For decades it was assumed that the diffraction of light meant that optical microscopy could not resolve features that were smaller than about the half the wavelength of the light being used to create an image. However, various ‘super-resolution’ methods have allowed researchers to overcome this diffraction limit for fluorescence imaging, which is the most popular form of microscopy used in the life sciences. This approach involves tagging the biomolecules of interest with fluorescent molecules, such as green fluorescent protein (GFP), so that they can be identified in cells. An excitation laser then drives the fluorescent molecule, which is also known as a fluorophore, into an excited state: after a short time, the fluorophore can return to its ground state by releasing a fluorescence photon. Images of the sample are built up by detecting these photons.

In STED super-resolution microscopy a second laser is used to instantly send the molecules from their excited or ‘on’ states back to their ground or ‘off’ states before any fluorescence can occur. The second laser beam is usually shaped like a doughnut, with a small region of low light intensity surrounded by a region of much higher intensity. STED microscopy is able to beat the diffraction limit because the second laser turns all the fluorophores ‘off’ except those in the small sub-wavelength region at the centre of the doughnut. The image is build up by scanning both lasers over the sample so that the small region in which the fluorophores are ‘on’ probes the entire cell.

RESOLFT is a similar technique that employs fluorescent molecules with ‘on’ and ‘off’ times that are much longer than those used in STED microscopy. In particular, RESOLFT uses fluorescent molecules that can be rapidly switched back and forth between long-lived ‘on’ and ‘off’ states many times by the two lasers. The fact that both these states are long-lived states means that RESOLFT requires much lower laser intensities than STED, which makes it attractive for imaging biological samples over large areas or long times.

RESOLFT demonstrated its suitability for bioimaging for the first time last year, with a protein called rsEGFP (reversibly switchable enhanced GFP) being employed as the fluorophore. However, the time needed to switch this protein between the ‘on state’ and the ‘off state’ was relatively long, and it took about an hour to record a typical image. Now, Grotjohann et al. have modified this protein to make a new fluorophore called rsEGFP2 with a shorter switching time, and have used it to image various structures—including Vimentin, a protein that forms part of the cytoskeleton in many cells, and organelles called peroxisomes—inside live mammalian cells. They were able to record these images some 25–250 times faster than would have been possible with previous RESOLFT approaches. The combination of RESOLFT and rsEGFP2 should allow researchers to image a wide variety of structures and processes in living cells that have not been imaged before.

While for many decades lens-based optical microscopy could not resolve features finer than half the wavelength of light (λ/2 > 200 nm), the recent development of fluorescence nanoscopy or super-resolution imaging methods boosted its resolution potential (Hell, 2003, 2009; Huang et al., 2010). In all these methods, the diffraction barrier is overcome by employing a fluorophore transition between two states, typically a fluorescent on- and a non-fluorescent off-state (Hell, 2009) in order to separate neighboring features. The method called RESOLFT differs from stochastic single fluorophore on-off-switching methods by the fact that a doughnut or a line pattern is scanned across the sample, determining at any point in time the nanosized coordinate range where the fluorophores are in the on-state. Using long lifetimes of the on- and off-states reduces the light intensities required for optical switching by orders of magnitude over the related STED approach, making RESOLFT attractive for extended live-cell or large area imaging.

Although it was suggested almost a decade ago (Hell, 2003; Hell et al., 2003) and its viability shown in principle (Hofmann et al., 2005; Dedecker et al., 2007; Schwentker et al., 2007) the wider applicability of RESOLFT nanoscopy to biological imaging has been demonstrated only recently (Brakemann et al., 2011; Grotjohann et al., 2011; Rego et al., 2012). The reason is that fluorescent protein based RESOLFT critically relies on reversibly switchable fluorescent proteins (RSFPs) providing many on-off cycles, such as the recently designed rsEGFP (Grotjohann et al., 2011). Unfortunately, in these demonstrations, the still relatively slow switching kinetics of rsEGFP entailed pixel dwell times of 10–20 ms at switching light intensities of ~1 kW/cm². Hence images of 10 µm × 10 µm in size and a pixel step size of 20 nm required recording times of about an hour. In this study we describe the generation, characterization and application of a novel RSFP, namely rsEGFP2, which facilitates faster switching and, together with modifications of the initial switching scheme, enables 25–250 faster RESOLFT recordings in living cells.

