Photosensitive fluorophores for single-molecule localization

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Review

Photosensitive fluorophores for single-molecule localization microscopy Fadi M. Jradi, and Luke D. Lavis ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00197 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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ACS Chemical Biology

The chemistry of photosensitive fluorophores for singlemolecule localization microscopy# Fadi M. Jradi and Luke D. Lavis* Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA, USA #Dedicated

to Professor Ronald T. Raines, on the occasion of his 60th birthday.

*Corresponding author: [email protected]

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ABSTRACT The development of single-molecule localization microscopy (SMLM) has sparked a revolution in biological imaging, allowing ‘super-resolution’ fluorescence microscopy below the diffraction limit of light. The last decade has seen an explosion in not only optical hardware for SMLM but also the development or repurposing of fluorescent proteins and small-molecule fluorescent probes for this technique. In this review, written by chemists for chemists, we detail the history of single-molecule localization microscopy and collate the collection of probes with demonstrated utility in SMLM. We hope it will serve as a primer for probe choice in localization microscopy as well as an inspiration for the development of new fluorophores that enable imaging of biological samples with exquisite detail.


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INTRODUCTION Fluorescence microscopy is a powerful tool for examining biological systems. The vast majority of molecules in a cell are not fluorescent and this simple fact, combined with straightforward filtering of excitation light, allows visualization of fluorescent molecules in complex environments with superb sensitivity. Nevertheless, fluorescence microscopy does suffer from constraints: (1) restricted depth penetration due to light scattering; (2) autofluorescence from endogenous chromophores; and (3) a limitation on the resolution due to the diffraction of light. Fortunately, this ‘diffraction limit’—described at length below—can be circumvented using physics and chemistry. Here, we detail one method to overcome the diffraction limit, single-molecule localization microscopy (SMLM), and collate the chemistry and properties of the photoactivatable protein and small-molecule probes used in this powerful technique. HISTORY Although the diffraction limit was first formulated by Ernst Abbe in 1873,1 methods to circumvent it arrived over a century later in the 1990s; this watershed decade forever changed optical microscopy. Stefan Hell pioneered stimulated emission depletion (STED) microscopy, which uses high-intensity light to selectively deplete fluorophores around a sub-diffraction focal spot.2 Heintzmann and Gustafsson advanced structured illumination microscopy (SIM) which can achieve a two-fold increase in resolution using conventional fluorophores.3 In 1995, Eric Betzig proposed a theoretical method that would eventually become SMLM, where the diffraction limit is circumvented by separating individual fluorophores along another axis (e.g., emission wavelength or time), measuring their position with nm precision (vide infra), and then combining these localizations into a reconstructed probability density map or ‘super-resolution image’. This



allows one to recover the optical information stored in discrete fluorophores that are densely occupying a region in a sample.4 Concurrent to these advances was the emerging ability to image individual fluorescent molecules, pioneered by W. E. Moerner, Betzig, and others. Nevertheless, SMLM requires both the ability to visualize individual molecules and specialized switchable fluorophores; as a result, it was the emergence of switchable fluorescent proteins and small-molecule dyes a decade later that propelled SMLM into practice. Eric Betzig and Harald Hess used photoactivatable fluorescent proteins such as PA-GFP—developed by Patterson and Lippincott–Schwartz5—and called the technique photoactivated localization microscopy (PALM).6 Likewise, Sam Hess also used PAGFP and gave it a similar name: fPALM.7 In contrast, Xiaowei Zhuang used antibodies labeled with pairs of CyDyes, which she had discovered previously as an innovative switching system, to develop stochastic optical reconstruction microscopy (STORM).8 Along the same lines, Hochstrasser used the fluorogenic binding of Nile Red to lipid bilayers to determine the coordinates of individual fluorophores with nm precision in what they dubbed points accumulation imaging in nanoscale topography (PAINT).9 These four simultaneous, independent reports in 2006 produced super-resolution images of circular DNA plasmids, unilamellar vesicles, lysosomal transmembrane proteins, mitochondrial proteins, and cytoskeletal components, thereby demonstrating the applicability of this method to biological samples and spurring great interest in developing new SMLM labels and techniques.10 In particular, Sauer followed his previous discovery of cyanine-based switches by demonstrating the use of single cyanine dyes as superresolution labels, in a technique he termed ‘direct STORM’ (dSTORM).11 The initial reports of super-resolution imaging usually sampled a single type of protein with limited z-resolution, largely due to limitations in optical hardware and the dearth of different color labels. Rapid improvements in both microscopes and fluorophores soon enabled multicolor ACS Paragon Plus Environment

