Review pubs.acs.org/CR
Single-Molecule Localization Microscopy in Eukaryotes Markus Sauer*,† and Mike Heilemann*,‡ †
Department of Biotechnology & Biophysics, Julius-Maximilian-University of Würzburg, 97074 Würzburg, Germany Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, 60438 Frankfurt, Germany
‡
ABSTRACT: Super-resolution fluorescence imaging by photoactivation or photoswitching of single fluorophores and position determination (single-molecule localization microscopy, SMLM) provides microscopic images with subdiffraction spatial resolution. This technology has enabled new insights into how proteins are organized in a cellular context, with a spatial resolution approaching virtually the molecular level. A unique strength of SMLM is that it delivers molecule-resolved information, along with super-resolved images of cellular structures. This allows quantitative access to cellular structures, for example, how proteins are distributed and organized and how they interact with other biomolecules. Ultimately, it is even possible to determine protein numbers in cells and the number of subunits in a protein complex. SMLM thus has the potential to pave the way toward a better understanding of how cells function at the molecular level. In this review, we describe how SMLM has contributed new knowledge in eukaryotic biology, and we specifically focus on quantitative biological data extracted from SMLM images.
CONTENTS 1. Introduction 2. History of Single-Molecule Localization Microscopy 3. Principle of Single-Molecule Localization Microscopy 4. Photoswitchable and Photoactivatable Fluorescent Probes 4.1. Organic Fluorophores 4.1.1. Improving Performance of Organic Fluorophores for Single-Molecule Fluorescence 4.1.2. First Approaches to the Use of Organic Fluorophores as Photoswitches 4.1.3. Thiol-Induced Photoswitching Mechanism 4.1.4. Intrinsic Photoswitchable and Photoactivatable Fluorophores to Improve Localization Precision 4.2. Fluorescent Proteins 5. Super-Resolution Imaging in Eukaryotic Cells 5.1. Multicolor Imaging of Cellular Structures with SMLM 5.2. Imaging Proteins in the Eukaryotic Nucleus 5.3. Live-Cell SMLM with Organic Dyes 5.3.1. Imaging Cellular Dynamics by SingleMolecule Localization Microscopy 5.3.2. Photodamage in Live-Cell SMLM 5.4. Exploring Photobleaching, Transient Binding, and Fluorogenic Probes for SingleMolecule Localization Microscopy Methods 6. SMLM in Neuroscience 6.1. Molecular Organization of the Presynapse 6.2. Molecular Organization of the Postsynapse 7. Super-Resolution Imaging of Virus−Cell Interactions © 2017 American Chemical Society
7.1. Imaging HIV−Host-Cell Interactions 7.2. Imaging Other Virus−Cell Interactions 8. Extracting Quantitative Information from SuperResolution Microscopy Images 8.1. Extracting Copy Numbers from Fluorophore Blinking Statistics 9. Correlated Electron Microscopy with SMLM 10. Concluding Remarks Author Information Corresponding Authors ORCID Notes Biographies Acknowledgments References
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1. INTRODUCTION Images provide information. The level of detail is determined by the achievable image resolution, which, in turn, should match the feature size of the object studied. In cell biology, we encounter objects ranging in size from micrometers to nanometers, and we are interested in understanding how they are organized and how they interact with their environment. To obtain a comprehensive mechanistic picture of cellular processes, we need information at the level of cellular componentsorganelles, protein complexes and single proteinswhich range in size from a few to many nanometers. Understanding how biomolecules are organized into complexes and the dynamics of this organization is essential to understanding how a cell functions and which mechanisms
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Special Issue: Super-Resolution and Single-Molecule Imaging Received: September 30, 2016 Published: March 13, 2017
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molecules3 and realized the first successful detection of a single fluorophore in aqueous solution in 1990.4 In addition to his achievements in the field of diffraction-unlimited singlemolecule imaging by near-field scanning optical microscopy (NSOM),5 Betzig proposed in 1995 a general method for super-resolution microscopy exploiting different discrete features of otherwise-identical molecules within a focal region.6 That is, to make subdiffraction-resolution imaging possible, the fluorophores present in a focal region that label and define a structure have to exhibit spatially or temporally discrete spectroscopic characteristics. In the following years, scientists used this approach to push the limits of ultrahigh-resolution colocalization microscopy of two and more fluorophores exhibiting distinctive spectroscopic characteristics, for example, fluorescence spectra or lifetime.7−10 Already in the days of video microscopy, the positions of single beads conjugated to a protein were determined with nanometer precision using centroid algorithms. 