We observed that already the exchange of a single amino acid in the chromophore of the photostable and widely-used enhanced green fluorescent protein (EGFP) (Patterson et al., 1997; Tsien, 1998), namely replacing threonine 65 by alanine, transformed EGFP into a fast switching RSFP, which exhibited a ‘negative switching mode’. ‘Negative switching’ RSFPs switch from the on- into the off-state at the wavelength, here ~480 nm, that is used to elicit fluorescence. The switching from the off-state into the on-state is achieved by irradiation with a different wavelength, here ~405 nm. Protein solutions of EGFP(T65A) exhibited a high residual fluorescence (~50% of the on-state signal) when the protein solution was switched into the off-state. However, analysis of EGFP(T65A) demonstrated fast switching and low switching fatigue, indicating that the different chromophore (an Ala-Tyr-Gly chromophore instead of a Thr-Tyr-Gly chromophore) might provide additional possibilities for the generation of fast switching RSFPs. Hence, we also introduced the mutation A206K to ensure that the protein is a true monomer (Zacharias et al., 2002) and introduced, alone or in combination, the four mutations that discriminate rsEGFP from EGFP. We screened this collection of EGFP variants for those exhibiting a high resistance against switching fatigue in combination with fast switching at light intensities previously used for RESOLFT (few kW/cm²). We found that introducing two mutations was sufficient to transform EGFP(T65A, A206K) into a novel RSFP with improved properties when compared to rsEGFP (Figure 1). We named this new variant (EGFP[T65A, Q69L, V163S, A206K]) (Figure 1—figure supplement 1) reversibly switchable EGFP2 (rsEGFP2). Note that rsEGFP has a TYG chromophore, whereas rsEGFP2 has an AYG chromophore.

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Adopting the fluorescent state at equilibrium, rsEGFP2 has its fluorescence excitation and emission maximum at 478 nm and 503 nm, respectively (Figure 1A). In the fluorescent state, a solution of rsEGFP2 exhibits an extinction coefficient of ε ≈ 61,000 M⁻¹cm⁻¹ and a fluorescence quantum yield of Φ_Fl = 0.3 (Table 1). Hence the brightness (given by ε Φ_Fl) of rsEGFP2 is similar to that of rsEGFP and about 60% of that of EGFP at low light intensity irradiation. Single-molecule spectroscopy measurements performed at higher light intensities (5 to 100 kW/cm²) showed that the single molecule brightness of rsEGFP and rsEGFP2 were ~52% and ~44% of that of EGFP, respectively (Figure 1—figure supplement 2). The slight difference between brightness values determined at low and high light intensities may be attributed to the increased population of unidentified dark states at higher intensities. rsEGFP2 exhibits a ‘negative’ switching mode, that is irradiation with light of around 480 nm induces fluorescence and, in a competing process, switches rsEGFP2 off. Subsequent irradiation with light of around 405 nm switches the protein back from the off- into the on-state (Figure 1B). Irradiation of a purified rsEGFP2 solution with light of ~480 nm leads to a decrease of the 483 nm absorption band, which presumably corresponds to the anionic (de-protonated) cis-chromophore, and to the onset of an absorption band peaking at 408 nm, presumably corresponding to the neutral (protonated) trans-chromophore state (Andresen et al., 2005; Andresen et al., 2007) (Figure 1C). The single molecule brightness, that is the number of emitted photons per time unit, of rsEGFP and rsEGFP2 are similar. However, because a single switching cycle of rsEGFP2 is on average shorter than a single switching cycle of rsEGFP, rsEGFP2 emits fewer photons in a single cycle than rsEGFP. The pK_a of the chromophore in the thermal equilibrium state is ~5.8 (Figure 1D). Hence the pK_a of rsEGFP2 is lower by 0.7 pH units than the pK_a of rsEGFP. As reported previously for similar RSFPs, the light driven switching of rsEGFP2 is likely due to a cis/trans isomerization of the chromophore, accompanied by a change of the chromophoric protonation state (Andresen et al., 2005; Andresen et al., 2007; Bourgeois and Adam, 2012).