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experiments, which revealed the relative organizational patterns of different types of protein and their assembly into higher-order structures. For example, multicolor SMLM resolved the structures of clathrin-coated pits (CCPs) and microtubules in a single cell12, 13 and allowed the study of the structural relationship between various proteins that are involved in adhesion complexes.14 Improvements in z-resolution initially used astigmatism to image the bowl-shaped, cage-like structure of CCPs.15 Further improvements in 3D reconstruction incorporate a number of optical strategies, including double-plane detection,16 two-photon activation by temporal focusing,17 double-helix point spread function,18 and interferometry,19 to enable image of samples with depths of a few microns. In addition to limitations on color and z-resolution, early SMLM experiments were performed exclusively on fixed cells, largely due to the long acquisition times (e.g., typically minutes to hours) required to collect enough localizations to generate an image. In general, SMLM is still most useful for fixed samples, but live-cell SMLM experiments have yielded biological insight, especially when studying relatively slow cellular processes such as the formation and evolution of adhesion complex proteins20 and nanoscale distribution and dynamics of hemagglutinin.21 A more fruitful application to live-cell imaging has come from combining the concept of SMLM with single-particle tracking (spt) experiments. This ‘sptPALM’ enables the imaging of the trajectories of thousands of individual proteins with densities up to 50 molecules/m2 by stochastically activating a subset of dyes, imaging them until they bleach, and repeating the process until the entire pool of labeled protein has been interrogated. These experiments generate spatially-resolved diffusion maps inside a cell, providing local context for individual molecules and shedding light on the dynamic heterogeneities present inside cells.22, 23 SMLM has moved beyond proof-of-principle experiments, enabling the discovery of new



biological information. Zhuang discovered novel ring-like actin filaments encircling the axon with 180 nm periodicity.24 Doksani visualized telomeres inside cells to assess the role of shelterin components in the t-loop formation.25 Ellenberg and Löschberger used SMLM to refine the molecular structure of the nuclear pore.26, 27 Although many more such discoveries lie in wait, the future of SMLM is likely in lock-step with electron microscopy (EM). As demonstrated in the original PALM paper,6 correlative light and electron microscopy (CLEM) is an exciting technique that combines the molecular information of light microscopy with the ultrastructural information of EM.28 New sample preparations,29 microscopy setups,30 and osmium tetroxide resistant photoconvertible fluorescent proteins31 portend the future of super-resolution imaging, where SMLM is used to color in the familiar grayscale EM images of cells and tissue. THE PRINCIPLES OF SMLM In a fluorescence microscope, the focused excitation light is transmitted via a high numerical aperture (NA) objective to the image plane to produce a diffraction pattern, referred to as the point spread function (PSF). This PSF takes the shape of a bright disk surrounded by dimmer higherorder diffraction rings (i.e., the Airy disk). The radius of the disk depends on the wavelength of the light source, and is approximately equal to 0.61/NA laterally, and ~2/NA2 along the optical axis, where  is the wavelength of excitation light, and NA is the numerical aperture of the objective. For example, imaging a single fluorophore (diameter ≈ 1 nm) using a common oil-immersion objective (NA = 1.4) and a standard excitation source ( = 488 nm) will produce a PSF ~200 nm in the lateral (x–y) dimension and ~500 nm in the axial (z) dimension. This disparity between the size of the molecule and the size of the PSF renders it fundamentally impossible to resolve two molecules that are within this distance of 0.61/NA (i.e., the ‘diffraction limit’). This


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makes it difficult to retrieve the information from individual fluorophores in a densely labeled sample. An SMLM imaging session begins with labeling a biological sample with a suitable switchable fluorophore, which is then activated stochastically with continuous fluorescence imaging. This switching is typically light-induced, but dyes that activate through enzymatic activity32 or binding events33 have been used in SMLM. Each frame of the resulting movie is then analyzed by software to localize individual molecules. These analyses perform a statistical fit using the microscope’s PSF to pinpoint the location of the fluorophore with greater precision than conventional imaging.34 The individual localizations are then combined to build up a high resolution map of molecular location: the ‘super-resolution’ image. As with all fluorescence microscopy, the photophysical properties of the fluorophore labels are crucial for SMLM. These are summarized in Tables 1–2 and include the absorption maximum (max), the fluorescence emission maximum (em), the extinction coefficient at max (ε), and the fluorescence quantum yield (). Properties specific to fluorophores activated with light include: the fluorescence contrast upon photoactivation (Fon/Foff), the turn-on (t½on) halftime, the photobleaching halftime (t½ PB) and the ‘photon yield’ of individual fluorophores before bleaching (N). The maturation halftime (t½ M) is an important consideration for fluorescent proteins. For caged dyes a critical factor is the photoactivation quantum yield ( PA). We note the switching between the dark and fluorescent states can be reversible or irreversible. Key properties of reversible photoswitchable dyes include the turn-off (t½off) and the fatigue resistance (FR; defined as the number of switching cycles needed to bleach 50% of the initial fluorescence). For small-molecule dyes that switch reversibly under specific conditions, key properties include the on/off ‘duty cycle’ (on/off)—the fraction of time a fluorophore spends in the fluorescent state vs.



