Probes for Single-Molecule Superresolution Microscopy
Several prominent fluorophore classes have emerged as the best candidates for labeling subcellular targets in PALM and related techniques. These include genetically-encoded fluorescent protein fusions, synthetic dyes, quantum dots, and hybrid systems that combine a genetically-encoded target peptide with a separate synthetic component that is membrane permeant (such as the FlAsH/ReAsH system). Each specific class of fluorescent probes has its particular strengths and weaknesses, however no single class or individual fluorophore has yet been developed that combines all the preferred characteristics of an ideal probe for any application in single-molecule superresolution microscopy. The fundamental consideration for developing superresolution probes is that they must be capable of being either photoactivated, photoswitched, or photoconverted by light of a defined wavelength band as a means to alter their spectral properties for the detection of selected subpopulations.
Among the most desirable attributes for singe-molecule superresolution probes are very high brightness and contrast levels, which are necessary to maximize the number of photons that can be detected per molecule before it photobleaches or reverts to a dark, non-fluorescent state. Brightness is determined by the product of the molar extinction coefficient (εabs) and the fluorescence quantum yield (φ). Thus, the best probes have high extinction coefficients and quantum yields and provide excellent contrast over the background. In general, the brightness levels of popular synthetic dyes, such as Cy5, ATTO 650, and Alexa Fluor 488, are very high due to extinction coefficients averaging 100,000 and quantum yields exceeding 0.90. Likewise, quantum dots have equally high brightness levels. In contrast, the optical properties for optical highlighter fluorescent proteins are much less optimal with extinction coefficients ranging from approximately 15,000 to 85,000 and quantum yields between 0.05 and 0.80. Clearly, there is a significant need to genetically engineer more useful fluorescent proteins for superresolution imaging.
In addition to having high brightness levels, the best probes for superresolution microscopy should exhibit spectral profiles for the active and inactive species that are sufficiently well separated and thermally stable so that spontaneous interconversion energies are very low compared with the light-controlled activation energy. Ideally, these probes should also exhibit high switching reliability, low fatigue rates (in effect, the number of survivable switching cycles), and switching kinetics that can be readily controlled. In terms of photobleaching or photoswitching to a dark state, the best probes are those whose inactivation can be balanced with the activation rate to ensure that only a small population of molecules is activated (to be fluorescent) for readout, and that these activated molecules are separated by a distance greater than the resolution limits of the camera system. Furthermore, each photoactivated molecule should emit enough photons while in an activated state to accurately determine their lateral position coordinates.
Besides displaying the necessary fluorescence emission and other photophysical properties, PALM superresolution probes must also be capable of localizing to their intended targets with high precision and exhibit the lowest possible background noise levels. Fluorescent proteins, hybrid systems, and highly specific synthetic fluorophores (such as MitoTrackers) are able to selectively target protein assemblies or organelles, but most of the cadre of synthetic dyes and quantum dots must first be conjugated to a carrier molecule for precise labeling. In many cases, the exact proximity of the fluorescent probe to the target is questionable, as is the number of actual fluorophore units involved, especially when small synthetic dye molecules or quantum dots are conjugated to large antibodies. Also, variations in photophysical properties (such as fluorescence quantum yield) induced by environmental fluctuations or intermolecular interactions can complicate data analysis. Finally, regardless of whether localization analysis is performed on fixed or living cells, autofluorescence arising from fixatives and transfection reagents can often produce excessively high background signal, thus reducing the localization accuracy.
A compilation of properties of the most useful fluorescent proteins and synthetic dyes for superresolution microscopy is presented in Table 1. Along with the common name and/or acronym for each fluorophore, the peak excitation (Ex) and emission (Em) wavelengths, molar extinction coefficient (EC), quantum yield (QY), relative brightness, number of photons emitted per molecule (N Photons), and physiologically relevant quaternary structure are listed. C-Rhodamine and C-Fluorescein refer to caged derivatives. The computed brightness values were derived from the product of the molar extinction coefficient and quantum yield, divided by the value for EGFP. The designation ND indicates the values have not been determined, whereas NA means that the value is not applicable for the listed probe. This listing was created from scientific and commercial literature resources and is not intended to be comprehensive. The excitation and emission peak values listed may vary in published reports due in some cases to the broad spectral profiles. In actual fluorescence microscopy investigations, the experimental brightness of a particular fluorophore may differ (in relative terms) from the brightness provided in this table. Among the many potential reasons for these differences are wavelength-dependent variations in the transmission or reflectance of microscope optics and the efficiency of the camera.
