), impeding the move to very low intensities. Isomeric states may be stabilized (and τ prolonged) for other molecules such as reversible switchable fluorescent proteins (RSFPs) or spiro-compounds, and/or by fixation in rigid environments (Ando et al. Reference Ando, Mizuno and Miyawaki2004; Bossi et al. Reference Bossi, Foelling, Dyba, Westphal and Hell2006; Dickson et al. Reference Dickson, Cubitt, Tsien and Moerner1997; Feringa, Reference Feringa2001; Irie et al. Reference Irie, Fukaminato, Sasaki, Tamai and Kawai2002; Lukyanov et al. Reference Lukyanov, Fradkov, Gurskaya, Matz, Labas, Savitsky, Markelov, Zaraisky, Zhao, Fang, Tan and Lukyanov2000; Sakata et al. Reference Sakata, Yan and Marriot2005). In RSFPs the differently isomerized states are usually stabilized by the protein barrel surrounding the chromophore (and often involve differently protonated states) (Andresen et al. Reference Andresen, Wahl, Stiel, Grater, Schafer, Trowitzsch, Weber, Eggeling, Grubmuller, Hell and Jakobs2005, Reference Andresen, Stiel, Trowitzsch, Weber, Eggeling, Wahl, Hell and Jakobs2007; Habuchi et al. Reference Habuchi, Dedecker, Hotta, Flors, Ando, Mizuno, Miyawaki and Hofkens2006; Wilmann et al. Reference Wilmann, Petersen, Devenish, Prescott and Rossjohn2005). It was therefore an RSFP, asFP595 (Lukyanov et al. Reference Lukyanov, Fradkov, Gurskaya, Matz, Labas, Savitsky, Markelov, Zaraisky, Zhao, Fang, Tan and Lukyanov2000), that was first used to experimentally prove the viability of the general RESOLFT concept in 2005 (Hofmann et al. Reference Hofmann, Eggeling, Jakobs and Hell2005). asFP595 can be reversibly switched between a dark trans and a bright cis state with light of 405–460 and 560 nm, respectively, and the thermal lifetimes of τ > ms in aqueous solution enabled for the first time the use of ultra-low intensities (<kW/cm²) for super-resolution microscopy. Similar sub-diffraction images were realized when using other RSFPs such as Dronpa (Ando et al. Reference Ando, Mizuno and Miyawaki2004; Bock, Reference Bock2008; Dedecker et al. Reference Dedecker, Hotta, Flors, Sliwa, Uji-I, Roeffaers, Ando, Mizuno, Miyawaki and Hofkens2007; Hofmann, Reference Hofmann2007), or when switching between an open and a closed form of a photochromic organic compound of the furyl-fulgides family in a polymeric matrix (Bossi et al. Reference Bossi, Foelling, Dyba, Westphal and Hell2006). In the latter, the difference in fluorescence signal between the two forms had been realized by the photochromic compound serving as a reversible switchable energy acceptor for a fluorescent compound. Unfortunately, the uses of both asFP595 and the photochromic organic compound for cellular RESOLFT nanoscopy were limited. First of all, specific labeling with either was not feasible, since asFP595 was only present in a tetrameric form, and the organic compound could not successfully be functionalized. Furthermore, under the final imaging conditions the signal could not be switched off completely due to cross-talk between laser lines (e.g. >30% of the ON-state fluorescence in the case of asFP595), requiring computational post-processing of the recorded image (through, e.g. deconvolution) in order to fully extract the sub-diffraction image contents – a procedure which can be prone to noise and biases. In addition, the switching speed was rather slow, entailing scanning dwell times of >100 ms. Finally, and most importantly, both labels exhibited considerable switching fatigue. Therefore, deliberate screening for fast and high-contrast switching monomeric RSFPs which should hardly show spontaneous decay of the involved states and, most importantly, survive >1000 and more photoswitching cycles was performed (Stiel et al. Reference Stiel, Trowitzsch, Weber, Andresen, Eggeling, Hell, Jakobs and Wahl2007). This screen ended in a RSFP variant of GFP (rsGFP: reversible switchable GFP), and its use in RESOLFT nanoscopy realized down to 40 nm spatial resolution in live-cell imaging (Grotjohann et al. Reference Grotjohann, Testa, Leutenegger, Bock, Urban, Lavoie-Cardinal, Willig, Eggeling, Jakobs and Hell2011). Similarly, the RSFP Dreiklang was created and applied to RESOLFT imaging, where suitable wavelengths for on and off switching and fluorescence excitation were completely disentangled, minimizing any action cross-talk (Brakemann et al. Reference Brakemann, Stiel, Weber, Andresen, Testa, Grotjohann, Leutenegger, Plessmann, Urlaub, Eggeling, Wahl, Hell and Jakobs2011). Applying laser intensities of only 1 kW/cm², the RESOLFT concept applying RSFPs is highly suited for live-cell applications. RESOLFT nanoscopy has in the meantime enabled the recording of live-cell dynamics even in 3D (Testa et al. Reference Testa, Urban, Jakobs, Eggeling, Willig and Hell2012). Further improvements have come with the development of RSFPs with faster photoswitching times and decreased switching fatigue (Grotjohann et al. Reference Grotjohann, Testa, Reuss, Brakemann, Eggeling, Hell and Jakobs2012), as well as with additional emission wavelength ranges (Stiel et al. Reference Stiel, Andresen, Bock, Hilbert, Schilde, Schönle, Eggeling, Egner, Hell and Jakobs2008) for multi-color observations. Phototoxic effects of the (often UV) photoswitching light may be avoided by the use of IR light in a two-photon mode (Denk, Reference Denk1996).

