Stimulated Emission Depletion Microscopy - Chemical Reviews (ACS

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Stimulated Emission Depletion Microscopy Hans Blom† and Jerker Widengren*,‡ †

Royal Institute of Technology (KTH), Dept Applied Physics, SciLifeLab, 17165 Solna, Sweden Royal Institute of Technology (KTH), Dept Applied Physics, Albanova Univ Center, 10691 Stockholm, Sweden

‡

ABSTRACT: Despite its short history, diffraction-unlimited fluorescence microscopy techniques have already made a substantial imprint in the biological sciences. In this review, we describe how stimulated emission depletion (STED) imaging originally evolved, how it compares to other optical superresolution imaging techniques, and what advantages it provides compared to previous golden-standards for biological microscopy, such as diffraction-limited optical microscopy and electron microscopy. We outline the prerequisites for successful STED imaging experiments, emphasizing the equally critical roles of instrumentation, sample preparation, and photophysics, and describe major evolving strategies for how to push the borders of STED imaging even further in life science. Finally, we provide examples of how STED nanoscopy can be applied, within three different fields with particular potential for STED imaging experiments: neuroscience, plasma membrane biophysics, and subcellular clinical diagnostics. In these areas, and in many more, STED imaging can be expected to play an increasingly important role in the future.

CONTENTS 1. Introduction 2. Super-Resolution Fluorescence Microscopy, How It Evolved and Put in Context to Other Technologies 2.1. Introduction/Historical Rés umé: Optical Imaging, Microscopy, and the Diffraction Limit 2.2. Early Attempts to Challenge the Diffraction Limit 2.2.1. Far-Field Optical Imaging Methods 2.2.2. Near-Field Imaging Methods 2.2.3. Other Nonoptical Approaches for HighResolution Imaging 2.3. Far-Field Optical Microscopy Techniques “Breaking” The Diffraction Limit 2.3.1. Coordinate-Targeted Super-Resolution Methods 2.3.2. Overview of Realizations Incorporating Coordinate-Targeted SRM 2.3.3. Coordinate-Stochastic Approaches for Super-Resolution Imaging 2.3.4. Specific Features of PALM/STORM Imaging vs STED Imaging 2.3.5. Other Diffraction Unlimited Optical Imaging Approaches 3. The Triad of Prerequisites for STED Imaging 3.1. Photophysical and Photochemical Considerations 3.2. Instrumentation for STED Microscopy 3.2.1. STED Lasers 3.2.2. STED Wavefront Tuning 3.2.3. Optical Paths, Mirrors, and Scanning Devices

© XXXX American Chemical Society

3.2.4. Microscope Objectives 3.2.5. Detector Systems 3.2.6. Calibration and Performance Testing 3.3. Sample Considerations for STED Imaging 3.3.1. Choice of Fluorophores 3.3.2. Choice of Labeling 3.3.3. Optimization of Labeling Density, Fixation, Permeabilization, And Mounting Medium 4. STED Imaging Combined with Spectroscopy 4.1. Multicolor STED Imaging 4.2. STED-FCS 5. Applications of STED Imaging 5.1. STED Imaging in Neuroscience 5.2. Plasma Membrane Organization and Dynamics 5.3. Diagnostics and Understanding Disease Mechanisms at the Nanoscale 6. Conclusions Author Information Corresponding Author ORCID Notes Biographies Acknowledgments References

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Special Issue: Super-Resolution and Single-Molecule Imaging Received: September 23, 2016

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DOI: 10.1021/acs.chemrev.6b00653 Chem. Rev. XXXX, XXX, XXX−XXX

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1. INTRODUCTION It is reasonable to claim that light microscopy has over the last century been the most influential form of microscopy in biomedical research and in the life sciences in general. Light microscopy offers minimal invasiveness and is compatible with live cell imaging. High biomolecular specificity can be obtained by use of fluorescent markers. In spite of impressive progress in other microscopy technologies in the last years, such as in electron microscopy and imaging mass spectrometry, light microscopy is likely to remain the dominating microscopy method in the life sciences. An important reason is the advent of the fluorescence-based super-resolution microscopy (SRM) techniques. Stimulated emission depletion (STED) microscopy represents a major category of these SRM techniques and was the first SRM technique to be proposed and experimentally realized. Since its realization, the development of STED microscopy as well as of other SRM techniques has been remarkable, and fluorescence-based SRM techniques have already started to profoundly transform our view of cellular and molecular biology. In this review, it is described how STED microscopy evolved, in the perspective of the general development of light microscopy within life sciences, but with focus on the major steps in the STED microscopy development. The operating principles, major strengths, and limitations of STED microscopy are discussed and briefly also those of other SRM techniques, to allow a comparison of STED microscopy to these other SRM techniques, as well as to previous goldenstandards for biological microscopy, such as diffraction-limited optical microscopy and electron microscopy. A major section in this review discusses the major triad of prerequisites for successful STED imaging experiments, emphasizing the equally critical roles of instrumentation, sample preparation, and photophysics. It is described how these prerequisites come into play and need to be considered in STED imaging experiments in life science, how they set limits for the performance of STED imaging, and evolving strategies for how to push the limits of STED imaging even further. The intention is to provide key methodological and experimental aspects, useful for both newcomers getting started with STED imaging and as reference and outlooks for more experienced users. Finally, we provide examples of how STED imaging can be applied, within three different fields with particular potential for STED imaging experiments: neuroscience, plasma membrane biophysics, and subcellular clinical diagnostics. The range of applications of STED imaging is quickly expanding, and these three application areas clearly only represent a fraction of all emerging application areas of STED imaging. However, rather than referencing to applications in all areas, we find it more instructive to focus on three major areas. In our view, they are representative for the use and they illustrate the benefits of STED imaging when applied within the life sciences, also in respect of their interesting potential for further development. This review includes references spanning from the early history of light microscopy to recent work reporting on the latest applications and methodological progress of STED imaging. However, it is not much more than 20 years since the first-principles for STED imaging and SRM were presented1 and less than 20 years since the first experimental realizations.2 The vast majority of work referenced are thus from this time period and clearly with an emphasis on work from the last

years. Indeed, the increasing number of reports of the use and further development of STED imaging is only one of numerous indications of its usefulness and potential in life sciences, yet to be fully exploited.

2. SUPER-RESOLUTION FLUORESCENCE MICROSCOPY, HOW IT EVOLVED AND PUT IN CONTEXT TO OTHER TECHNOLOGIES In this section, we will give a historical background of the development of light microscopy, how it eventually led to the development of stimulated emission depletion (STED) imaging and super-resolution far-field microscopy techniques in general. The basic principles for STED imaging and how it evolved will be outlined, as well as how STED imaging compares to other SRM techniques. Also, the performance of STED imaging in a broader view, in comparison to other microscopy techniques in the life sciences, will be briefly covered. 2.1. Introduction/Historical Résumé: Optical Imaging, Microscopy, and the Diffraction Limit