The total number of switching cycles before bleaching is critical for the usability of a RSFP for RESOLFT microscopy (Hell, 2009). To compare rsEGFP2 with rsEGFP in this regard, we immobilized purified proteins in polyacrylamide layers (PAA). Applying light intensities that have previously been used for RESOLFT (few kW/cm²), we recorded the fluorescence during 6,000 on-off switching cycles (Figure 1E). The illumination times were adapted for the two proteins such that the signal reached a maximum or minimum in each cycle. We found that under these conditions the fluorescence was halved not before ~1,100 (rsEGFP) and ~2,100 (rsEGFP2) cycles, demonstrating that rsEGFP2 accommodates even more switches than rsEGFP.

In most negative switching RSFPs, including rsEGFP, the ensemble off-switching with blue light is slower by 2–3 orders of magnitude, compared to the on-switching with UV light at comparable light intensities, rendering the off-switching the time-limiting step in RESOLFT microscopy. We compared the off-switching half-time (time after which the fluorescence signal is reduced to 50%) of rsEGFP2 and rsEGFP as a function of the off-switching light intensity (491 nm) (Figure 1F). We found that the difference in the off-switching halftimes of rsEGFP2 and rsEGFP depends on the light intensities applied. The lesser the light intensities, the more pronounced is the speed advantage of rsEGFP2. At a light intensity of 5.5 kW/cm² the off-switching of rsEGFP2 was ~6.5 times faster than the off-switching of rsEGFP (Figure 1F, inset). Presumably the switching speed advantage for rsEGFP2 is even higher at lower intensities. For undesirable intensities of >100 kW/cm², the differences between rsEGFP and rsEGFP2 are negligible (Figure 1F, inset). These measurements also revealed that the level to which the fluorescence intensity of an ensemble of rsEGFP2 proteins can be reduced (the off-state fluorescence), depends on the intensities for off-switching (Figure 1—figure supplement 3). Hence the light intensities and the irradiation times used for off-switching influence both the switching speed as well as the lowest residual fluorescence. Furthermore, we observed that the buffer conditions, most notably the pH, and the cellular environments also influence the absolute switching speed, potentially requiring adaptations of the illumination protocol to the observed samples.

In mammalian cells, rsEGFP2 can be fused to histone H2B and alpha-tubulin, which require a truly monomeric fusion tag (Figure 2). Corroborating its monomeric nature, it migrates on semi-native gels in a single band of the expected size (Figure 2—figure supplement 1). rsEGFP2 maturates in vitro at 37°C with a halftime of ~20 min, which is shorter or comparable to most conventional fluorescent proteins (Shaner et al., 2008) including mCherry (15 min), tdTomato (60 min) or TagRFP (100 min). Its maturation time is superior to both rsEGFP (3 hr) (Grotjohann et al., 2011) and a maturation-improved Dronpa(M159T) variant (Stiel et al., 2007; Willig et al., 2011) (~50 min). Presumably because of its fast maturation time and due to the fact that its linker is identical to that of EGFP (which has been proven to be very suitable for the generation of fusion proteins) rsEGFP2 is well suited to tag proteins in living mammalian cells (Figure 2).

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The possibility to switch rsEGFP2 faster than rsEGFP, its higher switching stamina, and its faster maturation kinetics, suggested that this protein would be superior for fast live-cell RESOLFT imaging. We first expressed rsEGFP2 fused to the C-terminus of Vimentin in mammalian PtK2 (Potorous tridactylis) cells. RESOLFT imaging was performed with a home-built setup maintaining the cells at 35°C. Because the filaments formed by Vimentin-rsEGFP2 are relatively immobile, we selected an irradiation scheme encompassing low switching light intensities in combination with extended irradiation times. The living cells were imaged pixel-by-pixel, by first irradiating with 405 nm light (2 kW/cm^²) for 40 µs to switch most proteins into their fluorescent on-state. After a short illumination break of 10 µs, the doughnut shaped 491 nm beam (10 kW/cm^², 300 µs) was used to switch rsEGFP2 into the off-state, confining the on-state to the doughnut center. Finally, the rsEGFP2 fluorescence was probed for 30 µs by irradiation with 491 nm light (38 kW/cm^²). Hence the pixel dwell time was 380 µs, which is 25- to 50-fold faster than previously reported using rsEGFP on similar structures (Grotjohann et al., 2011). To enhance the image contrast, we also employed Richardson-Lucy restoration (Richardson, 1972; Hofmann et al., 2005). Movements of Vimentin-filaments in a living cell were recorded in 10 µm × 10 µm frames of 20 nm pixel size, every 100 s (Figure 3a; Figure 3—figure supplement 1; Movie 1). After the 20th image, the overall fluorescence still amounted to 65% of its initial value, underscoring the bleaching resistance of rsEGFP2.

