Photoswitchable Fluorescent Proteins: Do Not Always Look on the Bright Side Karin Nienhaus† and Gerd Ulrich Nienhaus*,†,‡,§,∥ †
Institute of Applied Physics, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany Institute of Nanotechnology, Karlsruhe Institute of Technology, 76344 Eggenstein-Leopoldshafen, Germany § Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76344 Eggenstein-Leopoldshafen, Germany ∥ Department of Physics, University of Illinois at UrbanaChampaign, Urbana, Illinois 61801, United States ‡
ABSTRACT: Photoactivatable fluorescent proteins (FPs) have become essential markers for nanoscopy on live specimens. In this issue of ACS Nano, Wang et al. present a reversibly photoswitching FP, GMars-Q, which they promote as an advanced marker for RESOLFT imaging because of its low residual intensity in the off state and low switching fatigue. Here, we explain the observed peculiar photobleaching behavior of GMars-Q by a mechanism that involves efficient shelving of proteins in dark states, resulting in low switching fatigue and low residual off intensity. There is a continuing demand for novel FP markers with properties optimized for specific imaging techniques. Endeavors to engineer such proteins can greatly benefit from increased efforts to acquire deeper mechanistic understanding of their photophysics and photochemistry. he development of fluorescence markers from the green fluorescent protein (GFP) family is largely driven by the quest for more powerful genetically encoded labeling tools with novel or improved properties for specific imaging applications. Optical fluorescence microscopy plays key roles in the life sciences because it enables researchers to study live specimens in all three spatial dimensions over extended periods of time in a minimally invasive fashion with high sensitivity. Superresolution fluorescence microscopy, or nanoscopy, methods circumvent the Abbe diffraction barrier in ingenious ways and enable optical imaging with essentially unlimited spatial resolution.1 Unlike conventional, diffraction-limited microscopy, these techniques do not rely on wave interference to form the image. Instead, the image is reconstructed from the positions of single fluorophores in random locations (stochastic imaging) or from the fluorescence emission of small ensembles at selected locations (targeted imaging). All fluorescence imaging relies on the availability of bright fluorescent markers with which biological structures can be labeled with high specificity and selectivity. These include synthetic dyes, nanoparticles, and fluorescent proteins (FPs).2 Although FPs are typically less bright and photostable than dyes and nanoparticles, they have become the markers of choice for live imaging because they can be genetically encoded and thus produced by the cell or organism under study. In typical applications, FPs are fused to a protein of interest at the DNA level, and the gene of the fusion construct can be expressed by the cell so that it synthesizes a fusion protein, which often maintains its biological function despite the
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additional marker domain (although this needs to be thoroughly checked).
In this issue of ACS Nano, Wang et al. present GMars-Q, a reversibly photoswitching GFP with advantageous properties for use in reversible saturable optical linear fluorescence transitions (RESOLFT) imaging, a special type of targeted nanoscopy. The development of FPs of the GFP family has been ongoing for more than two decades. After the first demonstration of GFP from the jellyfish Aequoria victoria as a genetically encoded fluorescent marker for live imaging,3 researchers began to modify the photophysical properties of the fluorescent chromophore, 4-(p-hydroxybenzylidene)-5-imidazolinone (pHBI), formed autocatalytically from three adjacent residues in the presence of dioxygen within the barrel-shaped protein, by genetically engineering the amino acids of the fluorophore and of its environment. As a result, more robust, brighter, and wavelength-shifted variants have become available to the microscopy community.4 An enormous expansion of the palette of naturally occurring FPs as marker proteins came with the discovery of FPs in other animal species, notably Published: October 10, 2016 9104
DOI: 10.1021/acsnano.6b06298 ACS Nano 2016, 10, 9104−9108
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anthozoa. Besides the long-sought-after red fluorescent proteins,5 a variety of photoactivatable FPs were discovered6,7 that enable control of the fluorescence emission intensity or wavelength via light irradiation at specific wavelengths. These FPs play essential roles as markers for certain types of nanoscopy, especially for studies of live specimens. Two types of photoactivation are commonly distinguished: (1) irreversible photoconversion, in which the FP marker protein is photochemically modified, resulting in a change from a nonfluorescent to a fluorescent chromophore or a change in the emission wavelength, and (2) reversible photoswitching between a fluorescent and a nonfluorescent state. In this issue of ACS Nano, Wang et al.8 present GMars-Q, a reversibly photoswitching GFP with advantageous properties for use in reversible saturable optical linear fluorescence transitions (RESOLFT) imaging, a special type of targeted nanoscopy (vide inf ra). This system adds yet another variant to the steadily expanding toolbox of interesting FP markers that may enable even more powerful imaging experiments. Targeted approaches place specific demands on the fluorescent markers. First and foremost, they require chromophores that can be switched between fluorescent and nonfluorescent forms many times. Stimulated emission depletion (STED) imaging, the earliest type of nanoscopy,1 utilizes stimulated emission, a physical property intrinsic to all fluorophores, to switch off the excitation. Thus, any fluorescent dye can serve as a STED marker. However, because excited-state