Optical microscopy has played an important role in science for centuries. The first microscopes appeared in The Netherlands and in Italy in the first half of the 17th century. In 1665, the English scholar Robert Hooke published his popular science book “Micrographia”, featuring detailed illustrations of various known items from the flora and fauna, viewed with a microscope providing approximately 20 times magnification. “Micrographia” boosted the development and inspired others to start using microscopes more extensively for research, among them Antonie van Leeuwenhoek, who took the microscope technology to new extremes. With his microscopes, consisting essentially of a carefully crafted glass ball, sandwiched between the holes of two metal plates, van Leeuwenhoek could achieve higher magnifications than ever before: up to 300 times.3 The microscopes allowed things to be seen at a cellular level, and van Leeuwenhoek could not only observe known things at greater detail but also things no one had ever seen before, such as muscle fibers, spermatozoa, and bacteria. Simple microscopes played an important role in science well into the 19th century. Then, major improvements such as achromatic lenses, improved specimen illumination4 and light control provided the basis for the compound microscopes. With microscopes based on scientifically engineered glassware, pioneered by Carl Zeiss, Otto Schott, and Ernst Abbe in the late 19th century, further steps were taken toward microscopes capable to stretch toward the resolution limit of light microscopy set by diffraction. In 1873, Abbe describes in words his famous (later mathematically expressed) formula:5 dmin =

λ 2 sin(α)

(1)

where dmin is the minimal resolvable distance, λ the wavelength of the light, and α the half aperture angle of the microscope’s objective. Following Abbe’s first article about the resolution limit, Hermann von Helmholtz published one year thereafter a detailed mathematical derivation of dmin,6 taking also the index of refraction of the sample, n, into account (for Abbe air was selected; i.e. n = 1): dmin =

λ 2n sin(α)

(2)

Although the articles from Abbe and von Helmholtz are the first ones dealing in detail with the resolution limits of B


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microscopes, the effects of diffraction and its implication for resolution were known earlier.7 In particular, Airy calculated already in 1835 the diffraction image of a point source when the limiting aperture is circular in shape,8 and although Airy did not explicitly state that the diffraction limits resolution, it can be assumed that he was aware of this fact.7 Recognizing that broadening of imaged structures is inevitable due to diffraction, different criteria to define a structure as “resolved” were formulated. Often referred to is the Rayleigh criterion, published in 1874,9 which regard a structure as resolved if the principal intensity maximum of one diffraction pattern coincides with the first minimum of the neighboring diffraction pattern (Figure 1a). As noted in a historical review on the diffraction barrier in microscopy,7 this definition was established in a time when the human eye, which cannot resolve arbitrarily small intensity differences, was the common photodetector. Preceding the strong development of the microscope optics in the late 19th century was the first reported observations of fluorescence by Herschel10 in 1845, followed by the more detailed observations of Stokes in 1852 from the same material (quinine),11 and at the beginning of the 20th century, the companies Carl Zeiss and Carl Reichert realized the first fluorescence microscopes.12−14 Microscopic observation of fluorescence emission has turned out to be a very important milestone. A vast majority of all microscopy applications in the life sciences today is performed with light microscopy, and by far with fluorescence as the dominating contrast mode. Strongly emitting fluorophore labels, such as fluorescein15 and rhodamine16 derivatives, were reported as early as the late 19th century and are still widely used. The high signal-tobackground ratios achievable by spectrally filtering the fluorescence from the excitation light11 was early on realized for microscopic studies of living organisms.17 The high specificity of labeling, with the development of fluorescent antibodies in the early 40’s,18 and the cloning of green fluorescent proteins (GFPs) in the 90’s,19−21 allowing cellular proteins of interest to be labeled by genetic encoding, has also strongly contributed to the strong stand of fluorescence microscopy in life science. Apart from serving as labeling agents, organic fluorophores and fluorescent proteins have also been developed to reflect environmental parameters via their emitted fluorescence (e.g., fluorescein and rhodamine derivatives sensing cytosolic calcium levels).22 It is also the combination of light microscopy and fluorescence detection which has brought about the development of methods, first pushing, finally overcoming the fundamental limits of resolution set by the diffraction, as will be further discussed below. The diffraction limit5,6,9 for microscopy states that it is impossible to resolve two elements of a structure which are closer than about half the wavelength (λ) in the lateral plane (eqs 1 and 2 and Figure 1a). A far-field fluorescence microscope (e.g., a wide-field or a confocal microscope) utilizes a lens system to excite and collect fluorescence in the sample and to image it onto a photon detector. By the lens system, the excitation as well as the fluorescence light is focused to interfere constructively at a certain point in space, the focal point. As a consequence of the diffraction limit, however, the focal point is not infinitely sharp but displays a light intensity pattern with a central maximum and a width whose full-width-at-halfmaximum (fwhm) is at best ≈ dmin (eq 2) in the lateral direction and along the optical axis

Figure 1. (a) Schematic representation of one-dimensional (1D), twodimensional (2D), and three-dimensional (3D) lateral focal profile of two barely resolved point source entities (i.e., added Airy patterns) graphically visualizing the Rayleigh criterion. If the light intensity drops by 26% between the two image peaks they are defined as resolved, which mathematically is equal to dmin = 0.61 λ/NA [where NA = n sin (α)]. (b) Taking the Fourier transform of the PSF (here the Airy pattern) yields the optical transfer function (OTF), which describes how the modulation strength of different spatial frequencies are in the specimen. The maximum bandwidth of a classical microscope is 2NA/ λ, corresponding to resolving a period of λ/2NA (i.e., the Abbe limit). Note: it is quite natural that the Rayleigh limit gives a somewhat more “pessimistic” value because it corresponds to having a certain minimum modulation in order to resolve the objects.

dzmin ≈

λ n sin 2 α

(3)

In both the lateral and axial direction, the resolution is thus governed by λ and by the focusing strength of the lens (given by its semiaperture angle α and the refractive index n of the object medium, where NA = n sin α is referred to as the numerical aperture of the objective lens).23 The threedimensional diffraction pattern of light emitted from an infinitely small point source in the specimen and transmitted to the image plane through a lens system is considered to be C