2.2.5 Parallelization

The image acquisition in coordinate-targeted nanoscopy can be accelerated significantly by employing multiple observation spots simultaneously. As already outlined in Section 2.1.6, this has been realized in STED nanoscopy with four parallel scanning spots using Wollaston prisms (Bingen et al. Reference Bingen, Reuss, Engelhardt and Hell2011), or 100 or even 2000 parallel spots using optical standing wave patterns (Yang et al. Reference Yang, Przybilla, Mestre, Trebbia and Lounis2014; Bergermann et al. Reference Bergermann, Alber, Sahl, Engelhardt and Hell2015). Unfortunately, the laser intensities required for STED are still too high to allow further parallelization with currently available laser powers. The required average laser intensities in GSD – 10⁵-fold lower compared to STED – allowed the use of larger numbers of intensity zeros in parallel (Schwentker, Reference Schwentker2007). SSIM has so far been experimentally shown with structured illumination, i.e. with a multitude of zero-intensity lines (Gustafsson, Reference Gustafsson2005). The high laser intensities were delivered by a laser with large transient pulse peak powers but very low repetition rate, which again slows down the image acquisition process and makes live-cell imaging less feasible. This restriction has been lifted by employing RSFPs: the ultra-low intensities required for the RESOLFT concept allowed scanning with several intensity zeros and thus several sub-diffraction sized observation areas in parallel for live-cell nanoscopy (Rego et al. Reference Rego, Shao, Macklin, Winoto, Johansson, Kamps-Hughes, Davidson and Gustafsson2012; Schwentker et al. Reference Schwentker, Bock, Hofmann, Jakobs, Bewersdorf, Eggeling and Hell2007). The combination of RESOLFT and structured illumination therefore promises <50–60 nm with sub-second image acquisition times of >50 × 50 μm² fields of view (Rego et al. Reference Rego, Shao, Macklin, Winoto, Johansson, Kamps-Hughes, Davidson and Gustafsson2012). A more advanced illumination pattern than using parallel lines of high intensities as in conventional SIM is the scanning with thousands of parallelized points as in spinning-disc microscopy (McCabe et al. Reference Mccabe, Fewer, Ottewill, Hewlett and Hegarty1996) or in multifocal SIM (York et al. Reference York, Parekh, Nogare, Fischer, Temprine, Mione, Chitnis, Combs and Shroff2012). Similarly, more than 100 000 doughnuts were generated simultaneously for ultra-fast live-cell RESOLFT imaging of large fields of view with down to 70 nm spatial resolution (Chmyrov et al. Reference Chmyrov, Keller, Grotjohann, Ratz, D'Este, Jakobs, Eggeling and Hell2013) (Fig. 10d). With the development of further optimized photoswitchers, RESOLFT is therefore a very promising tool for revolutionizing live-cell optical nanoscopy.

2.2.6 Nanoscale writing

Concurrently to the imaging of various nanostructures, the RESOLFT concept was extended to the writing of structures with sub-diffraction size and spacing using visible light. Proposed in the early times (Hell, Reference Hell2004), writing of nanostructures based on the STED and RESOLFT concept has been experimentally realized using photochromic materials (Andrew et al. Reference Andrew, Tsai and Menon2009; Fischer et al. Reference Fischer, Freymann and Wegener2010; Harke et al. Reference Harke, Bianchini, Brandi and Diaspro2012; Li et al. Reference Li, Gattass, Gershgoren, Hwang and Fourkas2009a; Scott et al. Reference Scott, Kowalski, Sullivan, Bowman and Mcleod2009; Wiesbauer et al. Reference Wiesbauer, Wollhofen, Vasic, Schilcher, Jacak and Klar2013; Wollhofen et al. Reference Wollhofen, Katzmann, Hrelescu, Jacak and Klar2013) or RSFPs (Grotjohann et al. Reference Grotjohann, Testa, Leutenegger, Bock, Urban, Lavoie-Cardinal, Willig, Eggeling, Jakobs and Hell2011). Analogously to imaging sub-diffraction features, an intensity distribution of a photoswitching laser exhibiting one or several intensity zeros is used to maintain molecules in a reactive ON-state (i.e. in a writable state that, in contrast to the OFF-state, can be transferred to a permanent state) only at the sub-diffraction spots defined by the zeros. Scanning then realizes writing of nanostructures. RESOLFT has thus evolved to a versatile concept to reach the nanoscale in far-field optical applications.