Chemistry of Photosensitive Fluorophores for Single-Molecule Localization Microscopy Fadi M

Reviews

Cite This: ACS Chem. Biol. 2019, 14, 1077−1090 pubs.acs.org/acschemicalbiology

Chemistry of Photosensitive Fluorophores for Single-Molecule Localization Microscopy Fadi M. Jradi and Luke D. Lavis*

Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20147, United States

ABSTRACT: 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 past 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.

■ INTRODUCTION recover the optical information stored in discrete fluorophores 4 Fluorescence microscopy is a powerful tool for examining that are densely occupying a region in a sample. biological systems. The vast majority of molecules in a cell are Concurrent to these advances was the emerging ability to image individual fluorescent molecules, pioneered by W. E. not fluorescent and this simple fact, combined with Moerner, Betzig, and others. Nevertheless, SMLM requires straightforward filtering of excitation light, allows visualization both the ability to visualize individual molecules and of fluorescent molecules in complex environments with superb specialized switchable fluorophores; as a result, it was the sensitivity. Nevertheless, fluorescence microscopy does suffer emergence of switchable fluorescentproteinsandsmall- from constraints: (1) restricted depth penetration due to light molecule dyes a decade later that propelled SMLM into scattering, (2) autofluorescence from endogenous chromo- practice. Eric Betzig and Harald Hess used photoactivatable phores, and (3) a limitation on the resolution due to the fluorescent proteins such as PA-GFPdeveloped by Patterson diffraction of light. Fortunately, this “diffraction limit” and Lippincott−Schwartz5and called the technique photo- described at length belowcan be circumvented using physics activated localization microscopy (PALM).6 Likewise, Sam and chemistry. Here, we detail one method to overcome the Hess also used PA-GFP and gave it a similar name: fPALM.7 Downloaded via Fadi Jradi on July 14, 2019 at 16:08:01 (UTC). diffraction limit, single-molecule localization microscopy In contrast, Xiaowei Zhuang used antibodies labeled with pairs (SMLM), and collate the chemistry and properties of the of CyDyes, which she had discovered previously as an photoactivatable protein and small-molecule probes used in innovative switching system, to develop stochastic optical this powerful technique. 8 reconstruction microscopy (STORM). Along the same lines, Hochstrasser used the fluorogenic binding of Nile Red to lipid See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ■ HISTORY bilayers to determine the coordinates of individual fluoro- ff fi phores with nanometer precision in what they dubbed points Although the di raction limit was rst formulated by Ernst 9 Abbe in 1873,1 methods to circumvent it arrived over a century accumulation imaging in nanoscale topography (PAINT). later in the 1990s; this watershed decade forever changed These four simultaneous, independent reports in 2006 optical microscopy. Stefan Hell pioneered stimulated emission produced super-resolution images of circular DNA plasmids, depletion (STED) microscopy, which uses high-intensity light unilamellar vesicles, lysosomal transmembrane proteins, to selectively deplete fluorophores around a subdiffraction mitochondrial proteins, and cytoskeletal components, thereby focal spot.2 Heintzmann and Gustafsson advanced structured demonstrating the applicability of this method to biological samples and spurring great interest in developing new SMLM illumination microscopy (SIM) which can achieve a twofold 10 increase in resolution using conventional fluorophores.3 In labels and techniques. In particular, Sauer followed his 1995, Eric Betzig proposed a theoretical method that would previous discovery of cyanine-based switches by demonstrating ff the use of single cyanine dyes as super-resolution labels in a eventually become SMLM, where the di raction limit is 11 circumvented by separating individual fluorophores along technique he termed direct STORM (dSTORM). another axis (e.g., emission wavelength or time), measuring their position with nanometer precision (vide infra), and then Received: March 11, 2019 combining these localizations into a reconstructed probability Accepted: April 17, 2019 density map or “super-resolution image”. This allows one to Published: April 17, 2019

a Table 1. Properties of Fluorescent Proteins Used in Single-Molecule Localization Microscopy

a λ fl λ The schematic in the second column indicates the laser excitation wavelength ( ex) and maximal uorescence 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 b than one bright state, spectral data are provided for both states separated by a semicolon. NR indicates value not reported. t1/2M values are ° c ff ff reported for 37 C. t1/2on, t1/2o and t1/2PB are reported at di erent illumination intensities; check cited literature for direct comparisons.