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of the PSF, by a factor of ≈ 2 for two-photon excitation and by ≈ 3 for three-photon excitation. On the other hand, since the wavelength of the excitation light is also increased in correspondence with the multiplicity of excitation, the minimal size of the diffraction-limited excitation spot (eq 2) increases approximately by a factor of ≈ 2 (two-photon excitation) or ≈ 3 (three-photon excitation), and as consequence, the spatial resolution in multiphoton microscopy is, all in all, typically poorer than for corresponding one-photon excitation microscopy.34 Like confocal microscopy, however, multiphoton microscopy offers 3D sectioning, the excitation is confined to the focal plane which reduces out-of-plane photobleaching, and the long excitation wavelengths used (typically 700−1100 nm) allow imaging at deeper penetration depths and with less scattering of the excitation laser light. Scattering of the fluorescence in the sample can also be better tolerated than in confocal microscopy, since no pinhole is required in the image plane and no signal is lost by spatial filtering. These properties make multiphoton microscopy a useful tool for in particular (in vivo) live cell and tissue imaging, and it is also a modality that with some benefits can be combined with STED imaging, as discussed below. Selective-plane illumination microscopy (SPIM), also referred to as light-sheet fluorescence microscopy (LSFM), is an imaging modality in which a sample is illuminated from the side, perpendicular to the optical axis, by a beam engineered into a wide and relatively thin “sheet”.35,36 From a resolution point of view, SPIM can offer a higher resolution in the axial plane, compared to conventional widefield microscopy, and also compared to CLSM if a lower NA (1000 μm2) can be imaged with 70 nm spatial resolution, with frame rates up to a few Hertz, providing interesting prospects for live cell or tissue imaging applications. Also, in contrast to parallelized STED imaging, the laser intensities required in each of the spots is much lower in this realization, so that the available total power of the laser to a far lesser extent set a limit for the number of donuts that can be generated in parallel. An additional photophysical aspect to consider is how the orientation of the fluorophores, and in particular the orientation of their excitation and emission dipole moments, may affect the transition rates of excitation and stimulated emission. Depending on the direction of the electric fields of the laser light and the dipole moments of the fluorophore, selection rules for excitation and depletion apply differently. In other words, the cross sections for excitation and stimulated emission, σS10 and σS01, are for most organic fluorophores not isotropic, and the effective excitation and STED rates can thus be different depending on the orientation of the fluorophores. To average out excitation/depletion selection competition as a function of fluorophore dipole direction(s), circularly polarized light is often applied in STED microscopy.221 An additional aspect is that fluorescence as a function of the orientation of the fluorophore dipole orientation plays an essential role on the localization accuracy (i.e., the determination of where in space a labeled target is situated and from where emission occurred). Symmetric emission occurring from freely rotating fluorophore molecular dipoles can faithfully map image space with sample space. However, rotation-impaired fluorophores may as a function of focal position be shifted in image space. In particular in single molecule localization microscopy, this can possibly introduce systematic errors, which require iterative calibration measurements for correction (i.e., replacing imaged emission PSFs back to correct sample space locations).222,223 In STED imaging, however, such emission shifts are minimized, because the PSF of the depletion beam is determined by a predefined position generated by the (zero-intensity) depletion focus.224 Finally, in all fluorescence imaging situations, one needs a signal reaching above the background to detect the “unknown”. If however the contrast of the image is deteriorating as the resolution is improved, one soon reaches a situation where a gain in resolution is not a gain in image quality any longer. Basically, the images structure one wants to resolve then “drowns” in a too high background. This situation is a common concern in STED imaging, as well as in single molecule STED spectroscopy, as increasingly smaller depletion focal volumes will contain increasingly fewer signaling fluorophores.225 Theoretically, STED imaging resolution is unlimited, and it is only a matter of applying higher depletion intensities (eq 4). In practice, however, the attainable resolution is limited by the possibility to keep enough contrast (i.e., trying to avoid drowning in noise).226 This noise can originate from scattering of laser light, sample autofluorescence, background fluorescence from nonspecific labeling, or can be generated intrinsically from the stochastic photon generation/detection processes, from thermal and mechanical drift, as well as from optical aberrations distorting the imaging. Maximizing signal and minimizing background is thus the ever repeated golden mantra of fluorescence STED microscopy, once more emphasizing the central role of photophysics.

Since the invention of STED imaging, with the suggestion of overcoming the diffraction limit by employing stimulated emission to deplete the fluorescence process in the outer regions of the excitation PSF,1 its proper experimental realization has been a constant subject of discussion. A suitable specification of depletion lasers, properly adapted to the fluorophore characteristics, is technically the most important first step to consider when designing a STED microscope. Rearrangement of the STED depletion irradiance profile and full control of its spatial and temporal overlay with a suitable excitation profile is another consideration. Several additional issues must also be technically considered to optimize a STED microscope to its full potential. Below, we discuss these issues for the major categories of equipment making up a STED microscope. 3.2.1. STED Lasers. The workhorse of a STED imaging system is the depletion laser. In the first publication on STED imaging in the early 1990’s,1 mode-locked dye lasers were identified as suitable depletion lasers, providing picosecond pulses with repetition rates of the order of 100 MHz. For blue/ green fluorescent dyes having excitation maxima around 500 nm, these lasers were considered to have the necessary technical specifications to allow depletion with a (tunable) STED wavelength around 600 nm, making fluorescence microscopy with 50 nm resolution theoretically possible with visible fluorophores.1 The choice of picosecond pulses, and not shorter or even longer pulses, the choice of the repetition rate and the red-shifted emission wavelength of the STED laser (i.e., the technical specification of the lasers and how they should be operated to optimize STED depletion)226 is essentially determined by the characteristics of the reporting fluorophores, and in particular by their photophysical properties. With reference to Figures 2 and 5 and the preceding section on Photophysical and Photochemical Considerations, we will therefore more in detail dwell on how these properties influence the choice of lasers for STED imaging. 3.2.1.1. STED with Pulsed Lasers. Pulsed STED depletion lasers should have pulses shorter than the fluorescence lifetime, otherwise a major part of the laser irradiation intended for depletion will lose out and miss the chance to optically stimulate the decay (i.e., induce stimulated emission). This is basically a waste of laser photons and results in unnecessary light exposure of the sample. The STED pulse should obviously be timed just after the excitation-induced absorption to the excited state, and also after the vibrational decay within the electronic excited state (S1) has occurred, typically within tens of picoseconds, see Figures 2 and 5. If the STED pulse arrives too early in relation to the excitation pulse depletion will be inefficient because of a large fraction of yet not excited fluorophores, or because the excited S1 fluorophores have not yet vibrationally relaxed to an energy level matching the photon energies of the STED pulse. If too late, spontaneous emission will not be suppressed, as intended. In practice, major reasons to delay the STED pulse with some 10 ps after the excitation pulse are also the excitation pulse width and the timing jitter between the excitation and STED pulses. In the pioneering publication from 1994,1 a depletion laser pulse of around a few hundred picoseconds was found to be suitable to achieve as high as 30 nm resolution by STED imaging (at very short STED wavelengths, in the range of 400 nm). Further shortening of the duration of the pulses of the STED laser counteracts performance by two inter-related R


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considerations in designing a STED microscope. Instead of mode-locked dye lasers, the (initially) preferred laser system used in STED since 1999/2000 has been the solid-state, pulsed Ti:sapphire laser.2,92 The laser system has fixed (cavity) repetition rates of around 76−80 MHz, which are possible to change externally by using a bulky, complex, and expensive pulse-picker system to facilitate D- or T-Rex modalities.166 The typical wavelength tunability applied with the Ti:sapphire system is within the range of 740−780 nm, suitable for STED imaging of fluorophores emitting in the red/far-red spectra (>600 nm). Still today, several custom-built STED systems are designed with this laser system, as was the first commercial system sold by Leica Microsystems in 2007. This laser system is for excitation commonly combined with pulsed laser diodes, and in order to temporally align excitation and depletion, either an electronic synchronization unit is applied or the optical path lengths of the lasers beams are adjusted to ensure correct timing. To allow STED imaging of fluorophores emitting in the blue/green spectral range (500−580 nm), the pulsed STED depletion laser wavelength must be in the yellow/orange/red wavelength range (i.e., around 590−660 nm). As there is still a limited availability of modern suitable pulsed STED laser sources in this range, custom designs make use of optical parametric oscillators (OPO’s), which are pumped by, for example, a Ti:sapphire laser, to convert depletion to shorter wavelengths.183,230 The latter system is presently also rather bulky, complex, and expensive. Given the relatively high cost of a high-power Ti:sapphire laser (>200000 USD) and that the total laser system (including a pump-laser, cooling unit, and the laser cavity itself) is somewhat bulky, options have been presented to simplify this laser system. One way to reduce costs and size is to sacrifice wavelength tunability, applying a depletion laser with a fixed, single wavelength (with pico- or nanosecond pulse lengths). For STED imaging, applying this kind of depletion lasers, fluorescence images with 20 nm resolution can be obtained, as for example displayed on the official Nobel Price poster in Chemistry honoring super-resolution imaging in 2014. Along this strategy, depleting fluorescence of commonly used red/farred emitting fluorophores with a compact (frequency-doubled) fiber laser, emitting 1.2 ns pulses at 775 nm with a repetition rate of 20 MHz, has proven to offer an attractive combination of features, considering the points discussed above: low reexcitation because of the long depletion wavelength; longer pulses that reduce depletion probability somewhat but also diminish photobleaching, which all in all result in a gained image contrast.226 Furthermore, a small T-Rex effect might be obtained with the relatively low pulse repetition rate applied. Compact, high-power single-wavelength lasers of this kind are now increasingly applied for STED microscopy, especially in new commercially realized turn-key systems. Another line of laser development is the implementation of spectrally broader systems (i.e., white light laser sources or stimulated Raman scattering light sources) into STED imaging.231,232 With these broadband sources, excitation and depletion pulses in several spectral intervals can be generated from one and the same light source, thereby offering a convenient means for multicolor STED imaging. Pulsed depletion and excitation selection from the same spectrally broad-band laser offers the highest flexibility for wavelength optimization and also relaxes pulse synchronizing issues. Presently, such broadband laser systems have only been implemented in custom-designed STED systems in research laboratories, as a relatively simple and straightforward