the nonfluorescent ‘dark’ state—as well as the average number of switching cycles (SC) these dyes undergo before bleaching and the survival fraction (SF) of fluorophores after relatively harsh imaging conditions. Irreversible switching dyes are perhaps more useful in experiments where counting of single molecules is desired; reversibly switching dyes can also be effective but their use in counting requires additional calibration.35 Of course, since SMLM is based on the localization of individual molecules, the stochastic activation must be sparse enough to ensure that simultaneous activation of two molecules in the same diffraction limited spot is minimized. As a general rule, the fluorophore’s on-switching or activation rate should be much smaller than their off-switching or bleaching rate, with a duty cycle between 10–4 to 10–6.36 Another key metric in determining the quality of SMLM imaging is the precision of the localization (). This is inversely proportional to the square root of the number of photons detected per activated fluorophore:  ∝ s/(N½); where s is the standard deviation from the localization analysis and N is the photon yield or number of photons emitted by the fluorophore per localization event. This simplification is true only in thin samples with low background fluorescence, and many parameters determine the actual localization precision.37 In principle, it is possible to achieve a resolution that is two orders of magnitude higher than diffraction-limited imaging through the use of SMLM with high photon-yielding fluorophores; in general, a fluorophore must emit at least 100 photons to achieve super-resolution imaging. Finally, another important consideration for SMLM is labeling density. Labeling density is governed by the Nyquist criterion, which indicates that in order to achieve detailed structural information, the mean separation between fluorescent labels must be no greater than half of the desired spatial resolution.20, 38 FLUOROPHORES AND LABELING STRATEGIES Fluorophores employed in SMLM fall into two classes: fluorescent proteins (FPs; Table 1), and small-molecule fluorescent dyes (Table 2). FPs can be divided into the following classes: (1) ACS Paragon Plus Environment

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photoactivatable FPs (PA-FPs) that undergo irreversible switching between a dark off-state and a bright on-state; (2) photoswitchable FPs (PS-FPs) that undergo an irreversible transformation between two on-states of different colors; (3) photochromic FPs (PC-FPs) that undergo a reversible transformation between an off-state and an on-state, and (4) rarer, more complicated photoactivatable/photoconvertible FPs that undergo an irreversible transformation between two on-states, each of which can be reversibly switched to an off-state. In addition to the many modes of switching, FPs have the advantage that they are genetically encoded and can be expressed as a fusion to a protein of interest in a variety of cells and in vivo using the ever-growing molecular biology toolbox. Table 1 lists the photophysical properties of several widely-used FP-based labels for SMLM and Figure 1 illustrates the photochemistry behind several classes of fluorescent proteins, which can be categorized into the following mechanisms; decarboxylation, -elimination, oxidative ‘redding’, hydration/dehydration, or cis–trans isomerization. Small-molecule fluorescent dyes also fall into a relatively small number of categories: (1) activator–reporter dye pairs where an ‘activator’ dye placed within 1–2 nm of the fluorophore can modulate its photoactivation rate; (2) activator-free dyes that stochastically activate under continuous laser illumination; (3) spontaneously blinking dyes that stochastically and reversibly activate in the absence of light; (4) photochromic dyes that undergo light assisted reversible transformation between an on- and off-state; (5) photoactivatable or ‘caged’ dyes that irreversibly activate upon a photochemical reaction. Of course, chemical dyes cannot be genetically encoded and the most established and flexible labeling strategy is the use of antibodies.39 This is largely restricted to fixed samples, however, and lowers localization precision due to the large size (200 kD) of the antibody–dye conjugate. For live cell experiments, several hybrid approaches have been developed that exploit a specific interaction between genetically encoded proteins and smallmolecule dyes. These range from non-natural amino acids, streptavidin–biotin interactions,



enzyme-based self-labeling tags, and fluorophore ligase strategies.40 Table 2 lists the photophysical properties of dye labels for SMLM and Figure 2 details the chemistry behind their photochemical behavior. Our goal is to provide a primer for chemical biologists interested in SMLM and complement the excellent reviews from Vaughan and Johnsson.41, 42 FLUORESCENT PROTEINS Background and Photoactivatable Fluorescent Proteins. Green fluorescent protein (GFP) folds into a -barrel structure, which orients a three amino acid sequence—Ser-Tyr-Gly—to generate the GFP chromophore following cyclization, oxidation, and dehydration steps (Figure 1a).43 Additional colors can be generated by modifying this canonical chromophore structure (e.g., cyan fluorescent protein, CFP) or extending its conjugation (e.g., mOrange or Kaede; Figure 1b).44 The optical properties can also be altered by -stacking and/or electrostatic interactions with nearby amino acids, and modification of chromophore pKa. The advent of GFP and its ilk revolutionized live-cell imaging and sparked intense effort into the discovery and evolution of new FPs with novel properties. Several PA- and PS-FPs were described at the turn of the 21st century, shortly after the use of GFP became mainstream. The initial class of light-modulated fluorescent proteins included PA-GFP,5 Kaede45 and KFP1;46 These proteins were initially used as ‘optical highlighters’ as an alternative to fluorescence recovery after photobleaching (FRAP) experiments.47 PA-GFP5 is a particularly excellent label—it is monomeric and exhibits a high

PA48 and a reasonable photon yield (~300).36 Due to these properties, it remains—after almost two decades—one of the few reliable dark-to-green PA-FPs and was used in two of the three initial reports of SMLM.6, 21 The molecular basis behind the photoconversion of PA-GFP has been elucidated. Wild-type GFP exists as a mixture of two species that differ by the protonation state of the chromophore: a