The intrinsic ability of a subset of fluorescent proteins to alter their spectral properties upon exposure to light of a specific wavelength coupled with their excellent targeting specificity has been widely exploited in superresolution imaging (see the probes listed in Table 1). Although fluorescent proteins are known to undergo a variety of light-induced photoswitching characteristics, including the generation of distinct emissive and non-emissive states as well as on-and-off blinking behavior, the most useful properties are photoactivation, photoconversion, and photoswitching, properties that can be collectively termed optical highlighting (as described below). Photoactivatable fluorescent proteins are capable of being activated from a dark state to a bright fluorescent state upon illumination with ultraviolet or violet light, whereas photoconvertible fluorescent proteins can be optically transformed from one fluorescence emission bandwidth to another. Among the most useful members in the toolbox of the photoactivatable fluorescent proteins for superresolution imaging are PA-GFP and PA-mCherry1. photoconvertible fluorescent proteins that have found utility in PALM and related techniques are tandem dimer Eos (tdEos), mEos2, Dronpa, rsCherry (and the reverse derivative), and PS-CFP2.
Members of the potentially most useful class of fluorescent protein optical highlighters, those that are capable of photoswitching between a brightly fluorescent and dark state, have not been particular useful in single-molecule superresolution investigations due to low photon output in the bright state. Dronpa was used in dual-color PALM imaging, as will be subsequently discussed, but is marginal when compared to most of the photoactivatable and photoconvertible fluorescent proteins in terms of photon output per molecule. However, Dronpa features very high photoswitching contrast and emits fluorescence for an extended period, which can be advantageous for live-cell imaging (for example, using single-particle tracking as described below). Aside from the requirement that fluorescent proteins must display some type of optical highlighting behavior for superresolution imaging, they must also have sufficient brightness, chromophore maturation rates, and monomeric character to express the fusion without artifacts, such as poor targeting and dysfunction. In addition, oligomerization in fluorescent proteins can present a problem in stochastic superresolution microscopy as there is more than one chromophore per each localized probe molecule.
A rapidly growing number of synthetic organic fluorophores are emerging as excellent candidates for single-molecule superresolution imaging, including many of the traditional probes that have been used for several decades in preparing specimens for widefield and confocal fluorescence microscopy (see Table 1). Many compounds that were originally thought to be photostable have since been demonstrated to enter dark states under low oxygen conditions in the presence of aliphatic thiols. Therefore, reversible photoswitching is probably a more widespread phenomenon than was originally suspected, which opens the door to an even larger number of potential probe candidates. In addition, irreversible photoactivation can be achieved by "caging" fluorophores with a protective reactive moiety that is removed by irradiation with ultraviolet light. Unfortunately, however, there have been no reports to date of organic compounds or quantum dots that are capable of being photoconverted from one emission wavelength band to another.
Illustrated in Figure 6 are the ribbon structures, molecular models, and/or cartoon drawings of the various fluorescent probes that are potentially useful in single-molecule superresolution imaging. The synthetic carbocyanine dye, Cy5 (Figure 6(a)) is significantly smaller than a yellow quantum dot (Figure 6(c)) or the typical beta-barrel can-shaped polypeptide architecture of a fluorescent protein (green ribbon structure; Figure 6(d)). Likewise, all of these probes are reduced in size compared to a primary or secondary IgG antibody, which can span 12 to 15 nanometers across (red ribbon structure; Figure 6(b)). Quantum dots must be treated with antibody fragments, streptavidin, phalloidin, or some other functional moiety in order to target specific sub-cellular regions. In many cases, the combined size of a quantum dot conjugated to one or more secondary antibodies can range up to 25 to 30 nanometers, which dramatically exceeds the size of a monomeric fluorescent protein. The smallest fluorescent probes are the synthetic dyes (Figure 6(a)), but these feature relatively poor targeting efficiency and generally generate large background signal.
The primary advantage of using synthetic fluorophores and quantum dots over fluorescent proteins for superresolution imaging is their high intrinsic brightness, excellent photostability, good contrast, and greater fatigue resistance. For example, tdEos yields approximately 750 photons per molecule in contrast to the over 6000 photons typically observed for the photoswitchable fluorophore combination of Cy3 and Cy5. Furthermore, the carbocyanine dyes can undergo over 200 switching cycles before photobleaching. The primary disadvantage of using quantum dots and synthetic fluorophores is the difficulty in targeting specific locations and high background signal when compared to fluorescent proteins. The most reliable targeting strategy for fluorophores in this class is conjugation to a primary or secondary antibody, although several new synthetic dyes have been demonstrated to localize in specific organelles independently.
The downside of using antibodies and immunofluorescence techniques to label intracellular structures for superresolution imaging is that the proteins are too large to permeate membranes and are therefore only useful in fixed and permeabilized cells unless the target is displayed on the outer region of the plasma membrane. Labeling with antibodies is also relatively low in efficiency and adds 10 to 20 nanometers to the localization uncertainty between the label and target. In addition, conjugates of quantum dots to antibodies do not perform on par with analogous conjugates using synthetic dyes such as the Alexa Fluors and carbocyanines. In perspective, however, the use of antibodies to target synthetic probes for PALM and related techniques provides a much higher signal level than fluorescent proteins, which are more useful in live-cell imaging. Unfortunately, the oxygen scavengers and thiols necessary to produce photoswitching with popular synthetic dyes are incompatible with living cells, so until a workaround is developed, fluorescent proteins will be the probes of choice for live-cell superresolution microscopy.