The initial reports of super-resolution imaging usually which revealed the relative organizational patterns of different sampled a single type of protein with limited z-resolution, types of protein and their assembly into higher-order largely due to limitations in optical hardware and the dearth of structures. For example, multicolor SMLM resolved the different color labels. Rapid improvements in both micro- structures of clathrin-coated pits (CCPs) and microtubules scopes and fluorophores soon enabled multicolor experiments, in a single cell12,13 and allowed the study of the structural

1078 DOI: 10.1021/acschembio.9b00197 ACS Chem. Biol. 2019, 14, 1077−1090 ACS Chemical Biology Reviews

a Table 2. Properties of Dyes for Single-Molecule Localization Microscopy

a λ fl λ The schematic in the second column indicates the laser excitation wavelength ( ex) and maximal uorescence 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. NR indicates value not reported. bPhoton statistics reported in the presence of the GLOX (glucose oxidase with catalase) oxygen scavenging system and β- mercaptoethanol. cΦ increases upon rigidification. dΦ increases upon binding to protein. relationship between various proteins that are involved in dynamics of hemagglutinin.21 A more fruitful application to adhesion complexes.14 Improvements in z-resolution initially live-cell imaging has come from combining the concept of used astigmatism to image the bowl-shaped, cage-like structure SMLM with single-particle tracking (spt) experiments. This of CCPs.15 Further improvements in 3D reconstruction “sptPALM” enables the imaging of the trajectories of incorporate a number of optical strategies, including double- thousands of individual proteins with densities up to 50 plane detection,16 two-photon activation by temporal focus- molecules/μm2 by stochastically activating a subset of dyes, ing,17 the double-helix point spread function,18 and inter- imaging them until they bleach, and repeating the process until ferometry,19 to enable image of samples with depths of a few the entire pool of labeled protein has been interrogated. These microns. experiments generate spatially resolved diffusion maps inside a In addition to limitations on color and z-resolution, early cell, providing local context for individual molecules and SMLM experiments were performed exclusively on fixed cells, shedding light on the dynamic heterogeneities present inside largely due to the long acquisition times (e.g., typically minutes cells.22,23 to hours) required to collect enough localizations to generate SMLM has moved beyond proof-of-principle experiments, an image. In general, SMLM is still most useful for fixed enabling the discovery of new biological information. Zhuang samples, but live-cell SMLM experiments have yielded discovered novel ring-like actin filaments encircling the axon biological insight, especially when studying relatively slow with 180 nm periodicity.24 Doksani visualized telomeres inside cellular processes such as the formation and evolution of cells to assess the role of shelterin components in the t-loop adhesion complex proteins20 and nanoscale distribution and formation.25 Ellenberg and Löschberger used SMLM to refine

1079 DOI: 10.1021/acschembio.9b00197 ACS Chem. Biol. 2019, 14, 1077−1090 ACS Chemical Biology Reviews

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 λ chromophore. (g) Photoconversion of Dreiklang through hydration/dehydration. The colored shading of the structures indicates em. the molecular structure of the nuclear pore.26,27 Although ■ THE PRINCIPLES OF SMLM many more such discoveries lie in wait, the future of SMLM is In a fluorescence microscope, the focused excitation light is likely in lock-step with electron microscopy (EM). As transmitted via a high numerical aperture (NA) objective to demonstrated in the original PALM paper,6 correlative light the image plane to produce a diffraction pattern, referred to as and electron microscopy (CLEM) is an exciting technique that the point spread function (PSF). This PSF takes the shape of a ff combines the molecular information on light microscopy with bright disk surrounded by dimmer higher-order di raction the ultrastructural information on EM.28 New sample rings (i.e., the Airy disk). The radius of the disk depends on the 29 30 wavelength of the light source and is approximately equal to preparations, microscopy setups, and osmium tetroxide 0.61λ/NA laterally and 2λ/NA2 along the optical axis, where λ fl 31 resistant photoconvertible uorescent proteins portend the is the wavelength of excitation light and NA is the numerical future of super-resolution imaging, where SMLM is used to aperture of the objective. For example, imaging a single color in the familiar grayscale EM images of cells and tissue. fluorophore (diameter ≈ 1 nm) using a common oil-

1080 DOI: 10.1021/acschembio.9b00197 ACS Chem. Biol. 2019, 14, 1077−1090 ACS Chemical Biology Reviews

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 “ ” λ lactams and benzoxazines. (d) Exemplar caged dyes and their photoactivation. The colored shading of the structures indicates em. immersion objective (NA = 1.4) and a standard excitation activated stochastically with continuous fluorescence imaging. source (λ = 488 nm) will produce a PSF ∼200 nm in the This switching is typically light-induced, but dyes that activate − ∼ 32 33 lateral (x y) dimension and 500 nm in the axial (z) through enzymatic activity or binding events have been dimension. This disparity between the size of the molecule and used in SMLM. Each frame of the resulting movie is then the size of the PSF renders it fundamentally impossible to λ analyzed by software to localize individual molecules. These resolve two molecules that are within this distance of 0.61 / fi ’ NA (i.e., the diffraction limit). This makes it difficult to retrieve analyses perform a statistical t using the microscope s PSF to fl pinpoint the location of the fluorophore with greater precision the information from individual uorophores in a densely 34 labeled sample. than conventional imaging. The individual localizations are An SMLM imaging session begins with labeling a biological then combined to build up a high resolution map of molecular sample with a suitable switchable fluorophore, which is then location: the super-resolution image.