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neutral species that absorbs in the UV (max = 397 nm) and a red-shifted anionic species (max = 475 nm) with a pKa of 6.3. Exposing GFP to strong UV illumination elicits a photoinduced decarboxylation at Glu222 (Figure 1c),49 which results in a rearrangement of the hydrogen bonding network around the chromophore in a manner that stabilizes the anionic form, thereby increasing the absorption at 475 nm. The pKa of wild-type GFP is relatively high leading to a nonzero absorption at 475 nm, making the on-off contrast insufficient for SMLM. PA-GFP was developed by introducing a mutation (T203H) that further shifts the equilibrium to the neutral species and increases the Fon/Foff contrast to 60-fold.5 Given the initial success with PA-GFP and the need for multicolor imaging, efforts were directed towards developing a monomeric red-shifted equivalent. Founded on the heroic development of mRFP1,50 the first monomeric orange photoactivatable FP, PAmRFP1, was developed;51 it was followed by variants with improved photon yields, including the orange PAmCherry152 and PATagRFP,53 as well as the red PAmKate.54 The mechanism behind activation of PAmKate and PAmCherry1 is similar to PA-GFP—decarboxylation of an analogous Glu side chain;55 PATagRFP functions through a completely different mechanism involving oxidative ‘redding’ (Figure 1d). In contrast to GFP, the chromophore in PATagRFP features two aromatic systems that are separated by a saturated methylene spacer. Upon photoactivation, PATagRFP generates the chromophore via two one-photon mediated oxidative steps; the first photon catalyzes the oxidization of the N-acylamine moiety, extending the chromophore’s conjugation, while the second photon generates the DsRed-like chromophore by oxidizing the Tyr methylene unit.53 In addition to the desirable red-shifted absorption and emission wavelengths, the orange PAmCherry1 and PATagRFP and red PAmKate also exhibit higher on-off contrasts and photon yields when compared to PA-GFP, enabling high quality SMLM imaging in cells.36, 52-54, 56



Photoswitchable Fluorescent Proteins. Photoswitchable fluorescent proteins (PS-FPs) that convert between two fluorescent states of different colors are also useful for SMLM. A general disadvantage with using PA-FPs in SMLM is the absence of fluorescence in the dark state, which makes it difficult to identify a field of view containing cells that are expressing the PA-FP prior to committing to a long imaging experiment. PS-FPs circumvent this problem since their initial, preactivated form is fluorescent. Activation with ultraviolet or blue light substantially shifts the absorption maxima of these PS-FPs to the red, providing the requisite Fon/Foff contrast for high quality SMLM. Three different types of PS-FPs are (1) the GFP-like cyan-to-green FPs such as PS-CFP2; (2) the Kaede-like green-to-orange/red FPs Dendra2, mEos3.2, mClavGR2, and mMaple3 and (3) the DsRed-like green-to-far red protein, PSmOrange. In its preactivated state, PS-CFP2 emits in the cyan (em = 468 nm). Illumination with violet light causes a decarboxylation that changes the structure of the chromophore and elicits a bathochromic shift to 511 nm, aligning it with PA-GFP.57 The Fon/Foff fluorescence contrast of PSCFP2 is ~20 times higher compared to PA-GFP, but it exhibits a lower quantum yield and smaller photon yield than PA-GFP. Despite this poor brightness and photostability, PS-CFP2 exhibits an excellent on/off duty cycle (10−6), which is three orders of magnitude lower than that of PA-GFP.36 Cells labeled with PS-CFP2 can tolerate significantly higher labeling densities in SMLM and are compatible with red FPs.14 The PS-FPs mEos3.2, Dendra2, mClavGR2, and mMaple3 are part of the Kaede family, which contain a His residue instead of the Ser residue present in GFP. This substitution allows formation of a chromophore with extended conjugation and red-shifted wavelengths (Figure 1b). In the preactivated state, this family of PS-FPs contains a GFP-like chromophore but upon irradiation, the excited-state quinone methide intermediate undergoes cleavage at the N-C bond of the His


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to eliminate a carboxamide containing peptide. The subsequent loss of a proton yields a trans double bond between the C and C of the His, leading to extended conjugation and red-shifted wavelengths (Figure 1e).58 The highly evolved mEos3.2 is the result of numerous mutations to reduce the formation of multimers, maturation time at physiological temperatures, and localization of protein fusions in the cell. mEos3.2 is a highly monomeric, bright, and rapidly maturing protein that exhibits high photon yields and excellent contrast for SMLM with high labeling density.59 It is currently considered the best green-to-red PS-FP and one of the most-used FPs in SMLM, with demonstrated localization accuracies up to 12 nm in the lateral dimension.59 Although mEos3.2 has 2× the photon yield of mClavGR2,60 and 1.2× higher yields than Dendra2,57 and mMaple3,36 these related PSFPS have unique properties that make them valuable alternatives to mEos3.2. Dendra2 can be activated by longer wavelength, less phototoxic 488 nm light, facilitating live-cell SMLM. mMaple3 exhibits a very low on/off (10−7), which is an order of magnitude lower than mEos3.2 and Dendra2 and is useful in densely labeled samples.36 The red form of mClavGR2 exhibits high photostability, which could be useful in single particle tracking experiments. Finally, the DsRed-like PSmOrange61 and its enhanced version PSmOrange262 switch from an orange state to a far-red state upon excitation with blue-green light, emitting at 662 nm. This farred emission is distinct from other FP-based SMLM labels, making it a good imaging partner in multicolor super-resolution microscopy. It can also be activated efficiently with two-photon excitation, allowing for spatial control of activation in thicker samples. Similar to PATagRFP, PSmOrange generates the red chromophore via oxidative reddening, where two consecutive oxidation steps cleave the peptide backbone and prompt the formation of an atypical oxazolone ring (Figure 1e), which is red-shifted relative to the DsRed chromophore.61 This desirable far-red