11 This approach allowed the movement of motor proteins to be tracked at the molecular scale and their functioning to be understood.12,13 In a similar way, the positions of single fluorophores can be determined precisely by fitting the emission pattern with suitable model functions. Here, the localization precision is determined by the number of detected photons N and the standard deviation σ of the point-spread function (PSF); it can be estimated as ∼σ/√N for negligible background.14 Organic fluorophores exhibit a high brightness and photostability, which translates into a high number of photons emitted per fluorophore. They were used to sitespecifically label motor proteins and allowed their movement to be followed and the mechanism of individual stepping to be revealed with a precision approaching 1 nm.15 However, the number of discernible spectroscopic states of fluorophores is limited and, accordingly, does not allow for the super-resolution imaging of structures that are typically labeled by more than a handful of different fluorophores. Hence, it became clear that other methods for separating the fluorescence emission of an ensemble of fluorophores are required to realize super-resolution microscopy. It turned out that fluorescence intermittency or blinking might provide a possibility for separating the fluorescence emission in time, if it could be controlled experimentally in an appropriate way. Even the first single-molecule fluorescence experiments revealed on/off switching of individual fluorophores independent of the irradiation conditions due to accidental transitions into nonabsorbing and nonemitting states.16,17 Later, the observation of fluorescence blinking was used to demonstrate the detection of a single fluorophore: Two or more independent fluorophores could show blinking only if perfectly synchronized. In many situations, blinking originates from intersystem crossing into triplet states, which exhibit lifetimes of microseconds under aqueous conditions at room temperature. This occurs following the cycling of a fluorophore between the singlet ground state and the first excited singlet state: Depending on the intersystem crossing yield, the fluorophore can populate a long-lived triplet state where it is parked for several microseconds before the emission cycle is started again by reverse intersystem crossing, for example, through a collision with oxygen.18 The first effort to exploit the temporal separation of the fluorescence emissions of single emitters for super-resolution microscopy used semiconductor nanocrystals, so-called quantum dots, which are known to exhibit excitation-intensity-
occur in the case of a dysfunction or disease. In this context, a spatial resolution in the range of the size of biomolecules is needed. The spatial resolution of conventional fluorescence microscopy using visible light is physically limited to about 200 nm. This is largely sufficient for the study of the large-scale features of cells and tissues, and much of our current understanding of how cells are organized and biomolecules interact has been gained by light microscopy techniques. The development of fluorescence microscopy techniques that achieve a substantially better spatial resolution down to a few nanometers (“superresolution microscopy”) has opened another window to the study of cellular processes: Now, it is possible to investigate how biomolecules are organized and how they interact at the nanometer scale and, from such investigations, to understand how a misconfiguration of the molecular architecture and intermolecular interactions can lead to various forms of disease. Certainly, super-resolution microscopy techniques are just about to enter topics of health and disease, and much more work in different directions is needed. However, one can anticipate that, from a “nanoscopic” inspection of how proteins organize and interact inside a living cell, significant information will be obtained for an improved understanding and treatment of diseases. This review covers the technology of single-molecule localization microscopy (SMLM), which is one representative technique from the toolbox of super-resolution microscopies. Specifically, we describe how SMLM has been used to study cellular processes in eukaryotic cells. After first covering studies on nuclear proteins in fixed and live cells, we review how SMLM is used to address questions in neurobiology. Finally, we introduce the use of SMLM as a tool for elucidating pathogen− host-cell interactions using the example of human immunodeficiency virus (HIV) infection. SMLM emerged from the field of single-molecule imaging. We thus begin with a short historic review of the ideas that were developed before and contributed to SMLM. This is followed by a discussion of how SMLM can be realized in the different relevant conditions of a sample, for example, a fixed cell and a living cell. We elaborate on the importance of photoswitchable fluorescent probes that are required to conduct an SMLM experiment, particularly in view of the challenges and requirements for imaging eukaryotic cells. This aspect is crucial for the design of SMLM experiments in eukaryotic cells: A robust understanding of photophysical properties and how they depend on the cellular environment or a particular label are indispensable for interpreting the imaging data correctly. Finally, we review some image analysis tools that are specific for SMLM data and allow the extraction of quantitative data on protein copy numbers, clustering, stoichiometries, and interactions with other biomolecules.