mechanisms: (i) too short pulses cannot deplete as much fluorescence, because some excited state (S1) fluorophores will not have the time to vibrationally decay in S1 within the duration of the STED laser pulses (i.e., they get missed out for stimulated depletion). (ii) With the average power of the STED laser kept at the same level, shortening the duration of its pulses will increase the density of stimulating photons within the pulses. Due to nonlinear effects this will induce increased photobleaching, similar to that found to occur in the excitation volume for two-photon microscopy.227 A “solution” to achieve similar depletion probabilities would then be to use a shorter STED wavelength, inducing STED on the excited state fluorophore at an earlier stage of its vibrational relaxation within the S1 state. Any increase in STED will then however be accompanied and counteracted by an increased possibility of reexcitation from S0 and/or S1 by the depletion laser, resulting in an increased photobleaching of the fluorophore. This scenario has also been shown experimentally; blue-tuning the STED wavelength could indeed be shown to increase the depletion probability, but a decaying contrast of the image quality due to re-excitation was also induced.228 Red-tuning of the STED laser (with respect to the maximum excitation wavelength) is thus on first principle selected to avoid re-excitation/photobleaching. In principle, efficient depletion and minimized re-excitation could be achieved, if the STED pulse is applied to the sample with the blue edge of the STED pulse spectrum arriving first, and with the trailing red edge following after vibrational relaxation within S1. Thereby, a more efficient initial depletion may be achieved. Cryo-cooling of the fluorescent sample to nitrogen temperatures to maximize the efficiency of stimulated emission and to minimize photobleaching has further been tested.229 However, obviously this suggestion makes live-cell super-resolution STED studies highly unfeasible. Another technical specification to be considered for the pulsed STED depletion is the laser repetition rate. This parameter may on the one hand set an upper limit for the imaging speed, but on the other hand it also influences the contrast and the fluorescence image quality. The frequency and total number of emitted photons that one can obtain from a fluorophore depend on how fast and how many times one can cycle it between its ground and excited state, respectively. In an ideal situation, a very fast repetition rate (>1 GHz) would not be rate-limiting for the turnover rate of the excitation−emission (depletion) cycle. Ideally, as soon as an excitation event has occurred, STED depletion of excited S1 fluorophores would then take place. With several excitation−emission (depletion) cycles per imaged pixel, and subsequent scanning, a fluorescence STED microscopy image would then be built up. However, as discussed above, the signal strength and the observation time in fluorescence experiments are ultimately limited by the photostability (i.e., the photon budget of the fluorophore),157,158,163 and excited-state fluorophores may cross over to long-lived, metastable dark states (i.e., triplet or radical states) further reducing the fluorescence emission rates and promoting photobleaching. All in all, higher laser repetition rates would thus deteriorate signal strength and reduce possible experimental observation times. To avoid populating metastable states or allow these states to decay before another laser excitation−emission, or excitation−depletion, cycle takes place, fast scanning or low-repetition pulse rates can be applied166 (i.e., D- or T-Rex as discussed above). Wavelength selection, pulse duration, and repetition rates of the pulsed STED laser are thus important technical S


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way to realize a first generation instrumentation. In commercial systems, broadband lasers have so far only been applied as excitation sources, together with selected single-wavelength laser sources for STED. One reason for this is that the broadband laser sources are still somewhat high cost. In addition, because of insufficient pulse energies across the full emission spectrum of the lasers, they cannot be used for depletion at all desired depletion wavelengths but mainly in the far-red/near-infrared spectral range. 3.2.1.2. STED with Continuous-Wave Lasers. Given that high-power, pulsed lasers suitable for STED are not available within the full emission wavelength range of interest (within a reasonable level of cost and complexity), continuous-wave (CW) lasers can also offer alternatives for depleting fluorescence in STED microscopy. Basically all laser wavelengths of relevance for STED are available via compact CW laser units. From the sections above, discussing pulsed laser sources and their technical specifications, one may think that STED imaging is only possible by pulsed depletion. Historically, this is how all STED microscopes initially were designed. However, there is no such limitation. Switching off fluorescence by stimulated emission depletion is effectuated with light, and any mode of delivering the depletion light onto the signal-generating fluorophores should in principle work. To “fully” deplete (i.e., to switch off neighboring fluorescent entities), there is a need of a lot of far-red-shifted depletion light around the focus, which can successfully compete with spontaneous emission so that it does not occur. High-power CW lasers should thus also be feasible as depletion lasers in STED microscopy, and they would offer a relaxed instrumentation complexity with no laser-pulse preparation and control required. Using the Ti:sapphire laser in nonpulsing CW mode for a first test proved that CW-STED is feasible. In 2007, the first CW-STED imaging publications appeared, and CW-STED has thereafter also been made commercially available.233,234 While most pulsed STED microscopes use high-power nearinfrared light for depletion, the wavelengths used for depletion by high-power CW lasers are predominantly found in the visible range (in which range there is a lack of suitable lasers for pulsed STED). In CW-STED imaging, one may ask how an “infinitely” long, CW depletion pulse will affect the measurements and the sample. It can be noted that when using pulsed STED lasers, the excitation of the signaling fluorophores also needs to be pulsed. CW excitation will require CW-STED, since undepleted excitation events otherwise would occur in between the STED pulses. With CW-STED, however, both CW and pulsed excitation can be considered. How do then these different combinations apply to STED imaging? For CW excitation and CW-STED, a steady-state would emerge, with the excitation continuously working against the constant inhibition of the excited S1 state population by the depletion laser. The way depletion is achieved is in principle reflected in the STED saturation factor (Is) expressed in eq 4, which differs somewhat in pulsed and CW-based STED microscopy. In simplistic terms, for a pulsed STED system, the intensity of the depletion beam is concentrated into one pulse, aimed to deplete fluorescence immediately after a pulsed excitation event has occurred. In the CW excitation CW-STED approach, a fluorophore senses comparably lower depletion intensities when in its excited state because the depletion light is equally distributed over time and not concentrated to the fraction of time the fluorophore is in its excited state. Compared to a pulsed STED system (with ∼80 MHz pulse