emission is countered by PSmOrange’s low photostability and reduced photon yields, as well as the dependence of the photoconversion efficiency on redox environment. Photochromic Fluorescent Proteins. PC-FPs exhibit reversible switching between two states63 and those detailed below switch between a nonfluorescent dark state and a fluorescent bright state. The reversible transition means that a population of molecules can be activated thermally in the steady state, but also allows switching to the dark state using brief illumination prior to imaging. Although beyond the scope of this review, PC-FPs are useful in non-linear superresolution microscopy techniques such as reversibly saturatable optical fluorescence transition (RESOLFT)64 and non-linear structured illumination microscopy (NL-SIM).65 Unlike SMLM, these techniques involve multiple switching cycles and thus require the high fatigue resistance of fluorescent proteins like Skylan-S and Skylan-NS,66, 67 or the fast switching kinetics of rsFastlime and rsEGFP.68, 69 Most PC-FPs switch through a general cis–trans isomerization of the chromophore (Figure 1f).70 Prior to irradiation, Dronpa’s chromophore assumes a GFP-like cis-confirmation in the predominantly deprotonated bright state. Absorption of a blue photon elicits an isomerization to the trans-form which has a different absorption spectrum, lower quantum yield, a higher pKa, leading to low fluorescence. This isomerization can be reversed by absorption of a UV photon. Although other FPs exhibited hints of photochromism,71 the dark-to-green Dronpa was the first widely used PC-FP in cellular biology and super-resolution imaging.72 The monomeric Dronpa is 6× brighter than activated PA-GFP, exhibits a fast t½,on, a relatively slow t½,off, and a high photon yield. Over 90% of the expressed Dronpa proteins undergo only one blinking event, which enables counting of individual molecules using SMLM.73


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Dronpa underwent extensive protein engineering, producing variants with 45× faster switching kinetics (rsFastlime68), a positive switching mode (Padron),74 a broader absorption spectra (bsDronpa),74 and extended t½,off (mGeos-M).73 The increased t½,off of rsFastlime decreases photon yield but enables faster SMLM experiments69 as do other fast switching PC-FPs such as rsEGFP and rsEGFP2.75 Padron maintains the rapid switching kinetics and brightness of rsFastlime, adding the highest Fon/Foff contrast (150×) of extant PC-FPs.74 In contrast, mGeos-M73 exhibits a t½,off that is 52% longer than Dronpa, which enhances the photon yield and thus localization precision. A limitation of Dronpa and its variants is low photostability; bacterial colonies expressing Dronpa lose half of their initial fluorescence after only 4 switching cycles and only rsFastlime shows modestly better photostability. Stable PC-FPs do exist, however, as bacterial colonies expressing rsEGFP, EGFP2, and Skylan-S maintain half their initial fluorescence after 1200, 2100 and 7000 switching cycles, respectively. As mentioned above, most PC-FPs use cis–trans isomerization to modulate fluorescence. An exception is Dreiklang, which uses a reversible, light-mediated hydration reaction at the imidazolinone ring (Figure 1g).76 Irradiation with violet light facilitates water attack, converting the imidazolinone ring into a 2-hydroxyimidazolidinone, which disrupts the chromophore conjugation and decreases fluorescence; UV light reverses this hydration reaction. This unique property decouples the imaging light from the photoswitching light, alleviating a serious limitation in PC-FPs whose fluorescence readout and off-switching are interlocked. This unique switching modality, coupled with a high photon yield of the on-state, allowed high resolution SMLM of microtubules and keratin with 15 nm precision. Nevertheless, this requisite UV light and the temperature dependence of Dreiklang’s photophysics can complicate imaging of live cells. Finally, red-shifted PC-FPs such as rsCherry and rsCherryRev77 have also been developed, showing good photon yields and fast switching kinetics. Their photophysics is complex, however, ACS Paragon Plus Environment