2. HISTORY OF SINGLE-MOLECULE LOCALIZATION MICROSCOPY The development of single-molecule spectroscopy and imaging techniques in the 1990s paved the way for single-molecule localization microscopy. The single-molecule fluorescence era started in 1989−1990 with the independent detection of single dopant molecules in host crystals at cryogenic temperatures by Moerner and Kador1 and Orrit and Bernard.2 At the same time, Keller and co-workers proposed a method for the sequencing of an individual DNA molecule by laser-induced fluorescence detection of single fluorescently labeled mononucleotide 7479
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dependent blinking.18 The method was termed “pointillism” because the super-resolved image is finally reconstructed from individual localized points. In contrast to organic fluorophores whose blinking kinetics can be described with a characteristic time, quantum dots show dark periods (off times) ranging from milliseconds to seconds.19 The probability density distribution of these off times (as well as that of the on times) follows a power law. As a consequence, many short blinking events oppose a few long blinking events, which imposes a limitation for their use in SMLM. The question that remained open was how the unpredictable blinking of fluorophores can be controlled or, in other words, how fluorophores can be switched reliably between the on state and the off state. The realization of single-molecule localization microscopy required other ideas and findings of single-molecule fluorescence spectroscopy and imaging, respectively. On one hand, photoactivatable and photoconvertible variants of green fluorescent proteins were developed by Patterson and Lippincott-Schwartz that allowed the visualization of transport processes in living cells by selective imaging and tracking of a subset of labeled proteins.20 On the other hand, photochromic fluorophores were developed that can be switched between an on state and an off state as a result of photoisomerization by irradiation with light of an appropriate wavelength.21,22 Whereas photochromic compounds such as arylethenes, spiropyrans, and fulgides exhibit useful switching performances in polymers and organic solvents, their use as photoswitches in the SMLM of cellular structures in aqueous environments remains difficult. At the same time, single-pair fluorescence resonance energy transfer (spFRET), originally introduced by Weiss and colleagues to study the conformational dynamics of single molecules,23,24 was optimized to eliminate blinking and improve the photostability of FRET pairs such as Cy3/Cy5. For this purpose, thiol compounds that act as triplet quenchers, such as β-mercaptoethanol and β-mercaptoethylamine, in combination with enzymatic oxygen scavengers,25,26 were used successfully to minimize blinking and increase the photostability of FRET pairs. Interestingly, in some spFRET experiments, it was observed that the FRET acceptor Cy5 entered very long photoinduced off states with lifetimes of up to several seconds. Because the off state of the acceptor does not absorb in resonance with the donor’s emission, the FRET efficiency fluctuates accordingly. This finding was investigated and in the next step used advantageously by two groups independently in 2005 to demonstrate reliable photoswitching of the carbocyanine dye Cy5 in the absence27 and presence of a FRET-donor dye, later called the activator dye,28 in an aqueous buffer containing 100 mM β-mercaptoethylamine (MEA) and an enzymatic oxygen scavenger. Ultimately, this accidental finding cleared the way for single-molecule localization microscopy with photoswitchable organic fluorophores. Very shortly thereafter, SMLM was realized with photoactivatable fluorescent proteins in photoactivated localization microscopy (PALM)29 and fluorescence photoactivation localization microscopy (FPALM)30 and with organic fluorophores in stochastic optical reconstruction microscopy (STORM)31 and direct STORM (dSTORM).32
pixel-wise scanning, SMLM builds on the detection of single fluorophores and the precise determination of their centers of mass. The necessary separation of fluorophores is achieved by the temporal confinement of fluorescence emission using fluorophores that exhibit stochastic photoswitching or bind reversibly to a target structure. From the collection of positional coordinates, a super-resolved image is generated computationally33 (Figure 1).
Figure 1. (A) Fluorophores that can be photoswitched between a dark state and a fluorescent state (left scheme) or photoactivated state (right scheme) allow for the selective and controlled highlighting of a subset of molecules. (B) At low activation densities (i.e., low emitter densities), single fluorophores can be observed with a camera (left), and their positions can be determined with high precision. The collection of single coordinates can be used for the reconstruction of an artificial image that provides subdiffraction spatial resolution (right).