frequency), CW mode STED thus needs about 4- to 5-fold increased average power of the STED laser to reach the same resolution enhancement.96,226 This increased laser power may increase photobleaching as well as unwanted re-excitation. An additional drawback is the possibility of fluorescence emission without depletion, arising as a consequence of the dynamic equilibrium established between (stochastic) continuous excitation and depletion. Residual fluorescence thus often remains within the depletion focus, which lowers the achievable contrast and resolution in CW-STED imaging compared to pulsed STED imaging. A benefit of using CW excitation is that the fluorescence signal can be increased by the “infinitely” fast repetition rate, with no intervals in between pulses when excitation is not possible. This enables fast imaging with CWSTED, and in combination with fast beam-scanning microscopy, it is well-suitable for live-cell fluorescence imaging. The fast modulation introduced decreases photobleaching by not allowing the fluorophore to enter the environmentally reactive triplet or radical states, and as the signaling fluorophore is generally only switched between the ground and excited states (i.e., absorbing and emitting photons), the effective fluorescence per unit time is increased. As an alternative to CW excitation and CW-STED, pulsed laser excitation with CWSTED can offer additional benefits. Pulsed excitation and CW depletion basically merges the protocols discussed before, and by introducing time-gating of the detectors, it is possible to temporally filter out a higher spatial resolution and receive better image contrast, compared to applying CW-STED with CW excitation.210 Most realizations of pulsed excitation/CW STED imaging this far are based on one-photon excitation in the visible wavelength range, but also two-photon excitation has been proven fully feasible.235 Even without detector gating, pulsed excitation with CW-STED is beneficial, as it may reduce residual, undepletable, fluorescence that can now occur only in direct conjunction to the short duration of the excitation, thereby improving contrast compared to CW-STED with CW excitation. Furthermore, adding temporal gating of the signal, referred to as gated CW-STED, allows additional contrast improvements as one can suppress unwanted background. Practically, by opening the detector after the excitation pulse has passed, the detected signal is free of background contamination coming from scattered excitation light. By closing it after fluorescence has seized allows blocking any depletion laser-induced (re)excitation which again improves image contrast. From the discussion above on pulsed STED (together with pulsed excitation), it is clear that in order to avoid unwanted background, the fluorescence signal should be detected after the short excitation and longer depletion pulses have passed. However, with CW-STED, the depletion laser is continuously on, so when should one then initiate the signal detection? Again, this depends on laser specifications coupled to fluorophore characteristics, as well as on the STED depletion profile. The pulsed excitation should typically be shorter than the fluorescence lifetime, which is in the range of 1−4 ns for most fluorophores. Moreover, the depletion induced by the STED laser lowers the time fluorophores are in the excited state by inducing rapid stimulated emission. With more or less depletion power, shorter or longer decay times will be present. Selecting a long detector delay, significantly longer than the excitation pulse, thus allows only fluorescence from undepleted fluorophores in the very center of the null-intensity STED profile to be detected. In other words, temporal filtering allows a more precise spatial coordinated separation of closely located T


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Figure 6. Schematic overview (from left to right) of essential components in a conventional STED microscope setup. Work horse in the setup is an appropriate STED laser, either combined with a separate excitation laser or a multiwavelength laser unit (from which spectral separation and beam routing occurs). After selection of appropriate depletion (red) and excitation (blue) wavelengths, the laser beams are spatially filtered in a singlemode fiber, beneficially improving and controlling the wavefront, polarization, and pulse length (if necessary). The size of the laser beam (including collimation) can also be controlled with this unit. Following the fiber unit is a polarization control of the excitation beam. It is necessary to clean up polarization or change its directions to have the appropriate excitation conditions to match depletion and the selection rules of absorbing and emitting fluorophores. Simultaneously, in the depletion beam, an appropriate optical unit resculpturing the STED profile is added, either being a spiraling phase plate (i.e., vortex plate) and/or a phase ring that introduces spatial phase differences over the STED beam to generate lateral and/or axial depletion donuts. A segmented phase plate (easy STED) modifying the polarization across the cross section of the STED beam can also be selected.173 After recombining both laser beams (or having them already colinear), they are launched into a scanner and the microscope objective, to sequentially excite and deplete the labeled sample. Detection of fluorescence is simultaneously achieved through the scanner. Fluorescence passes a spatial pinhole (to eliminate out-of-focus light) and is filtered with appropriate bandpass filters (blocking laser light), before finally reaching the photodetector (several for multicolor STED). Note that several optical parts like metallic mirrors, dichroic beam splitters, additional telescope units for beam size expansion/reduction, etc. have not been added. Additionally, electronics units for laser pulsing synchronization control or a fluorescence emission counting, for example, all mounted in a computer with an appropriated software interface, is also missing.

fluorescent species with similar features. As can be realized, this possibility to increase resolution comes at a cost: a reduced detected signal-to-background ratio. This is because gated detection also suppresses valuable signal from the focal center, occurring within the time the detector is gated off. Another issue is that the detector electronics for gated CW-STED must be possible to gate and pulse in a controlled, synchronized manner in relation to the pulsed excitation laser, which adds some instrumental complexity and cost to the system. The benefits regarding resolution and image contrast is however large compared to CW-STED with CW excitation, as discussed above. Gated CW-STED systems are thus, together with pulsed STED systems, the preferred systems, now also offered by several microscopy venders. 3.2.2. STED Wavefront Tuning. As pointed out above, the workhorse of a STED microscope is the depletion laser (CW or pulsed) in combination with an excitation laser (including detector system and additionally needed electronics). Figure 6 shows a schematic of a basic STED configuration, where most essential optical elements have been included. For STED imaging to work, the beam profile of the depletion laser in the microscope focus must be resculptured into a beam profile with zero-intensity light in some parts of its cross-sectional area. The extent to which this zero-intensity requirement can be reached critically determines the resolution achievable, but even a contrast of 100:1 between the maximum intensity and the null of the STED beam can be sufficient for achieving an eight- to

ten-fold better spatial resolution than the diffraction limit.46,226 By overlapping such a STED laser beam profile onto an excitation laser beam profile, targeted switching becomes possible. Fluorescent entities are then switched off by stimulated emission in the outer focal areas and are allowed to generate a detectable fluorescence signal from the central focal spot. Scanning the coaligned STED and excitation beams over the fluorescent sample and detecting sequentially each nanoscale pixel can then finally build up a super-resolution raw STED image (following the principle outlined in Figure 2). A very appealing benefit of STED nanoscopy is the direct generation of a super-resolved image. No mathematical image processing is necessary, in contrast to, for example, PALM/ STORM and SIM. Image processing is optional, but normal deconvolution of the STED image generates improved contrast, which helps in the later data analyses.236 3.2.2.1. One- and Two-Dimensional Wavefront Tuning. Historically, the first experimental proof of a working STED principle for imaging was done without any dedicated wavefront tuning, by overlapping an unmodified STED profile to the side of an excitation focus.92 Thereby, fluorescence subdiffraction resolution imaging of nanocrystals by use of STED imaging could be demonstrated, albeit with a relatively modest resolution improvement factor of 1.3. After this pioneering work, wavefront tuning by phase-plates was introduced,2 and as the extension of a diffraction-limited focus is basically 3−4 times larger in the axial direction than in U