with these proteins showing intensity-dependent switching and brightness.52 One useful PC-FP that avoids this complex switching behavior is the dark-to-orange rsTagRFP,78 which avoids UV or violet activation light, is substantially brighter than rsCherry (2.5×) or rsCherryRev (10×), and has 2× Fon/Foff contrast due to lower residual fluorescence in the off state. While most red-shifted PC-FPs possess inferior photophysical properties compared to the green PC-FPs, they can still prove useful in two-color super-resolution imaging as demonstrated with imaging non-raft microdomains in the plasma membrane using Dronpa and rsTagRFP.79 Photoactivatable/Photochromic FPs. Occupying a class of their own, mIrisFP80 and NijiFP81 combine the photoswitching behavior of both PC- and PS-FPs. These proteins can be photoactivated from dark-to green using UV light and then undergo further photoconversion from green-to-red upon excitation. This combination allows sophisticated experiments that combine super-resolution microscopy and dynamic imaging in living cells. In such an experiment, the protein-of-interest is expressed as a fusion with a PA/PC-FP. Live-cell SMLM images are generated by activating the green form of the FP. This is followed by switching a subset of the FPprotein fusion to the red form and monitoring the migration of these proteins by switching the red form on and off. Both mIrisFP and NijiFP are at least as bright as PA-GFP, and mIrisFP exhibits a relatively high photon yield that allows for high-quality SMLM. SMALL-MOLECULE FLUOROPHORES Background. Small-molecule dyes activated by light predate SMLM by decades, being used for optical recording media, organic electronics, and unraveling the dynamic processes in cells.82, 83

Small-molecule fluorophores were also the focus of ground-breaking single-molecule

microscopy experiments by Moerner at cryogenic temperatures,84 and later examples by Betzig at room temperature using near-field optical microscopy.85 Over time, improvements in optical


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hardware allowed for such imaging to be performed in aqueous media under ambient conditionsl.86 Despite this availability of both light-activated dyes and the ability to image single molecules, SMLM was founded largely on activatable FPs due to their ease of use and facile optimization through directed evolution. Overall, small-molecule dyes for SMLM have lagged behind FPs in breadth, characterization, and publications; this field remains an active and important area of chemistry research. When compared to their protein counterparts, fluorescent small-molecules typically possess higher brightness, photostability, and photon yields. Small-molecule fluorophores are also more versatile in that they allow the use of the entire chemical lexicon rather than the meager 20 proteogenic amino acids. Improvements in chemistry and a growing understanding of structureactivity relationships in chemical dyes allow the design and synthesis of rationally designed derivatives with finely tuned properties. Nevertheless, small-molecule fluorophores have several severe disadvantages compared to fluorescent proteins. Because small molecule dyes cannot be genetically encoded and fused to a protein of interest, they necessitate chemical synthesis and specialized labeling strategies.40 They also can suffer from nonspecific binding and cellular toxicity, which can be detrimental to a sensitive imaging technique like SMLM. An excellent exhaustive review on small-molecule fluorophores for SMLM was recently published.87 Activator–reporter dye pairs. One of the first classes of small-molecule fluorophores for SMLM were based on the cyanine-based ‘CyDyes’. Zhuang showed that biomolecules labeled with two different cyanine dyes such as Cy3 and Cy5 showed a switching phenomenon where the primary Cy5 ‘reporter’ fluorescence could be modulated by excitation of the secondary Cy3 ‘activator’ dye.88 This switching behavior was initially proposed as a complement to Förster Resonance Energy Transfer (FRET) due to the higher distance dependence of this phenomenon. The utility of this system for SMLM was soon realized, however, and a number of pairs were ACS Paragon Plus Environment


identified using shorter wavelength activator dyes (e.g., Cy2, Cy3, or the pyrene-based Alexa Fluor 405) and longer wavelength reporter dyes such as Cy5, Cy5.5, Cy7, and the Cy5 analog Alexa Fluor 647. This dual label system could be easily implemented by using antibodies labeled with combinations of activator and reporter dyes, enabling multicolor, three-dimensional SMLM.8, 12, 15 dSTORM dyes (i.e., ‘activator-free’ dyes). Concurrent with the development of activator– reporter pairs, Sauer discovered that single cyanine dyes could be photoswitched under reducing conditions without an activator dye, albeit with substantially higher laser powers.11, 89 This was later expanded to other small molecule fluorophore types, yielding the general dSTORM method for SMLM using commercial fluorophores.90,

91

An extensive survey of the properties of 26

commercially available fluorophores was published92 that identified the best dyes in four spectral windows: ATTO 488 (green), Cy3B (yellow), Alexa Fluor 647 (red) and DyLight750 (near infrared). In general, far-red dyes perform best in SMLM with Alexa Fluor 647 exhibiting the highest photon yields (~5200) making it the best dye for dSTORM and the most commonly used SMLM label; we note the use of single-dye label systems has largely replaced the earlier activator– reporter pairs. dSTORM requires a specific chemical environment to elicit switching behavior and the imaging buffer typically contains a cocktail of additives: oxygen scavengers and triplet quenchers along with reducing agents such as primary thiols93 and phosphines.94-96 For some rhodaminebased dyes, the mechanism of photoswitching relies on the formation of transient, nonfluorescent radicals as evidenced by EPR studies;97 these species can revert back to a fluorescent form spontaneously or in a light-mediated manner (Figure 2a). For cyanine dyes, the photoswitching could stem from direct reaction of the reducing agents with the fluorophore. For example, irradiation of Cy5 in the presence of primary thiols produced dark photoproducts presumably arising from thiol addition to the polymethine bridge (Figure 2a).93 Likewise, water soluble ACS Paragon Plus Environment