1081 DOI: 10.1021/acschembio.9b00197 ACS Chem. Biol. 2019, 14, 1077−1090 ACS Chemical Biology Reviews

As with all fluorescence microscopy, the photophysical of different colors, (3) photochromic FPs (PC-FPs) that properties of the fluorophore labels are crucial for SMLM. undergo a reversible transformation between an off-state and These are summarized in Tables 1 and 2 and include the laser an on-state, and (4) rarer, more complicated photoactivatable/ λ fl excitation wavelength ( ex), the uorescence emission photoconvertible FPs that undergo an irreversible trans- λ ffi λ ε maximum ( em), the extinction coe cient at max ( ), and formation between two on-states, each of which can be the fluorescence quantum yield (Φ). Properties specificto reversibly switched to an off-state. In addition to the many fluorophores activated with light include: the fluorescence modes of switching, FPs have the advantage that they are contrast upon photoactivation (Fon/Foff), the turn-on (t1/2on) genetically encoded and can be expressed as a fusion to a halftime, the photobleaching halftime (t1/2PB), and the photon protein of interest in a variety of cells and in vivo using the yield of individual fluorophores before bleaching (N). The ever-growing molecular biology toolbox. Table 1 lists the maturation halftime (t1/2M) is an important consideration for photophysical properties of several widely used FP-based labels fluorescent proteins. For caged dyes, a critical factor is the for SMLM, and Figure 1 illustrates the photochemistry behind Φ fl photoactivation quantum yield ( PA). We note the switching several classes of uorescent proteins, which can be categorized between the dark and fluorescent states can be reversible or into the following mechanisms: decarboxylation, β-elimination, irreversible. Key properties of reversible photoswitchable dyes oxidative “redding”, hydration/dehydration, or cis−trans ff ff include the turn-o (t1/2o ) and the fatigue resistance (FR; isomerization. defined as the number of switching cycles needed to bleach Small-molecule fluorescent dyes also fall into a relatively 50% of the initial fluorescence). For small-molecule dyes that small number of categories: (1) activator−reporter dye pairs switch reversibly under specific conditions, key properties where an “activator” dye placed within 1−2 nm of the include the on/off “duty cycle” (on/off)the fraction of time a fluorophore can modulate its photoactivation rate, (2) fluorophore spends in the fluorescent state vs the non- activator-free dyes that stochastically activate under continuous fluorescent “dark” stateas well as the average number of laser illumination, (3) spontaneously blinking dyes that switching cycles (SC) these dyes undergo before bleaching and stochastically and reversibly activate in the absence of light, the survival fraction (SF) of fluorophores after relatively harsh (4) photochromic dyes that undergo light assisted reversible imaging conditions. Irreversible switching dyes are perhaps transformation between an on- and off-state, (5) photo- more useful in experiments where counting of single molecules activatable or “caged” dyes that irreversibly activate upon a is desired; reversibly switching dyes can also be effective but photochemical reaction. Of course, chemical dyes cannot be their use in counting requires additional calibration.35 Of genetically encoded, and the most established and flexible course, because SMLM is based on the localization of labeling strategy is the use of antibodies.39 This is largely individual molecules, the stochastic activation must be sparse restricted to fixed samples, however, and lowers localization enough to ensure that simultaneous activation of two precision due to the large size (200 kDa) of the antibody−dye molecules in the same diffraction limited spot is minimized. conjugate. For live cell experiments, several hybrid approaches As a general rule, the fluorophore’s on-switching or activation have been developed that exploit a specific interaction between rate should be much smaller than their off-switching or genetically encoded proteins and small-molecule dyes. These bleaching rate with a duty cycle between 10−4 and 10−6.36 range from non-natural amino acids, streptavidin−biotin Another key metric in determining the quality of SMLM interactions, enzyme-based self-labeling tags, and fluorophore imaging is the precision of the localization (σ). This is ligase strategies.40 Table 2 lists the photophysical properties of inversely proportional to the square root of the number of dye labels for SMLM, and Figure 2 details the chemistry photons detected per activated fluorophore: σ ∝ s/(N1/2); behind their photochemical behavior. Our goal is to provide a where s is the standard deviation from the localization analysis primer for chemical biologists interested in SMLM and and N is the photon yield or number of photons emitted by the complement other recent reviews on protein and small- fluorophore per localization event. This simplification is true molecule probes for super-resolution microscopy.41,42 only in thin samples with low background fluorescence, and many parameters determine the actual localization precision.37 ■ FLUORESCENT PROTEINS In principle, it is possible to achieve a resolution that is two Background and Photoactivatable Fluorescent Pro- ff orders of magnitude higher than di raction-limited imaging teins. Green fluorescent protein (GFP) folds into a β-barrel throughtheuseofSMLMwithhighphoton-yielding structure, which orients a three amino acid sequence (Ser-Tyr- fl fl uorophores; in general, a uorophore must emit at least Gly) to generate the GFP chromophore following cyclization, 100 photons to enable super-resolution imaging. Finally, oxidation, and dehydration steps (Figure 1a).43 Additional another important consideration for SMLM is labeling density. colors can be generated by modifying this canonical Labeling density is governed by the Nyquist criterion, which chromophore structure (e.g., cyan fluorescent protein, CFP) indicates that to achieve detailed structural information, the or extending its conjugation (e.g., mOrange or Kaede; Figure mean separation between fluorescent labels must be no greater 44 π 20,38 1b). The optical properties can also be altered by -stacking than half of the desired spatial resolution. and/or electrostatic interactions with nearby amino acids, and fi modi cation of chromophore pKa. The advent of GFP and its ■ FLUOROPHORES AND LABELING STRATEGIES ilk revolutionized live-cell imaging and sparked intense effort Fluorophores employed in SMLM fall into two classes: into the discovery and evolution of new FPs with novel fluorescent proteins (FPs; Table 1) and small-molecule properties. Several PA- and PS-FPs were described at the turn fluorescent dyes (Table 2). FPs can be divided into the of the 21st century, shortly after the use of GFP became following types: (1) photoactivatable FPs (PA-FPs) that mainstream. The initial class of light-modulated fluorescent undergo irreversible switching between a dark off-state and a proteins included PA-GFP,5 Kaede,45 and KFP1.46 These bright on-state, (2) photoswitchable FPs (PS-FPs) that proteins were initially used as “optical highlighters” as an undergo an irreversible transformation between two on-states alternative to fluorescence recovery after photobleaching