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Figure 7. Common phase-plates and their generated depletion patterns. (Top) 1D Lateral phase-plates where the cross-section of the STED beam is optically delayed from left-to-right as well as from below-to-above; in the wave-picture a phase-shift over the cross-section generates crest and trough overlaps after focused by the objective lens, thus annihilating depletion intensity along a lateral line. Schematic STED patterns of this so-called “bun” intensity distributions are shown together with their line profiles. Moreover, the 2D pattern generated by crossing two buns generating a depletion pattern resembling a donut as seen from above is also shown. (Middle) Axial phase-plate (i.e., phase-ring) where the cross section of the STED beam is optically delayed from the external-to-internal, which when focused by the objective lens generates an axially hollow depletion intensity distribution. The reason for this is a simplistic picture that the internal delayed part is focused by under-filling the lens (thus producing a large focus), whereas the external parts are focused by the full aperture thus generating a smaller focus. The overlap with different phase shifts again annihilates depletion intensity inside the STED focus. In the middle and to the right, the schematic and experimental depletion profiles of applying an axial phase-plate are also shown [40 nm gold bead scanned through the focus (i) laterally and (ii) axially]. (Bottom) 2D Lateral phase-plate with a spiraling shift of the phase around the cross section of the STED beam (i.e., a vortex plate), which produces a hollow cylindrical depletion intensity distribution. Opposite sides of the STED beam sees a delay that is the same generating the 1D bun intensity distribution. An “overlay” of a continuum of slightly shifted buns generates the hollow cylinder, which when experimentally scanned laterally over a scattering gold bead looks like a donut, see (i). If sliced axially through its center, the donut in (i) displays as off-centered buns as seen in (ii).

indeed confirmed an improvement by a factor of 1.3 (i.e., in 1999,92 our remark). However, this moderate improvement was achieved along a single direction only, and it remained unclear whether it would be effective in biological imaging”. In ref 2, the phase-plate designed to modify the wavefront of the STED beam consisted of a planar glass substrate with a central ring of evaporated magnesium fluoride (Figure 7, middle). With a carefully selected diameter and thickness of the evaporated

its transverse (or lateral) dimension, effort was primarily focused on improving the resolution along the optical axis. In this follow-up work,2 it is interesting to quote the published words and opinion on how and what might then be achievable: ‘‘Engineering of the fluorescence spot, or point-spread-function (PSF) by stimulated emission depletion, was predicted to improve the resolution in the transverse direction (i.e., in 1994,1 our remark), and initial experiments with nanocrystals V


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STED depletion beams into “rings” of high laser intensity surrounding an area of zero intensity.231,238 Present day commercial STED microscopes apply both vortex plates and SLMs, with the latter technology being more complex and expensive but dynamically adjustable. The adjustability may allow fine-tuning and correcting aberration of the laser beam wave-fronts and allows imaging deeper into, for example, layers of highly scattering biological tissue (i.e., into highly optically “hostile”, distorting specimen that effectively degrade image contrast and spatial resolution). This wavefront approach falls under so-called adaptive optics (AO), applied for example in astronomy to correct for wavefront aberration induced by the atmospheric sky when imaging the universe. The application of AO to SRM is still in its infancy.153 By AO, however, using a dynamically reconfigurable optical element, such as an SLM or a programmable and deformable mirror array, it has been shown possible to restore operation of a STED microscope, also deeper into optically distorting specimens.239 In general, aberration and its influence of the STED focus can be analyzed with AO and can help in controlling the depletion profiles.240−242 3.2.2.2. Three-Dimensional Wavefront Tuning. As cellular biology is a three-dimensional microcosmos, the wish in STED imaging development was then to go from 2D to 3D STED microscopy. The initial “trick” was again to combine two depletion profiles to improve resolution both along the axial focal plane and in the lateral focal plane directions. Two STED beam paths were thus constructed and overlaid to have the 2D hollow cylinder depleting in the x, y-directions (i.e., the donut profile seen in the axial direction, Figure 7, bottom), together with an “overlaid” axial 1D distribution depleting along the z direction. Proof-of-principle 3D (2D+1D) STED imaging of fluorescent nanoscale bead assemblies and the immunofluorescent-labeled microtubular network in PtK2 cells generated focal spot precision of 43−45 nm and 108−125 nm in the lateral and axial extents, respectively.238,243 As is well-known, the performance of STED microscopy is dependent on several interrelated factors. First, the perfect minimum of the STED profile needs consideration as residual depletion intensity will dim the signal in the nanoscale image, and in the worst case, the signal may not be visible above the background at all. Second, the spatial overlap of the excitation and depletion beams is important, selecting centrally located fluorophores generating the sequentially detected signal (i.e., voxel by voxel). For “perfect” depletion performance, the central zero should really be a null-intensity area, and the overlap of excitation and depletion should basically not be off at all (for full contrast performance), and then this aligned overlap should stay stable over time, when the beams are scanned and during the entire STED imaging session. Such design challenges have been the driving force for implementing some of the reported “easy STED” approaches.244−246 In a first realization based on diffractive optical elements, a single beam-path STED system was constructed, which includes a phase plate that selectively modulates light in the wavelength range of the STED beam but leaves light with the excitation beam wavelength unchanged. In this configuration, the beams are aligned by design, coaligned into a single optical common element, and the alignment is hence insensitive to mechanical drift.245 Similarly, a segmented birefringent device has been designed, which produces a donutshaped focal spot with suitable polarization for the STED laser, while leaving the excitation spot virtually intact.246 For existing fluorescence confocal microscopes, equipped with suitable

optical layer, matching the STED wavelength and entrance pupil of the microscope objective, the sign of the depletion wavefront amplitude could be reversed with respect to the remaining ring-shaped area. Destructive interference was thereby generated at the focal point, with constructive STED intensity distributions axially (with a minor fraction also laterally) above and below this point. Applying the resulting STED intensity distribution for imaging of fluorescent beads and labeled membrane structures in yeast cells generated sub100 nm axial focal precisions, an improvement by a factor of about 5−6 over diffraction-limited imaging by confocal microscopy.2 By changing the wavefront of the STED beam at different parts over its profile it is obvious that also other depletion focal intensity distributions can be realized. Phase-shifting the wavefront “left-to-right” over the STED beam profile, in conjunction with unilateral y-polarization, results in an unidirectional valley distribution, with STED light split to the sides, resembling a hot dog bun (Figure 7, upper row). This STED light distribution squeezes the fluorescence focal spot along one lateral direction (i.e., along the x axis). This “bun” distribution is only suitable for 1D image improvements. However, its use with full depletion power generated sub-20 nm focal precision in one lateral direction.221 The combination of two “bun” phase-plates, adding an x-oriented, x-polarized counterpart to the one just described above, generates two lineshaped valley minima, which when properly overlapped can generate a donut-shaped STED light distribution. The “donut” STED distribution symmetrically squeezes the fluorescence focal spot from all lateral directions thus having a chance to improve conventional 2D imaging several-fold. Because of the somewhat tedious and complex work of aligning, as well as twice the cost for all optical components including optomechanics etc., other ways were sought to achieve a 2D STED microscope than the two crossed “bun” depletion pathways. A historical note in this context is that the very first commercial STED system (from Leica Microsystems) constructed in 2007 applied two “buns” for nanoscale imaging, achieved by reflecting half the depletion beam at a Brewster angle to introduce a π shift in the STED beam(s). The focusing of a laser beam with a high-numerical microscope objective can, depending on the polarization component of the wavefront (interfering constructively and destructively), generate a selected focal spot intensity distribution. If the wavefront amplitude of the depletion beam is “flipped” on one side of the STED beam and then merged in the focus, destructive interferences between the two beam sides result in a zero intensity depletion line as mentioned above. Delaying the wavefront by a helical phase ramp where each opposite side around the perimeter of the STED beam is “flipped” then yields a doughnut-shaped depletion spot, as in the crossed “bun” case above. With the use of polarization-sensitive liquid crystals, optically and electronically addressable in so-called spatial liquid crystal modulators (SLM’s), helical phase ramps were first imprinted into the reflected STED beam to convert it to a donut intensity distribution for 2D STED imaging.230,237 Soon afterward, it was discovered that cheap plastically replicated diffraction optics for laser beam profiling, with an imprinted helical phase ramp for a plethora of wavelengths, could also be applied to design 2D STED microscopes. This so-called vortex plate, with a phase singularity to generate a donut depletion beam, thereafter became the most applied tuning-element to optically sculpt W