The fluorescence signal of a single fluorophore appears as an extended blurry spot in the image of a camera. The full width at half-maximum (fwhm) of this spot is in the range of ∼λ/2, where λ is the imaging wavelength. This spot represents the PSF of the microscope and can be approximated by a twodimensional (2D) Gaussian function whose center represents the position of the fluorophore. The precision with which this position can be determined (often termed the “localization precision”) mainly depends on the standard deviation of the PSF, σ, and the number of photons emitted by the fluorophore, N, and can be estimated as ∼σ/√N. Additional parameters that affect the localization precision are the pixel size of the camera and the background signal.14 The localization precision is an important parameter that characterizes the quality of an SMLM image. The localization precision can be determined from the number of photons detected in the fluorescence spot of a single fluorophore.14 An alternative and straightforward strategy is based on determining the distances of nearest-neighbor fluorophores in adjacent frames.34 This approach builds on the theory of how precise distances in single-molecule FRET experiments can be determined35 and returns an experimental localization precision for an SMLM image. The localization precision has to be taken into account for any quantitative analysis and interpretation of the imaging data. Together with the labeling density, the localization precision determines the achievable theoretical spatial resolution. However, the spatial resolution of an SMLM image cannot be expressed with a single number and further depends on the local structural complexity of a particular cellular feature.36,37 Once a collection of single-fluorophore coordinates is in hand, a super-resolved image can be generated. This can be
3. PRINCIPLE OF SINGLE-MOLECULE LOCALIZATION MICROSCOPY Rather than detecting the fluorescence signal of all fluorophores in a sample at once by imaging with a camera or employing 7480
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Figure 2. Fluorescence trajectories of single ATTO647N-labeled DNA molecules immobilized by biotin/streptavidin binding in an aqueous environment on a coverslip (PBS, pH 7.4) at 10- and 1-ms temporal resolution, where oxygen was removed by an enzymatic oxygen-scavenging system: in the presence of (A) 1 mM methylviologen, (B) 1 mM ascorbic acid, and (C) 1 mM mehtylviologen and 1 mM ascorbic acid. The fluorescence signal of the fluorophore was split into short- and long-wavelength paths by a dichroic mirror (gray and black, respectively). In addition, the upper right insets show the corresponding second-order autocorrelation functions G(t) of the trajectories with monoexponential fits. Samples were excited at 635 nm with an average excitation intensity of approximately 2 kW cm−2.57
be attributed to triplet states. This can be explained by the dual role of molecular oxygen, which is not only a potent oxidant but likewise a very efficient triplet-state quencher. On the other hand, it was shown that addition of β-mercaptoethanol (BME) or β-mercaptoethylamine (MEA) to the oxygen-scavenging system partially suppresses the millisecond fluctuations in the fluorescence intensity of Cy5.54 Interestingly, it was observed that Cy5 molecules entered very long photoinduced off states with lifetimes of up to several seconds when thiols (BME, MEA) were added at higher concentrations. Other studies introduced alternative antifading reagents such as propyl galate, ascorbic acid, and glutathione,55 but none of these reagents outperformed Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), an analogue of vitamin E. Trolox was found to quench the triplet state efficiently without causing Cy5 blinking.53,56 Knowledge about the exact photophysical mechanisms behind the triplet-state quenching (i.e., blinking suppression) of Trolox, however, remained elusive until the introduction of a reducing and oxidizing system (ROXS).57 Use of a ROXS constitutes a generally applicable method for tuning the fluorescence characteristics of organic fluorophores. It is based on the idea that, in many cases, triplet quenching does not repopulate the singlet ground state but rather reduces or oxidizes the fluorophore into a radical anion or cation. To back-transfer the fluorophore to the singlet ground state and feed the absorption/emission cycle again, a second redox reaction is required. This is exactly the principle on which ROXS builds: Following triplet quenching, a reducing agent such as ascorbic acid generates a radical anion that is oxidized by methylviologen, for example, in a second step to repopulate the singlet ground state. Whether the fluorophore is first oxidized and then reduced to repopulate the singlet ground state or first reduced and then oxidized is controlled by the reactivity of the fluorophore. Regardless of which reaction occurs first, efficient repopulation of the singlet manifold enables the recording of extended fluorescence trajectories of up to several minutes from individual fluorophores uninterrupted by blinking (Figure 2). In subsequent years, it was shown that the photostabilization and antiblinking effect of Trolox can also be explained by the ROXS mechanism.58 Because of the reducing and oxidizing properties of Trolox and a quinone derivative of Trolox present in aerated aqueous Trolox solutions, respectively, the initially formed radical anion is oxidized, and the singlet ground state is recovered. The next step was to bypass the concentration-dependent quenching of triplet states by direct attachment of protective additives (photostabilizers) such as Trolox, cyclooctatetraene