1082 DOI: 10.1021/acschembio.9b00197 ACS Chem. Biol. 2019, 14, 1077−1090 ACS Chemical Biology Reviews

(FRAP) experiments.47 PA-GFP5 is a particularly excellent elicits a bathochromic shift to 511 nm, aligning it with PA- Φ 48 57 fl ∼ label: it is monomeric and exhibits a high PA and a GFP. The Fon/Foff uorescence contrast of PS-CFP2 is 20 reasonable photon yield (∼300).36 Due to these properties, it times higher compared to PA-GFP but exhibits a lower remains, after almost two decades, as one of the few reliable quantum yield and smaller photon yield than PA-GFP. Despite dark-to-green PA-FPs and was used in two of the three initial this poor brightness and photostability, PS-CFP2 exhibits an reports of SMLM.6,7 excellent on/off duty cycle (10−6), which is 3 orders of The molecular basis behind the photoconversion of PA-GFP magnitude lower than that of PA-GFP.36 Cells labeled with PS- has been elucidated. Wild-type GFP exists as a mixture of two CFP2 can tolerate significantly higher labeling densities in speciesthatdiffer by the protonation state of the SMLM and are compatible with red FPs.14 λ chromophore: a neutral species that absorbs in the UV ( max The PS-FPs mEos3.2, Dendra2, mClavGR2, and mMaple3 λ = 397 nm) and a red-shifted anionic species ( max = 475 nm) are part of the Kaede family, which contain a His residue with a pKa of 6.3. Exposing GFP to strong UV illumination instead of the Ser residue present in GFP. This substitution elicits a photoinduced decarboxylation at Glu222 (Figure allows formation of a chromophore with extended conjugation 1c),49 which results in a rearrangement of the hydrogen and red-shifted wavelengths (Figure 1b). In the preactivated bonding network around the chromophore in a manner that state, this family of PS-FPs contains a GFP-like chromophore stabilizes the anionic form, thereby increasing the absorption at but upon irradiation, the excited-state quinone methide − 475 nm. The pKa of wild-type GFP is relatively high leading to intermediate undergoes cleavage at the Nα Cα bond of the a nonzero absorption at 475 nm, making the on−off contrast His to eliminate a carboxamide containing peptide. The insufficient for SMLM. PA-GFP was developed by introducing subsequent loss of a proton yields a trans double bond between a mutation (T203H) that further shifts the equilibrium to the the Cα and Cβ of the His, leading to extended conjugation and 5 58 neutral species and increases the Fon/Foff contrast to 60-fold. red-shifted wavelengths (Figure 1e). Given the initial success with PA-GFP and the need for The highly evolved mEos3.2 is the result of numerous multicolor imaging, efforts were directed toward developing a mutations to reduce the formation of multimers, maturation monomeric red-shifted equivalent. Founded on the heroic time at physiological temperatures, and localization of protein development of mRFP1,50 the first monomeric orange fusions in the cell. mEos3.2 is a highly monomeric, bright, and photoactivatable FP, PAmRFP1, was developed;51 it was rapidly maturing protein that exhibits high photon yields and followed by variants with improved photon yields, including excellent contrast for SMLM with high labeling density.59 It is the orange PAmCherry152 and PATagRFP53 as well as the red currently considered the best green-to-red PS-FP and one of PAmKate.54 The mechanism behind activation of PAmKate the most-used FPs in SMLM, with demonstrated localization and PAmCherry1 is similar to PA-GFPdecarboxylation of an accuracies up to 12 nm in the lateral dimension.59 Although analogous Glu side chain;55 PATagRFP functions through a mEos3.2 has 2× the photon yield of mClavGR260 and 