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To determine the obtainable resolution, we imaged cells expressing Keratin19-rsEGFP2 filaments using similar illumination conditions as before. Evaluating the intensity profiles of these filaments in the focal plane revealed a resolution <50 nm in raw image data (Figure 3B).

Next, we targeted rsEGFP2 to the lumen of the endoplasmic reticulum (ER). Since the ER is a fast moving structure we increased the recording speed further by doubling the light intensities (on: 405 nm, 4 kW/cm^², 20 µs; off: 491 nm, 20 kW/cm^², 50 µs; read-out: 491 nm, 76 kW/cm^², 5 µs) thus cutting the pixel dwell time down to 75 µs, which is ~250 times faster than reported previously using rsEGFP. We did not introduce an irradiation break within a single switching cycle. Because we aimed at capturing fast movements, we also increased the pixel size to 40 nm × 40 nm, thus covering a field of 100 µm² in 5.9 s (Figure 3C). Repeated imaging revealed fast changes occurring in the range of seconds in the highly interconnected ER. To visualize movements of the ER in the sub-second range, we reduced the field of view to ~9 µm² corresponding to 0.5 s frame times. Figure 3D shows six RESOLFT images out of a time lapse movie containing 100 images (Movie 2) revealing changes in the ER structure occurring in <1 s. Since the ER-tubules are too large to determine the obtained resolution, we imaged again Keratin19-rsEGFP2 expressing cells using a similar dwell time and established <90 nm in raw data (Figure 3—figure supplement 2), meaning that speed was obtained at the expense of resolution.

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Likewise, when rsEGFP2 was fused to the protein Pex16 to highlight the peroxisomes in living PtK2 cells, we could follow the movement of individual peroxisomes in the RESOLFT-mode in a 3 µm × 2 µm field of view at 2 Hz (Figure 3E; Movie 3). Since the fluorescent proteins are confined to individual peroxisomes and do not diffuse over large distances, labeled peroxisomes are well suited for a direct comparison of rsEGFP2 and rsEGFP bleaching. The comparison showed that the overall photobleaching of rsEGFP2 was substantially lower than that of rsEGFP when the above fast RESOLFT imaging conditions were applied (Figure 3—figure supplement 3).

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Phototoxicity is a major concern in (superresolution) fluorescence microscopy of living cells. Among the established methods, STED-microscopy entails relatively large intensities, (5–200 MW/cm²), depending on the desired resolution. However, since the wavelengths (560–800 nm) used for STED are in the comparatively benign long-wavelength part of the spectrum (Hell, 2009), STED microscopy can image living cells and tissues, including neurons in the cerebral cortex of a living mouse (Berning et al., 2012). In the RSFP-based RESOLFT microscopy demonstrated here, the employed light intensities (1–80 kW/cm²) are several orders of magnitude lower than in STED-microscopy and comparable to those used in live-cell confocal fluorescence microscopy. Stochastic single-molecule based approaches, such as the methods called PALM (Manley et al., 2008; Shroff et al., 2008), STORM (Jones et al., 2011; Shim et al., 2012), and GSDIM (Fölling et al., 2008), typically use similar light intensities for imaging living cells (0.1–100 kW/cm^² depending on the exposure time or camera frame rate), but they apply these intensities (i.e. temporal and spatial photon densities) continuously to all points in the imaged area. Since our RESOLFT approach has been implemented as a point-scanning system, the intensities employed are applied only for a brief duration on a small, sub-micrometer sized region of the imaged area. Hence any pixel is only illuminated during a small fraction of the recording time of the image. In the stochastic methods, the whole imaged area is irradiated for the entire time of recording, that is for a couple of seconds or minutes. Therefore, in the RESOLFT microscopy demonstrated here, the total light dose impinging on the cell is lower by 3–4 orders of magnitude compared to the stochastic single-molecule based approaches. Concretely, for recording the shown RESOLFT images, 2–10 J/cm^² were applied for switching into the on-state, and 25–300 J/cm^² for eliciting fluorescence and switching the protein off. PALM live-cell imaging reportedly requires light doses of 1,000–100,000 J/cm^² for on-switching with UV (405 nm) light and 25,000–300,000 J/cm^² for fluorescence excitation (Manley et al., 2008; Shroff et al., 2008). Live-cell GSDIM experiments using a yellow fluorescent protein required even larger irradiation doses (100,000–900,000 J/cm^²) (Fölling et al., 2008; Testa et al., 2010). Although RESOLFT microscopy may induce phototoxicity after extended exposure, similarly to live-cell confocal microscopy, it currently is the superresolution method entailing the lowest light dose. Therefore, perhaps not surprisingly, RESOLFT microscopy has been used to image neurons in living organotypical hippocampal cultures over several hours without noticeable photodegradation (Testa et al., 2012). Note that the comparatively low-dose/low-intensity requirement of the RESOLFT concept is due to the fact that it uses long-lived on- and off-states in combination with the fact that it does not require fast emission of many photons for establishing molecular coordinates (localization). Since the applied intensities are largely determined by the on-off switching kinetics, the concept offers ample room for accommodating novel proteins with switching kinetics entailing even lower light doses and intensities.