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pixel-by-pixel (or for 3D imaging voxel-by-voxel) detect undepleted fluorescence and to eventually build up a nanoscale image; either the sample is moved step-by-step and all optical beam paths are kept fixed onto the optical axis (stage-scanning) or the laser beams are swept over the sample by a scanner (beam-scanning). The most common beam-scanning solution is a mirror pair, driven by galvanic motors to generate small rotations and thus offsetting of the beams at the back aperture of the microscope objective lens, which subsequentially induces a focal spot shift. The largest technical difference between stage-scanning and beam-scanning is the speed at which imaging acquisition can occur. As beam-scanning physically does not move a heavy mass (i.e., the scanning stage holding the sample), it can achieve line-by-line sweeping of several hundreds to thousands of lines per seconds, over a field of view of typically 25 × 25 μm, as used in STED imaging. This is fast enough for many live-cell fluorescence imaging applications. Improved imaging speeds can be achieved by increasing line frequencies, up to ten thousand lines per second, by driving the mirrors in resonance. Thereby, fast video-rate STED imaging becomes possible, but only for a smaller field of view as long as the scanning along one axis is still performed by stagescanning.247 For a full 2D (x- and y-direction) resonant mirror approach, however, video-rate imaging also for larger fields of view should be possible. Moreover, acousto-optical deflectors allow line rates up to a few hundred thousand sweeps per second, but wavelength-dependent deflection angles and beam distortions render the system complex, which however can be circumvented by the use of electro-optical scanners.207 The latter technique can sweep the focal spots so fast that fluorescence imaging becomes temporally stochastic (i.e., the pixel dwell times are on the order of the fluorescence lifetime of the fluorophore). Thus, this ultrafast STED modality has the potential to detect dynamic processes at higher imaging rates, while providing increased photon yield compared to slow scanning (avoiding population of reactive states of the fluorophores166). Given the often challenging and laborious preparation of biological samples for SRM, the need to make the technical aspects of STED more rugged and simple, to bring it easier within reach of a broader community of life scientists, is a continuous driving force for STED development. Technical improvements will basically forever continue to catch up with ever more complex biological imaging wishes. A drawback even with conventional diffraction-limited confocal imaging is the point-by-point sweeping approach of collecting images. As is well-known, point scanning becomes slow when trying to image larger areas and especially large volumes. Various technical solutions to this have been presented and also commercialized. Scanning several foci as done in spinning disk confocal microscopy248,249 allows improved imaging speed because a single focus builds an image sequentially, one pixel at a time, while an array of foci can simultaneously illuminate the entire specimen field and in parallel detect generated fluorescence. For STED, the first parallelized system quadrupled the number of excitation and STED focal points, providing up to 4-fold faster image acquisition.205 A further increase when imaging larger areas have been achieved by switching to camera-based detection (i.e., multiplexing the detector side by abandoning point detection), combined with gridlike illumination with 100 or even 2000 equivalent donut spots.206,250 3.2.4. Microscope Objectives. Ever since the late 19th century, it has been theoretically and experimentally realized

lasers, addition of optical elements of this kind can easily turn them into STED microscopes. The segmented birefringent device generates a somewhat steeper null intensity of the STED profile, better tolerates misalignment on the optical axis into the microscope objective, including deviations of the designed wavelength, and compared to a vortex phase-plate design, it tolerates a higher imperfection of the input polarization.244 For a vortex helical phase-plate minute remanences of wrong depletion polarization will introduce nonzero intensities in the center of the depletion beam.46 Furthermore, a multiplate segmented birefringent device can be designed to generate a rugged optical element for 3D STED microscopy. 244 Programming a dynamically reconfigurable SLM also allows a single beam path for 3D STED microscopy to be designed, for both the excitation and depletion laser beams, with lateral and axial depletion profiles. 3.2.3. Optical Paths, Mirrors, and Scanning Devices. The standard configuration of a STED microscope is a modified confocal or two-photon microscope to which one or several suitable depletion or excitation lasers have been added, including a wavefront tuning selection system (see sections above). A very important issue when designing a STED microscope, with a sharp zero intensity minimum of the depletion beam(s), is to have full control on polarization and wavefront issues of these beam(s) after passing all optical elements. Wrong selection of beam path optics having unsuitable surface quality and nonflat coatings (often induced from too thin glass substrates) can seriously distort the depletion wavefront(s). Flat surface optics, with deviations of less than a quarter of the wavefront in selected wavelength(s) (i.e., λ/4 or less) are thus selected. As a rule of thumb in STED microscopy, the optical elements with the highest possible flatness are typically selected. To help in “cleaning up” wavefronts, the STED beam is often launched via single mode optical fibers, which also preserve polarization. In pulsed STED, the physical length of the fiber is additionally adapted to properly stretch the temporal extent of the depletion pulse, typically to hundreds of picoseconds. An equally, or even more important issue is the polarization control of the depletion beam as it passes through normal mirrors, dichroic mirrors, filters, and lenses. With dependence on the polarization component of the wavefront, the focal spot intensitydistribution can be tuned, as discussed above. Reflecting mirrors with metal-coated glass substrates are thus most often used, since they preserve polarization better than dielectric mirrors when guiding the depletion beam through the optical pathway of the microscope. Dielectric mirrors, put at exactly correct angles to reflect with maintained polarization components of the wavefront, may however also be possible to use at less critical parts of the pathway. To help to get full control of polarization issues, polarizers are added into the beam path, especially before and after optical elements merging different beam paths used in multicolor STED imaging. Furthermore, as discussed in the section above, to shape the depletion beam, STED wavefront tuning is achieved with a combination of amplitude and/or polarization control, by adding a phase-ring, vortex plate, easySTED plate, or by reflection via a dynamically controllable SLM. More than ten years of STED publications show that all these alternatives work, and that they produce experimentally similar resolutions, irrespective of selected elements. For STED imaging, there are basically two alternatives for how to move the focal spot over the sample, to sequentially, X