1.2× completely different mechanism involving oxidative “redding” higher yields than Dendra257 and mMaple3,36 these related (Figure 1d).IncontrasttoGFP,thechromophorein PS-FPS have unique properties that make them valuable PATagRFP features two aromatic systems that are separated alternatives to mEos3.2. Dendra2 can be activated by longer by a saturated methylene spacer. Upon photoactivation, wavelength, less phototoxic 488 nm light, facilitating live-cell PATagRFP generates the chromophore via two one-photon SMLM. mMaple3 exhibits a very low on/off (10−7), which is mediated oxidative steps; the first photon catalyzes the an order of magnitude lower than mEos3.2 and Dendra2 and is oxidization of the N-acylamine moiety, extending the useful in densely labeled samples.36 The red form of chromophore’s conjugation, while the second photon gen- mClavGR2 exhibits high photostability, which could be useful erates the DsRed-like chromophore by oxidizing the Tyr in single particle tracking experiments. methylene unit.53 In addition to the desirable red-shifted Finally, the DsRed-like PSmOrange61 and its enhanced absorption and emission wavelengths, the orange PAmCherry1 version PSmOrange262 switch from an orange state to a far-red and PATagRFP and red PAmKate also exhibit higher on−off state upon excitation with blue-green light, emitting at 662 nm. contrasts and photon yields when compared to PA-GFP, This far-red emission is distinct from other FP-based SMLM − enabling high quality SMLM imaging in cells.36,52 54,56 labels, making it a good imaging partner in multicolor super- Photoswitchable Fluorescent Proteins. PS-FPs that resolution microscopy. It can also be activated efficiently with convert between two fluorescent states of different colors are two-photon excitation, allowing for spatial control of activation also useful for SMLM. A general disadvantage with using PA- in thicker samples. Similar to PATagRFP, PSmOrange FPs in SMLM is the absence of fluorescence in the dark state, generates the red chromophore via oxidative reddening, which makes it difficult to identify a field of view containing where two consecutive oxidation steps cleave the peptide cells that are expressing the PA-FP prior to committing to a backbone and prompt the formation of an atypical oxazolone long imaging experiment. PS-FPs circumvent this problem ring (Figure 1d), which is red-shifted relative to the DsRed because their initial, preactivated form is fluorescent. chromophore.61 This desirable far-red emission is countered Activation with ultraviolet or blue light substantially shifts by PSmOrange’s low photostability and reduced photon yields the absorption maxima of these PS-FPs to the red, providing as well as the dependence of the photoconversion efficiency on the requisite Fon/Foff contrast for high quality SMLM. Three redox environment. different types of PS-FPs are (1) the GFP-like cyan-to-green Photochromic Fluorescent Proteins. PC-FPs exhibit FPs such as PS-CFP2; (2) the Kaede-like green-to-orange/red reversible switching between two states,63 and those detailed FPs Dendra2, mEos3.2, mClavGR2, and mMaple3; and (3) below switch between a nonfluorescent dark state and a the DsRed-like green-to-far red protein, PSmOrange. fluorescent bright state. The reversible transition means that a λ In its preactivated state, PS-CFP2 emits in the cyan ( em = population of molecules can be activated thermally in the 468 nm). Illumination with violet light causes a decarbox- steady state but also allows switching to the dark state using ylation that changes the structure of the chromophore and brief illumination prior to imaging. Although beyond the scope