lifetimes are short (typically 1−5 ns), a highly intense STED beam is needed to de-excite fluorophores efficiently before they emit light. The generalization of this basic principle, denoted RESOLFT nanoscopy, utilizes chromophores that can be reversibly switched between a fluorescent and nonfluorescent state via light irradiation, for example, reversibly photoswitching FPs.9,10 For optimal performance, thermally activated switching transitions should be so infrequent as to become negligible, so that the depletion beam for RESOLFT can be orders of magnitude less intense than for STED. This principle is depicted in Figure 1. Like raster scanning confocal microscopy (Figure 1a), RESOLFT microscopy also uses a diffractionlimited beam to irradiate the sample. At every location, this beam switches an ensemble of marker chromophores to their fluorescence emitting state (Figure 1b). Subsequently, a precisely spatially overlaid “donut” beam, with its intensity increasing from zero in the center to high values outside, selectively switches off markers in the periphery of the excitation spot, so that only markers within the central region can still emit fluorescence. As a result, an image can be collected with a much sharper scanning probe than in conventional, diffraction-limited confocal microscopy. To achieve high spatial resolution, the probing light needs to be raster-scanned across the sample in much finer steps than in confocal microscopy. As a rule of thumb, a 10-fold resolution improvement requires 10 switching cycles per spatial dimension and thus 102 (103) cycles for imaging in two (three) dimensions. Typical photoswitching FPs can be cycled between on and off states only a few times before they photobleach. However, researchers have made great strides in recent years to generate FP variants that, in RESOLFT imaging, can undergo more than 1000 switching cycles (in an ensemble, not as a single chromophore) prior to complete photobleaching.10 In proteins of the GFP family, reversible photoswitching is usually based on photoinduced isomerization coupled with protonation/deprotonation of the hydroxyphenyl group of the
Figure 1. (a) Confocal microscopy. Fluorophores are excited by a tightly focused light beam that is raster-scanned across the sample to yield the image. (b) RESOLFT nanoscopy. Chromophores are switched on by irradiation with a focused beam of 405 nm light. Subsequently, a donut-shaped depletion spot of 488 nm light is applied. Molecules near the center are not irradiated and remain in their fluorescent states and can still emit photons, whereas those in the periphery are switched back to their nonfluorescent off state. An image is acquired by repeating this pulse sequence while scanning across the sample. Reproduced with permission from ref 10. Copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
p-HBI chromophore (or variants thereof). Accordingly, the effect depends on the interplay between four chromophore species, the neutral (protonated) chromophores, CH and TH, and the anionic (deprotonated) chromophores, C− and T−, in their cis and trans isomeric states, respectively (Figure 2a). Different proton affinities in the two isomeric states govern the switching process as well as the type of photoswitcher. Negative photoswitchers with a p-HBI chromophore are switched off by (typically blue, ∼488 nm) light used for exciting the anionic, green fluorescent chromophore species and switched on by (typically violet, ∼405 nm) light absorbed by the neutral species. Positive photoswitchers turn their fluorescence on upon irradiation into the anionic chromophore band. Because the novel GMars-Q is a negative photoswitcher, we will not dwell on positive photoswitchers here and only refer to recent reviews.7,11,12 The “ideal” switching mechanism of negative photoswitchers is sketched in Figure 2b. The C− state, the only one of the four that can fluoresce after photon absorption, has the lowest free energy and is thus completely populated in thermal equilibrium in the dark at physiological pH (∼7). Accordingly, the cis isomer has a low proton affinity; pKa (cis) 9105
DOI: 10.1021/acsnano.6b06298 ACS Nano 2016, 10, 9104−9108
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two of the four states can be appreciably populated, C− by 405 nm and TH by 488 nm irradiation. A popular strategy to create novel FP markers is to begin with an established FP that has advantageous properties and to exchange specific amino acids, eliciting the desired effects in other FPs. Following this route, Wang et al.8 employed the bright green-to-red photoconvertible protein, mMaple3,13 as a template to develop GMars-Q. They transferred an amino acid modification to mMaple3 that was known to endow the greento-red photoconverter EosFP14,15 with reversible photoswitching capability.16 In a second step, the histidine residue of the chromophore tripeptide, which appears to be essential for green-to-red photoconversion,17 was replaced by glutamine to obtain a pure photoswitcher. GMars-Q is reported to be monomeric and fast-maturing, and its spectroscopic properties resemble those of its predecessor mMaple3. Two photophysical properties make this FP variant attractive for RESOLFT microscopy. (1) The residual off-state fluorescence is very low. This property is of utmost importance for nanoscopy because, considering the high density of labels required to fulfill the sampling (Nyquist) criterion, even very weak signals from many inactivated fluorophores may overwhelm strong signals from a few activated fluorophores. (2) The fatigue resistance is very high, although this property comes with a truly peculiar photobleaching behavior observed upon repetitive photoswitching. Unlike a reversibly switchable enhanced GFP (rsEGFP) variant,9 which fades roughly exponentially during repetitive cycling between on and off states, Wang et al.8 report that the intensity of GMars-Q initially drops much faster, but after 300 cycles, it flattens out at a level of