In conclusion, rsEGFP2 is a bright, monomeric, photostable, quickly maturating, and fast switching alternative to rsEGFP with comparatively low photobleaching. We expect it to outperform other green fluorescent RSFPs (Ando et al., 2007; Stiel et al., 2007) because of its faster maturation and good usability for functional protein tagging. An implementation of quick illumination sequences allowed us to realize up to 250-fold faster recordings as compared to previous reports, thus facilitating live-cell RESOLFT nanoscopy with pixel dwell times down to 70 µs. Since one can adjust both the duration and the illumination intensity, as well as the optical switching scheme, RESOLFT nanoscopy allows one to adapt speed and resolution within a certain range, to the sample needs.

Finally, we note that in the point-scanning scheme used here, the total recording time of the image scales with the area of recording. Parallelization of the scanning procedure with an array of doughnuts or lines (so-called ‘structured illumination’) (Gustafsson, 2005; Schwentker et al., 2007; Rego et al., 2012) overcomes this dependence on the field of view and cuts down the recording time by the degree of parallelization. Owing to the low-light level operation, the degree of parallelization can easily amount up to a factor of 100–1000. For this reason, given the short pixel dwell times attained herein, parallelized RESOLFT versions should enable video-rate nanoscopy across the whole field of view of the objective lens.

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Author details

1. Tim Grotjohann

   Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   TG, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Ilaria Testa

   Competing interests

   No competing interests declared.

2. Ilaria Testa

   Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   IT, Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   Tim Grotjohann

   Competing interests

   No competing interests declared.

3. Matthias Reuss

   1. Abberior Instruments GmbH, Göttingen, Germany
   2. Science for Life Laboratory, Advanced Light Microscopy, Karolinska Institutet, Solna, Sweden

   Contribution

   MR and TB, Acquisition of data

   Competing interests

   MR and SWH: Own shares of the Abberior Instruments GmbH start-up supplying RESOLFT microscopy kits. The latter also benefits through IP secured by the Max Planck Society.

4. Tanja Brakemann

   Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   MR and TB, Acquisition of data

   Competing interests

   No competing interests declared.

5. Christian Eggeling

   Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   CE, Conception and design

   Competing interests

   No competing interests declared.

6. Stefan W Hell

   Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   SWH, Conception and design, Drafting or revising the article

   For correspondence

   shell@gwdg.de

   Competing interests

   MR and SWH: Own shares of the Abberior Instruments GmbH start-up supplying RESOLFT microscopy kits. The latter also benefits through IP secured by the Max Planck Society.

7. Stefan Jakobs

   1. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
   2. Department of Neurology, University of Göttingen, Göttingen, Germany

   Contribution

   SJ, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   sjakobs@gwdg.de

   Competing interests

   No competing interests declared.

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