1083 DOI: 10.1021/acschembio.9b00197 ACS Chem. Biol. 2019, 14, 1077−1090 ACS Chemical Biology Reviews of this review, PC-FPs are useful in nonlinear super-resolution which avoids UV or violet activation light, is substantially microscopy techniques such as reversibly saturatable optical brighter than rsCherry (2.5×) or rsCherryRev (10×), and has fl 64 × fl uorescence transition (RESOLFT) and nonlinear struc- 2 Fon/Foff contrast due to lower residual uorescence in the tured illumination microscopy (NL-SIM).65 Unlike SMLM, off state. While most red-shifted PC-FPs possess inferior these techniques involve multiple switching cycles and thus photophysical properties compared to the green PC-FPs, they require the high fatigue resistance of fluorescent proteins like can still prove useful in two-color super-resolution imaging as Skylan-S and Skylan-NS66,67 or the fast switching kinetics of demonstrated with imaging nonraft microdomains in the rsFastlime and rsEGFP.68,69 plasma membrane using Dronpa and rsTagRFP.79 Most PC-FPs switch through a general cis−trans isomer- Photoactivatable/Photochromic FPs. Occupying a class ization of the chromophore (Figure 1f).70 Prior to irradiation, of their own, mIrisFP80 and NijiFP81 combine the photo- Dronpa’s chromophore assumes a GFP-like cis-confirmation in switching behavior of both PC- and PS-FPs. These proteins the predominantly deprotonated bright state. Absorption of a can be photoactivated from dark-to green using UV light and blue photon elicits an isomerization to the trans-form which then undergo further photoconversion from green-to-red upon has a different absorption spectrum, lower quantum yield, and excitation. This combination allows sophisticated experiments fl higher pKa, leading to low uorescence. This isomerization can that combine super-resolution microscopy and dynamic be reversed by absorption of a UV photon. Although other FPs imaging in living cells. In such an experiment, the protein-of- exhibited hints of photochromism,71 the dark-to-green Dronpa interest is expressed as a fusion with a PA/PC-FP. Live-cell was the first widely used PC-FP in cellular biology and super- SMLM images are generated by activating the green form of resolution imaging.72 The monomeric Dronpa is 6× brighter the FP. This is followed by switching a subset of the FP- than activated PA-GFP, exhibits a fast t1/2on, a relatively slow protein fusion to the red form and monitoring the migration of ff ff t1/2o , and a high photon yield. Over 90% of the expressed these proteins by switching the red form on and o . Both Dronpa proteins undergo only one blinking event, which mIrisFP and NijiFP are at least as bright as PA-GFP, and enables counting of individual molecules using SMLM.73 mIrisFP exhibits a relatively high photon yield that allows for Dronpa underwent extensive protein engineering, producing high-quality SMLM. variants with 45× faster switching kinetics (rsFastlime68), a positive switching mode (Padron),74 a broader absorption ■ SMALL-MOLECULE FLUOROPHORES 74 ff 73 spectra (bsDronpa), and extended t1/2o (mGeos-M). The Background. Small-molecule dyes activated by light ff increased t1/2o of rsFastlime decreases photon yield but predate SMLM by decades, being used for optical recording 69 enables faster SMLM experiments as do other fast switching media, organic electronics, and unraveling the dynamic 75 PC-FPs such as rsEGFP and rsEGFP2. Padron maintains the processes in cells.82,83 Small-molecule fluorophores were also rapid switching kinetics and brightness of rsFastlime, adding the focus of ground-breaking single-molecule microscopy × 74 84 the highest Fon/Foff contrast (150 ) of extant PC-FPs. In experiments by Moerner at cryogenic temperatures and 73 ff contrast, mGeos-M exhibits a t1/2o that is 52% longer than later examples by Betzig at ambient temperature using near- Dronpa, which enhances the photon yield and thus localization field optical microscopy.85 Over time, improvements in optical precision. A limitation of Dronpa and its variants is low hardware allowed for such imaging to be performed in aqueous photostability; bacterial colonies expressing Dronpa lose half of media under ambient conditions.86 Despite this availability of their initial fluorescence after only 4 switching cycles and only both light-activated dyes and the ability to image single rsFastlime shows modestly better photostability. Stable PC-FPs molecules, SMLM was founded largely on activatable FPs due do exist, however, as bacterial colonies expressing rsEGFP, to their ease of use and facile optimization through directed EGFP2, and Skylan-S maintain half their initial fluorescence evolution. Overall, small-molecule dyes for SMLM have lagged after 1200, 2100, and 7000 switching cycles, respectively. behind FPs in breadth, characterization, and publications; this As mentioned above, most PC-FPs use cis−trans isomer- field remains an active and important area of chemistry ization to modulate fluorescence. An exception is Dreiklang, research.42,87 which uses a reversible, light-mediated hydration reaction at When compared to their protein counterparts, fluorescent the imidazolinone ring (Figure 1g).76 Irradiation with violet small-molecules typically possess higher brightness, photo- light facilitates water attack, converting the imidazolinone ring stability, and photon yields. Small-molecule fluorophores are into a 2-hydroxyimidazolidinone, which disrupts the chromo- also more versatile in that they allow the use of the entire phore conjugation and decreases fluorescence; UV light chemical lexicon rather than the meager 20 proteogenic amino reverses this hydration reaction. This unique property acids. Improvements in chemistry and a growing under- decouples the imaging light from the photoswitching light, standing of structure−activity relationships in chemical dyes alleviating a serious limitation in PC-FPs whose fluorescence allow the design and synthesis of rationally designed readout and off-switching are interlocked. This unique derivatives with finely tuned properties. Nevertheless, small- switching modality, coupled with a high photon yield of the molecule fluorophores have several severe disadvantages on-state, allowed high resolution SMLM of microtubules and compared to fluorescent proteins. Because small molecule keratin with 15 nm precision. Nevertheless, this requisite UV dyes cannot be genetically encoded and fused to a protein of light and the temperature dependence of Dreiklang’s photo- interest, they necessitate chemical synthesis and specialized physics can complicate imaging of live cells. labeling strategies.40 They also can suffer from nonspecific Finally, red-shifted PC-FPs such as rsCherry and rsCherryR- binding and cellular toxicity, which can be detrimental to a ev77 have also been developed, showing good photon yields sensitive imaging technique like SMLM. and fast switching kinetics. Their photophysics is complex, Activator−reporter Dye Pairs. One of the first classes of however, with these proteins showing intensity-dependent small-molecule fluorophores for SMLM were based on the switching and brightness.52 One useful PC-FP that avoids this cyanine-based “CyDyes”. Zhuang showed that biomolecules complex switching behavior is the dark-to-orange rsTagRFP,78 labeled with two different cyanine dyes such as Cy3 and Cy5