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phosphines such as tris(2-carboxyethyl)phosphine (TCEP) can quench the fluorescence of Cy5 and other cyanines through light-independent addition to the C4 of the polymethine bridge. UVillumination of the dye–phosphine adduct restored the cyanine’s fluorescence in a reversible fashion.94 In a related technique, treatment of rhodamine- or cyanine-labeled samples with sodium borohydride yields nonfluorescent ‘leuco’ dyes (Figure 2a). Illumination of these reduced fluorophores with UV or violet light allows SMLM with a high fraction of recovered molecules (40–66%)95, 96 and new rigidified cyanine dyes show excellent performance using this strategy.98 Spontaneously Blinking Dyes. dSTORM dyes rely on light and/or the presence of additives in the imaging buffer to induce photoswitching. Both of these complicate SMLM in live cells, although in some cases the reducing environment of the cytosol is sufficient to allow live-cell super-resolution imaging using conventional dyes.99, 100 To circumvent this problem, the Urano group set out to design a dye that would be in equilibrium between a nonfluorescent and fluorescent form and therefore blink spontaneously. They found that hydroxymethyl (HM) rhodamines were an excellent scaffold and devised methods to tune the equilibrium constant of intramolecular spirocyclization (Kcycl). This resulted in the development of hydroxymethyl-Si-rhodamine (HMSiR; Figure 2b) and other related compounds.101,

102

The transient lifetime of the ‘open’

fluorescent form (50–150 ms) allows for high-quality SMLM with excellent photon yields (~ 2600) even under substantially reduced illumination intensities.102 More recently, this compound class has been used for live-cell and two-color SMLM by exploiting changes in chemical environment to further modulate the blinking properties.101, 103 Photochromic Dyes. In addition to the discovery of new switching mechanisms and spontaneously blinking dyes, SMLM has also renewed interest in classic light-activated dye systems, including reversible photochromic dyes. Rhodamine lactams, first introduced in 1977,104 undergo transient (t½ ~ ms) lactam bond cleavage upon illumination with far-UV light. This ACS Paragon Plus Environment


fluorescent form thermally reverts back to the colorless lactam. This transient fluorophore formation is ideal for SMLM imaging and recent advances have extended the activation wavelength using phthalimide substituents on rhodamine B (RhB phthalimide, Figure 2c) allowing activation with near-UV (375 nm) or 2-photon excitation (747 nm). RhB phthalimide enabled optical sectioning and resolved the structure of microtubules with 55–70 nm localization precision and good photon yield (~900).105 This strategy has been extended to rhodamines with different spectral properties,106, 107 and use of a stilbene substituent allowed activation with visible light.108 Another classic photochromic system involves benzooxazine that preferentially adopts a nonfluorescent bicyclic oxazine form (e.g., in OA-2, Figure 2c). Upon illumination, a photoinduced excited-state electron exchange reaction opens the oxazine ring, restoring the fluorophore conjugation and eliciting a large increase in fluoresence.109 Although these systems exhibit relatively fast switching kinetics and high photostability—important for SMLM—the relatively low brightness, high hydrophobicity, and high background have limited their utility in super-resolution imaging.110 Finally, unlike the rhodamine lactams and benzooxazines, which thermally revert back to their nonfluorescent forms, diarylethenes can be driven in both directions with light. Like the benzoxazine compounds, diarylethenes have been limited by both water solubility and low fluorescence quantum yields. Steady improvements in both these properties have begun to yield results in SMLM.111, 112 Photoactivatable Dyes. Photoactivatable (or ‘caged’) small-molecule fluorophores incorporate a photolabile moiety that is removed or modified upon illumination with light. This irreversible photoactivation is especially useful for sptPALM experiments and the three most common chemical caging modalities are o-nitrobenzyl (o-NB) derivatives, 2-diazoketones, and azides. The o-NB moiety predates caged dyes, being used as a photolabile protecting group for diverse chemical functionalities.113, 114 To prepare caged fluorophores, the o-NB is attached to the ACS Paragon Plus Environment