achieved by histogramming the single-molecule localizations in “subpixels” with a pixel size in the range of the localization precision. An alternative approach is to plot each localization as a single point and to convolve the resulting plot with a Gaussian function whose standard deviation represents the localization precision.38 Different optical configurations were developed that allow for the determination of the positions of single fluorophores in three-dimensional (3D) space. A first approach introduced a cylindrical lens into the imaging path,39 leading to an asymmetric distortion of the PSF the extent of which reports on the axial position of a fluorophore. This concept was adapted for 3D SMLM.40 Other approaches employ multiple imaging planes,41,42 a helical PSF,43 an interferometric configuration,44 or a detection scheme using two opposing objective lenses.45,46 The various approaches for 3D localization differ in their experimental complexity, their achievable localization precision, and the axial range that can be covered in a single image.47 A variety of algorithms and software tools for localizing single molecules have been developed, and we refer to excellent reviews covering this topic.48,49
4. PHOTOSWITCHABLE AND PHOTOACTIVATABLE FLUORESCENT PROBES 4.1. Organic Fluorophores
4.1.1. Improving Performance of Organic Fluorophores for Single-Molecule Fluorescence. With the rise of single-molecule fluorescence imaging and spectroscopy, photobleaching and blinking of single fluorophores were identified as limiting factors. To increase the observation time and to minimize fluorophore blinking, different strategies have been developed. Because it was thought that molecular oxygen is the molecule primarily responsible for photobleaching by photo-oxidation, the first attempts to prolong the observation time of single fluorophores employed enzymatic oxygenscavenging systems. A prominent example is a mixture of glucose oxidase and catalase that converts glucose and oxygen into gluconic acid and water in a two-step process.50 Many of the early single-molecule fluorescence studies employed cyanine dyes (Cy3 and Cy5), as they exhibit high water solubilities and were available as chemically functionalized dyes, which allowed their conjugation to biomolecules.51,52 Oxygen depletion increases the photobleaching lifetime of cyanine dyes, but it was found in single-molecule experiments that this also induces the appearance of time spans without fluorescence, that is, millisecond off states,53 which can 7481
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(COT), or 4-nitrobenzyl alcohol (NBA) to the fluorophore, enabling an intramolecular “self-healing” reaction.59 Provided that the protective additives selectively quench the triplet state but not the excited singlet state of the fluorophore, the photostability is increased, and the blinking is reduced. Very likely, a similar ROXS mechanism might occur.60 For example, a fluorophore that enters a triplet state reacts with the attached reductant Trolox, forming a fluorophore radical anion and a Trolox radical cation, which is a strong oxidant. As the two radical species cannot be separated by diffusion, they ultimately collide and react again, restoring both the fluorophore and Trolox in their ground states. A similar mechanism of geminate recombination was excluded for nonconjugated Trolox− fluorophore interactions owing to fast diffusional separation.58 For other photostabilizers (e.g., COT), other effects such as triplet-state quenching by triplet−triplet energy transfer have to be considered as well. To simplify the use of self-healing fluorophores for the labeling of biomolecules, conjugates of a photostabilizer and a fluorophore using unnatural amino acids were introduced.61 Trifunctional unnatural amino acids are ideal scaffolds because they enable the linking of the fluorophore and the photostabilizer, as well as the mild covalent labeling of the conjugates to biomolecules with the third functional group using standard labeling chemistry. Alongside improving the photostability of fluorophores for SMLM, it is desirable to increase the fluorescence quantum yield. The motivation behind this goal is that a higher number of photons emitted per time interval improves the error in localizing a single fluorophore (i.e., the localization precision). On the other hand, a higher photon yield also allows for faster image acquisition and, thus, a higher temporal resolution. Replacing water in imaging buffers by heavy water, D2O, was demonstrated to increase the photon yields of organic fluorophores62,63 and fluorescent proteins.64 This simple solution to increase the brightness of fluorophores works best in the red and far-red spectral regions and matches the “spectral window” of water,62 which suggests that a possible mechanism might be energy