1084 DOI: 10.1021/acschembio.9b00197 ACS Chem. Biol. 2019, 14, 1077−1090 ACS Chemical Biology Reviews showed a switching phenomenon where the primary Cy5 Urano group set out to design a dye that would be in “reporter” fluorescence could be modulated by excitation of equilibrium between a nonfluorescent and fluorescent form the secondary Cy3 “activator” dye.88 This switching behavior and therefore blink spontaneously. They found that hydrox- was initially proposed as a complement to Förster resonance ymethyl (HM) rhodamines were an excellent scaffold and energy transfer (FRET) due to the higher distance dependence devised methods to tune the equilibrium constant of of this phenomenon. The utility of this system for SMLM was intramolecular spirocyclization (Kcycl). This resulted in the soon realized, however, and a number of pairs were identified development of hydroxymethyl-Si-rhodamine (HM-SiR; Fig- using shorter wavelength activator dyes (e.g., Cy2, Cy3, or the ure 2b) and other related compounds.101,102 The transient pyrene-based Alexa Fluor 405) and longer wavelength reporter lifetime of the “open” fluorescent form (50−150 ms) allows for dyes such as Cy5, Cy5.5, Cy7, and the Cy5 analogue Alexa high-quality SMLM with excellent photon yields (∼2600) even Fluor 647. This dual label system could be easily implemented under substantially reduced illumination intensities.102 More by using antibodies labeled with combinations of activator and recently, this compound class has been used for live-cell and reporter dyes, enabling multicolor, three-dimensional two-color SMLM by exploiting changes in chemical environ- SMLM.8,12,15 ment to further modulate the blinking properties.101,103 dSTORM Dyes (i.e., “Activator-Free” Dyes). Concurrent Photochromic Dyes. In addition to the discovery of new with the development of activator−reporter pairs, Sauer switching mechanisms and spontaneously blinking dyes, discovered that single cyanine dyes could be photoswitched SMLM has also renewed interest in classic light-activated under reducing conditions without an activator dye, albeit with dye systems, including reversible photochromic dyes. Rhod- substantially higher laser powers.11,89 This was later expanded amine lactams, first introduced in 1977,104 undergo transient fl ∼ to other small molecule uorophore types, yielding the general (t1/2 ms) lactam bond cleavage upon illumination with far- dSTORM method for SMLM using commercial fluoro- UV light. This fluorescent form thermally reverts back to the phores.90,91 An extensive survey of the properties of 26 colorless lactam. This transient fluorophore formation is ideal commercially available fluorophores was published92 that for SMLM imaging, and recent advances have extended the identified the best dyes in four spectral windows: ATTO 488 activation wavelength using phthalimide substituents on (green), Cy3B (yellow), Alexa Fluor 647 (red), and Dy- rhodamine B (RhB phthalimide, Figure 2c), allowing activation Light750 (near-infrared). In general, far-red dyes perform best with near-UV (375 nm) or 2-photon excitation (747 nm). RhB in SMLM with Alexa Fluor 647 exhibiting the highest photon phthalimide enabled optical sectioning and resolved the yields (∼5200), making it the best dye for dSTORM and the structure of microtubules with 55−70 nm localization most commonly used SMLM label; we note the use of single- precision and good photon yield (∼900).105 This strategy dye label systems has largely replaced the earlier activator− has been extended to rhodamines with different spectral reporter pairs. properties,106,107 and use of a stilbene substituent allowed dSTORM requires a specific chemical environment to elicit activation with visible light.108 switching behavior, and the imaging buffer typically contains a Another classic photochromic system involves benzooxazine cocktail of additives: oxygen scavengers and triplet quenchers that preferentially adopts a nonfluorescent bicyclic oxazine along with reducing agents such as primary thiols93 and form (e.g., in OA-2, Figure 2c). Upon illumination, a − phosphines.94 96 For some rhodamine-based dyes, the photoinduced excited-state electron exchange reaction opens mechanism of photoswitching relies on the formation of the oxazine ring, restoring the fluorophore conjugation and transient, nonfluorescent radicals as evidenced by electron eliciting a large increase in fluorescence.109 Although these paramagnetic resonance studies;97 these species can revert systems exhibit relatively fast switching kinetics and high back to a fluorescent form spontaneously or in a light-mediated photostability, which is important for SMLM, the relatively low manner (Figure 2a). For cyanine dyes, the photoswitching brightness, high hydrophobicity, and high background have could stem from direct reaction of the reducing agents with the limited their utility in super-resolution imaging.110 Finally, fluorophore. For example, irradiation of Cy5 in the presence of unlike the rhodamine lactams and benzooxazines, which primary thiols produced dark photoproducts presumably thermally revert back to their nonfluorescent forms, diary- arising from thiol addition to the polymethine bridge (Figure lethenes can be driven in both directions with light. Like the 2a).93 Likewise, water-soluble phosphines such as tris(2- benzoxazine compounds, diarylethenes have been limited by carboxyethyl)phosphine (TCEP) can quench the fluorescence both water solubility and low fluorescence quantum yields. of Cy5 and other cyanines through light-independent addition Steady improvements in both these properties have begun to 111,112 to the C4 of the polymethine bridge. UV-illumination of the yield results in SMLM. dye−phosphine adduct restored the cyanine’s fluorescence in a Photoactivatable Dyes. Photoactivatable (or “caged”) reversible fashion.94 In a related technique, treatment of small-molecule fluorophores incorporate a photolabile moiety rhodamine- or cyanine-labeled samples with sodium borohy- that is removed or modified upon illumination with light. This dride yields nonfluorescent “leuco” dyes (Figure 2a). irreversible photoactivation is especially useful for sptPALM Illumination of these reduced fluorophores with UV or violet experiments, and the three most common chemical caging light allows SMLM with a high fraction of recovered molecules modalities are o-nitrobenzyl (o-NB) derivatives, 2-diazoke- (40−66%),95,96 and new rigidified cyanine dyes show excellent tones, and azides. The o-NB moiety predates caged dyes, being performance using this strategy.98 used as a photolabile protecting group for diverse chemical Spontaneously Blinking Dyes. dSTORM dyes rely on functionalities.113,114 To prepare caged fluorophores, the o-NB light and/or the presence of additives in the imaging buffer to is attached to the phenolic oxygen of fluoresceins as an ether or induce photoswitching. Both of these complicate SMLM in live the aniline nitrogens of rhodamines as a carbamate link- − cells, although in some cases, the reducing environment of the age.115 117 Examples of fluorophores that were successfully cytosol is sufficient to allow live-cell super-resolution imaging caged with o-NB and utilized in SMLM include Oregon Green, 99,100 using conventional dyes. To circumvent this problem, the rhodamine 110 (Rh110), Q-rhodamine, carborhodamine 110,