Quantum dots are inorganic semiconductor nanocrystals composed of a cadmium selenide (CdSe) core surrounded by a zinc sulfide (ZnS) shell that exhibit fluorescent properties owing to confined exciton emission. A passivation layer and hydrophilic coating must be applied to quantum dots for biological applications, and they must also be conjugated to streptavidin or antibodies for targeting. The fluorescence emission profile of quantum dots is remarkably symmetrical and generally exhibits a large quantum yield, whereas their broad absorption profile enables them to be excited over an unusually wide wavelength range. The size of the CdSe core dictates the emission spectral profile, with smaller cores (ranging down to approximately 2 nanometers) emitting in the blue and cyan regions and larger cores (5 to 7 nanometers) emitting in the yellow and red wavelengths.
In general, the photostability for quantum dots dramatically exceeds that of all other known fluorophores, including synthetic fluorophores and fluorescent proteins, which creates a problem for stochastic superresolution imaging unless quantum dots can be converted into a photoswitchable state. Recently, investigators demonstrated that manganese doping of ZnSe quantum dots can be used to generate a species that can be reversibly photoswitched with high efficiency using light without the requirement for external activators or quenchers (effectors). Targeting remains a problem with quantum dots; however, continued advancements in quantum dot chemistry will undoubtedly lead to new and better probes in this class.
Among the synthetic reversibly photoswitchable probes that have found utility in superresolution imaging are rhodamine derivatives, carbocyanine dyes (Cy2 through Cy7), Alexa Fluors, and ATTO dyes, with new candidates being developed and tested on a continuous basis. The photoswitchable cyanine dyes have been the most extensively used synthetic organic probes in stochastic superresolution imaging. The near-infrared dye, Cy5, has seen the most duty and can be used without an effector, although combining Cy5 with a secondary chromophore (such as Cy3, Cy2, or Alexa Fluor 405) dramatically facilitates photoswitching. As an example application, combining Cy5 with Cy3 enables the use of a red laser (635 nanometers) to photoswitch Cy5 to a stable dark state, while exposure to 543-nanometer light converts Cy5 back to the fluorescent species. The rate of conversion back to the fluorescent species depends on the proximity of the secondary effector (Cy3).
Additional combinations of cyanine and Alexa Fluor dyes, such as Cy3 and Cy5.5 or Cy7 and Alexa Fluor 647 with Cy2 or Cy3, have been reported, greatly expanding the available color palette for superresolution imaging. Furthermore, a large number of commercially available synthetics, including most of the Alexa Fluor and ATTO dye families, have been demonstrated to reversibly photoswitch in fixed cells using a protocol that includes thiol-containing reducing agents (β-mercaptoethylamine, dithiothreitol, or glutathione) to generate a stable non-fluorescent state. Thus, the potential for generating synthetic organic photoswitchers for multi-color imaging, even though the switching mechanism has yet to be determined, currently surpasses the limited palette of FPs that undergo light-induced modulation. Presented in Figure 7 are the structures of several synthetic probes illustrating the wide diversity of fluorophores that have been found useful for superresolution imaging.
Similar to the photoactivatable FPs (PA-GFP and PA-mCherry), a large library of caged synthetics would be especially useful for stochastic superresolution imaging. Unfortunately, only a few promising candidates have been reported to date: caged versions of fluorescein and rhodamine, which have been demonstrated to act in a manner similar to PA-GFP in PALM imaging. In practice, the caged synthetics are liberated from their protective ester groups using irradiation with ultraviolet light to generate a fluorescent species exhibiting excellent contrast that can be localized with high precision and then photobleached. Until a larger variety of caged fluorophores emerges, this class will remain limited in superresolution imaging applications.
The future of superresolution imaging relies on increasing the specificity and labeling efficiency of very bright photoswitchable fluorophores while simultaneously decreasing the size of the targeting peptides. A wide spectrum of hybrid systems designed to couple synthetic fluorophores with genetically-encoded targets may one day be capable of achieving this goal. All of these systems utilize a peptide or protein sequence that is expressed in living cells and is capable of recruiting a small synthetic molecule to bestow fluorescence. The most developed candidate in this class utilizes a tetracysteine motif fused to a variety of genetic targets to recruit blue, green, or red fluorophores (CHoAsH, FlAsH, and ReAsH, respectively) capable of binding to the cysteine residues to generate a probe similar in specificity to FPs. The major disadvantage of these combinations is the inability to overcome the high background levels of unbound fluorophore that lower contrast. A number of other hybrid system candidates have been developed, but none have seen significant use in superresolution imaging.