transfer from the fluorophore to higher-level vibrational states of surrounding water molecules. For some farred fluorophores, a nearly 3-fold increase in fluorescence quantum yield was reported.62 4.1.2. First Approaches to the Use of Organic Fluorophores as Photoswitches. With the demand of SMLM for photoswitchable fluorophores, chemical compounds that promote the transitions of fluorophores into off states attracted interest. The influence of thiol compounds such as BME and MEA in combination with enzymatic oxygen scavengers on the fluorescence blinking behavior of fluorophores was investigated in more detail. In 2005, two groups demonstrated that the carbocyanine dye Cy5 can be switched reversibly between a fluorescent on state and a nonfluorescent off state upon irradiation at shorter wavelengths between 337 and 532 nm in oxygen-depleted thiol-containing (100 mM MEA) aqueous buffer (Figure 3).27,28 Both reports demonstrated that light-induced on/off switching can be performed more than 100 times with high reliability at room temperature and that each switching cycle enables the detection of several thousand fluorescence photons per on event. One study facilitated the restoration of the fluorescent state through the use of a green-absorbing and -emitting second dye (Cy3), termed the “activator”, in close proximity to the “reporter” Cy5;28 the other study demonstrated direct restoration in the absence of an activator upon irradiation at shorter wave-
Figure 3. (A) Thiol-induced photoswitching of individual Cy5 molecules in aqueous solvent (PBS, pH 7.4). Cy5-labeled DNA strands were immobilized on BSA/streptavidin-coated coverslips. Photoswitching was perform in the presence of 100 mM MEA. Oxygen was removed by the addition of an enzymatic oxygen scavenger. A single Cy5 strand was parked in the laser focus and transferred to the off state upon irradiation at 633 nm (red). Reactivation was performed at 488 nm (blue). Only one out of 21 switching events was unsuccessful. (B) Ensemble switching experiment of a 10−6 M aqueous solution of Cy5 (PBS, pH 7.4, 100 mM MEA) purged with argon. After irradiation of an oxygen-depleted solution at 647 nm (300 mW), the main absorption band at 650 nm decreased by approximately 50% (red). Irradiation at 488 nm (300 mW) for 30 min restored ∼40% of the absorption (blue). Recovery of the fluorescent on state was more effective at the single-molecule level, most probably because of the different experimental conditions such as a different excitation intensity and oxygen removal efficiency.
lengths.27 The activator−reporter-based photoswitching of Cy5 was later used for the development of stochastic optical reconstruction microscopy (STORM)31,40,65 in contrast to activator-less direct STORM (dSTORM).32,36,66 The activation rate constant from the off state to the fluorescent on state was found to be linear with respect to the laser power of the shorter-wavelength activation laser for both methods. Although a laser power that was ∼200 times higher was reported for the approach without an activator, Cy5 photoactivation was demonstrated for activation laser powers (514 nm) in the microwatt to milliwatt range (corresponding to irradiation intensities of 8.75 The triplet states of the fluorophores can also be 7483
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Figure 5. (A) Absorption spectra of an aqueous solution of Alexa Fluor 488 with 100 mM MEA at pH 9.3. Upon irradiation at 488 nm (5 min), the absorption at 488 nm (blue) decreased, and a new absorption band corresponding to the radical anion appeared at 396 nm (violet). (B) The anion radical of Alexa Fluor 488 is stable for several hours at room temperature. (C) EPR spectrum (inset) and temporal decay of the maximum of the EPR signal recorded from an aqueous 10−4 M solution of Alexa Fluor 488 with 100 mM MEA at pH 9.3, following irradiation at 488 nm for several minutes. The EPR signal decays exponentially at room temperature with a lifetime of approximately 100 min. Reproduced with permission from ref 75. Copyright 2011 The Royal Society of Chemistry.
the absence of oxygen, a typical rhodamine dye emits ∼500 fluorescence photons (assuming an intersystem crossing yield of 0.2%) before it it enters a triplet state and is reduced by a thiolate. Assuming an experimental detection efficiency of about 10%, only ∼50 photons would be detected. In the presence of oxygen, however, the triplet state would be depleted several times, enabling the detection of several hundred to several thousand photons before the thiolate reduces the dye into a long-lived off state. Thus, the triplet state and quenching by molecular oxygen belong to the “on state” in SMLM experiments (Figure 4). 4.1.4. Intrinsic Photoswitchable and Photoactivatable Fluorophores to Improve Localization Precision. The simplest way to increase the localization precision is to increase the number of photons detected from a single fluorophore per switching event. This can be achieved experimentally by doubling the collection efficiency of a microscope using a 4π detection scheme with two opposing lenses.44,86 By combining astigmatism imaging with a dual-objective scheme, Xu et al. successfully resolved the 3D ultrastructure of the actin skeleton by SMLM with a lateral resolution of