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phenolic oxygen of fluoresceins as an ether or the aniline nitrogens of rhodamines as a carbamate linkage.115-117 Examples of fluorophores that were successfully caged with o-NB and utilized in SMLM include Oregon Green, rhodamine 110 (Rh110), Q-rhodamine, carborhodamine 110, and Si-Q-rhodamine (SiRhQ, Table 2).116, 118-121 In general, caged dyes show excellent Fon/Foff contrast and good photon yields. For example, photoactivated Rh110 emitted an average of ~3500 photons, giving 16 nm precision.118 NVOC2-SiRhQ (Figure 2d) exhibited a photon yield 1.6× higher than Alexa Fluor 647 without the need for a dSTORM buffer; addition of antioxidants increased the photon yield to ~ 1.5 × 104. Similar to o-NBs, 2-diazoketone caging groups require UV or violet light to undergo photolysis. However, their smaller chemical footprint has some advantages: increased solubility and lower byproduct toxicity.122 This strategy is modular and relatively straightforward, allowing caging of a variety of rhodamines with different spectral properties.123 Despite the clear advantages of this method, however, 2-diazoketone-caged rhodamines have a severe limitation—complex photochemistry. Photolysis yields two major products: a fluorescent phenylacetic acid rhodamine and a ‘dark’ inden-1-one substituted dibenzooxepine.122 124 The fluorescent species results from a Wolff rearrangement whereas the dark product presumably results from a different rearrangement of the carbene intermediate (Figure 2c). To complicate this further, the fluorescent phenylacetic acid product can undergo a photoinduced decarboxlation under UV light to generate the o-tolyl substituted rhodamine, which is also fluorescent. The diazoketone strategy has been used to prepare the photoactivatable Janelia Fluor dyes: PAJF549 and PA-JF646. These compounds showed similar activation rates and duty cycles compared to mEos3.2 with higher photon yields; the far-red PA-JF646 enabled two-color SMLM.124 These were also the first diazoketone-caged dyes to be used in living cells, enabling sptPALM experiments of the transcription factor Sox2 and other proteins.23 We note the partitioning of the ACS Paragon Plus Environment


photoreaction between the fluorescent and ‘dark’ product is highly dependent on both the chemical structure of the dye and the chemical environment; in many cases the major product is the undesired nonfluorescent form. This sensitivity can be exploited, however, to prepare sophisticated molecular logic gates that rely on both light and enzyme activation.125 Occupying a class of their own, the dicyanodihydrofuran (DCDHF) ‘push–pull’ fluorophores have demonstrated utility in single-molecule imaging due to high photostability and fluorescence quantum yield.126 Transforming the amine donor in DCDHF into an azide (e.g., N3-DCDHF, Figure 2d) substantially changes the electronic structure of the chromophore, effectively quenching fluorescence. Irradiation with violet light produces an arylnitrene intermediate, which can undergo various reaction (reduction, bond insertion, etc.) to form a fluorescent product.127, 128 SMLM of microtubules labeled with N3-DCDHF gave cross-sections of ~85 nm, well below the 450 nm obtained from diffraction limited images.129 CONCLUSION In the last dozen years SMLM has become a fixture in modern cellular imaging, enabling new investigations on the organization of molecular components within cells. This field has not only been driven by improvements in optical hardware, but also by advances in both protein and smallmolecule labels. Although the vast majority of fluorophores used in SMLM are FP-based, it is also clear that small-molecule probes can exhibit substantially better properties, especially Fon/Foff contrast and photon yield. Moving forward, the challenge rests on organic chemists to create new molecules with improved performance inside living cells and devise new methods for activating molecules in a sparse and controlled manner. Continuing this productive marriage of optical physics, chemistry, and biology will unravel more molecular details underpinning living systems.


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TABLE LEGENDS AND FIGURE CAPTIONS Table 1. Properties of fluorescent proteins used in single-molecule localization microscopy. The schematic in the second column indicates maximal fluorescence excitation (ex) and emission (em) of the protein in different states as well as the switching wavelengths (hv); all values are in nm. All other properties are defined in the text. For proteins with more than one bright state, spectral data is provided for both states separated by a semicolon. at1/2 M values are reported for 37 C. bt1/2 on , t1/2 off and t1/2 PB are reported at different illumination intensities; check cited literature for direct comparisons. NR indicates value not reported. Table 2. Properties of dyes for single-molecule localization microscopy. The schematic in the second column indicates maximal fluorescence excitation (ex) and emission (em) of the dye in different states as well as the switching wavelengths (hv); all values are in nm. All other properties are defined in the text. aphoton statistics reported in the presence of the ‘GLOX’ (glucose oxidase with catalase) oxygen scavenging system and β-mercaptoethanol. b increases upon rigidification. c increases upon binding to protein. NR indicates value not reported. Figure 1. The chemistry of fluorescent protein chromophores and mechanisms of photoswitching. (a) Formation of the GFP chromophore. (b) Chemical structures of common FP chromophores with different spectral properties. (c) Mechanism of photoactivation of PA-GFP via photoinduced decarboxylation. (d) Mechanism of photoswitching of PS-mOrange and PA-TagRFP via an oxidative ‘redding’. (e) Mechanism of photoswitching of Dendra, mEos, mClavGR2, mIrisFP and mMaple via β-elimination. (f) The cis–trans isomerization of the Dronpa chromophore. (g) Photoconversion of Dreiklang through hydration/dehydration. The colored shading of the structures indicates em. Figure 2. The chemistry of small-molecule fluorophores and mechanisms of photoswitching. (a) Photoswitching of conventional fluorophores under reducing conditions and illumination. (b) A spontaneous blinking fluorophore HM-SiR. (c) Representative photochromic rhodamine lactams and benzoxazines. (d) Exemplar ‘caged’ dyes and their photoactivation. The colored shading of the structures indicates em.


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TABLE 1 Name

 × 104 (M–1cm–1)



Fon/Foff

t1/2Ma (min)

t1/2onb (s)

t1/2PBb (s)

N

 (nm)

ref.

Photoactivatable PA-GFP

1.74

0.79

60

Photosensitive fluorophores for single-molecule localization

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