1085 DOI: 10.1021/acschembio.9b00197 ACS Chem. Biol. 2019, 14, 1077−1090 ACS Chemical Biology Reviews

116,118−121 and Si-Q-rhodamine (SiRhQ, Table 2). In general, molecules in a sparse and controlled manner. Continuing this caged dyes show excellent Fon/Foff contrast and good photon productive marriage of optical physics, chemistry, and biology yields. For example, photoactivated Rh110 emitted an average will unravel more molecular details underpinning living ∼ 118 − of 3500 photons, giving 16 nm precision. NVOC2 SiRhQ systems. (Figure 2d)exhibited a photon yield 1.6× higher than Alexa Fluor 647 without the need for a dSTORM buffer; addition of ■ AUTHOR INFORMATION antioxidants increased the photon yield to ∼1.5 × 104. Corresponding Author Similar to o-NBs, 2-diazoketone caging groups require UV or *E-mail: [email protected]. violet light to undergo photolysis. However, their smaller ORCID chemical footprint has some advantages: increased solubility 122 Fadi M. Jradi: 0000-0001-7633-331X and lower byproduct toxicity. This strategy is modular and Luke D. Lavis: 0000-0002-0789-6343 relatively straightforward, allowing caging of a variety of rhodamines with different spectral properties.123 Despite the Notes clear advantages of this method, however, 2-diazoketone-caged Theauthorsdeclarethefollowingcompetingfinancial rhodamines have a severe limitation: complex photochemistry. interest(s): L.D.L. has filed patents and patent applications Photolysis yields two major products: a fluorescent phenyl- for various fluorophores mentioned in this review. Their value acetic acid rhodamine and a “dark” inden-1-one substituted could be affected by this publication. dibenzooxepine.122,124 The fluorescent species results from a Wolff rearrangement, whereas the dark product presumably ■ DEDICATION results from a different rearrangement of the carbene Dedicated to Professor Ronald T. Raines on the occasion of his intermediate (Figure 2d). 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