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around 30−50 nm.101,102 In these experiments, the fluorescent sample was depleted by the standing-wave focus of the two opposing objective lenses, while excitation and emission detection was done through the “common” one-sided focus, thus only improving axial resolution (of thin samples). Extending the 4Pi-STED system to have both lateral and axial depletion beams, together with a coherent standing-wave excitation and dual-objective fluorescence detection, generated isotropic STED resolution down to 30 nm and could be demonstrated to visualize mitochondrial cristae.257,258 To ensure that the lateral and axial depletion standing-waves did not interfere among themselves, different polarization directions were applied. As pointed out by the authors, circularly polarized STED light would be advantageous as it can better deplete fluorophores in the focal plane, irrespective of their molecular dipole orientation. With interferometric 4Pi-STED microscopy, however, full control of the coherent excitation and detection paths is possible. To allow for improved control of the 4Pi-STED interfering wavefront approaches, adaptive optics has also been applied.259 The technical benefit of using a dualobjective system has also been applied to single-molecule localization microscopy to improve 3D resolution in cellular imaging, by measuring the phase difference of emission on several detectors to map sample space (standing-wave) coordinates.260 3.2.5. Detector Systems. In the strive for high resolution in STED imaging, the effective excitation volume after depletion is made smaller, and there is thus often only a handful of fluorophores in this volume. As a consequence, the detectors used need to be sensitive. Single-photon sensitive avalanche photodiodes (SPADs or APDs) are the most commonly used photodetectors for STED imaging. APDs have been used in the single molecule spectroscopy field since the early 1990’s.261,262 They provide high quantum efficiencies (typically efficiency of above 50% in the green spectra, increasing to above 70% in the red), come in integrated packages that contain on-board amplifiers and cooling, and offer low dark counts (30% from green to red), high dynamic range, low dark noise, and fast timing response with a larger detection area.264 Presently, an increasing number of STED microscopes, especially from commercial vendors, are equipped with such hybrid photodetectors. Apart from the detectors themselves, detection electronics in the form of counter/timer cards are needed to store the detected signal and make it presentable on a computer screen.

that the microscope objective is the fundamental piece of an optical microscope, setting the achievable diffraction-limited resolution. Also for STED imaging, ever since the pioneering proof-of-concept studies,2,92 high-numerical oil immersion objectives, in principle handmade thus showing some “individuality”, have maintained their role as standard items. Oil objectives are well-suitable for imaging of single cells on top of a coverslip, where the cells have a refractive index around 1.37−1.42. They are even more suitable when the cells are mounted in a higher refractive media matching the refractive index of oil (and glass); this being around 1.51−1.52 at room temperature. For live-cell imaging, however, the surrounding medium is an aqueous buffer with a refractive index of 1.34. Imaging within thin flat cells (typical height around 3−5 μm) close to the coverslip surface then still allows “perfect” imaging conditions, also with an oil objective. Penetrating deeper into an index-mismatched aqueous solution, however, induces focal spot distortion lowering the optical resolution. Similarly, imaging deeper into cellular layers will also lower the reachable STED resolution for nanoscale visualization. The situation becomes even more severe trying to image deep into biological tissue, where on the one hand it would seem as if index matching might be “improved”; for example, fat cells have a refractive index around 1.5, but where the longer optical path into the highly scattering medium and the autofluorescence of the tissue lowers resolution and imaging contrast. The historical solution to this problem has been slicing, of plastically embedded or frozen tissue blocks, into ultrathin sections and then imaging of these slices, after which mathematical image alignment puts everything back in place. Such an approach has also been applied with STED.251 Recently, application of optical clearing, and mounting with index matching solution allowed deeper STED microscopy of nanoscale kidney morphology,252 with up to 30 μm imaging depths tested. This morphology has previously only been resolved by EM.252 To further increase penetration depth in tissue, which does not offer a good index match with an oil immersion objective (e.g., brain tissue has refractive around 1.38), selection of other immersion solutions can be applied. For brain tissue, a high-NA glycerol immersion objective lens makes it possible to reduce spherical aberrations stemming from the mismatch in refractive index between the immersion medium of the lens and the brain tissue, otherwise leading to degraded resolution below the tissue surface. STED microscopy with such an objective, finetuning the correction collar to minimize aberration mismatches, has allowed morphological nanoscale imaging to be performed deep inside brain tissue (at depths up to 120 μm),253 even in vivo imaging in a living mouse brain has been achieved.254 For brain tissue slices, it has been pointed out that selection of a water immersion objective lens might potentially allow STED microscopy at even higher penetration depths, as this would offer a higher degree of index matching.89 This far, however, no published work has presently been achieved with deep STED imaging based on a single water immersion objective. Combination of dual-objective systems for STED nanoscopy has actually explored the latter. The so-called 4Pi configuration (i.e., the use of two opposing objectives that by coherent addition of excitation or/and emission sharpens the PSF of the microscope) was implanted already in some of the very first STED microscopy setups, and then both oil and water immersion objectives were applied.255,256 Proof-of-concept studies with 4Pi-STED demonstrated the imaging of bacterial membranes and cellular microtubules, with axial precision of Y


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the STED microscope. Pulse durations down to roughly tens of picoseconds can be measured with a very fast photodiode in combination with a fast oscilloscope. Alternatively, the measurement of the STED pulse duration could be done within the STED microscope, sweeping the STED pulse over the excitation pulse by an electronic delay and monitoring the intensity signal from a fluorophore solution. With moderate STED power and a Gaussian STED profile, the signal reports the cumulative STED pulse energy after the excitation pulse. While the pulse length itself needs only to be checked once, a daily issue to check for pulsed STED is the fine-tuning of the temporal overlap of excitation and depletion laser beams, which needs to be tweaked to optimize resolution performance. Good temporal overlaps can be obtained, either electronically by tuning pulse delays between depletion and excitation or physically by tuning beam path length differences (where 3 cm free-space difference is assumed to represent a delay of 100 ps and allowing fairly good pulse overlaps to be set during instrument assembly). The preferred temporal alignment is however finally done with electronic delay shifting, as this does not physically move laser beams. At the stage of instrument assembly, it is also important to check that the wavefront flatness is perfect, after passing all mirrors, lenses, and all other optical components of the instrument, for example, by using an interferometric sheer-plate. Equally important to ensure best settings is to check the polarization state of the depletion beams, by a polarizer-analyzer in front of a detector for example. Polarization tends to change somewhat as the beam(s) transverse all optical elements in the microscope. The beams sometimes need to be polarized perfectly linear or perfectly circular (right-handed or left-handed) to generate the final intensity profiles for the excitation and depletion PSFs, in the latter crucially important for the generation of the zerointensities. In view of that the resolution in STED imaging scales with depletion intensity (eq 4), measurements of the power of the depletion laser are important, in particular of the actual depletion power of the laser when leaving the single mode fiber and going into the microscope. The output powers of the lasers are specifications provided by the manufacturer and can simply be measured with a power meter. Power measurements (after exit of the beam from the fiber) are necessary at least on a weekly basis. Reduced depletion power coupling can also be detected in deteriorated resolution in calibration samples, as discussed below. In a high-end super-resolution STED nanoscope, the alignment of various critical components and parameters are highly interrelated. A major parameter typically well reflecting the overall performance of a STED instrument is the power drift over time of the fiber in-coupled laser light. This typically gives a good measure of how well several critical parameters of the optical hardware are adjusted with respect to each other. When drift does occur, for example, of a fiber core having a diameter of a few micrometers, manifested in a lowering of the available depletion powers and/or excitation powers, reoptimization might induce small changes in the set polarization. In a worst case scenario, pulse delays and wave-fronts might be slightly skewed as well, requiring readjustments. As a consequence, several things need to be controlled and checked iteratively before reaching the best performance possible. However, before being able to check many of these interrelated matters, a STED microscope setup is first aligned through all optical elements, to achieve just conventional PSFs, with the PSF of the depletion beam(s) overlaid onto the excitation

Such electronics are central, for example, for gated CW-STED imaging,265 as described above. Time-gated detection (i.e., filtering of the signal in a certain time-window with respect to a periodic pulsed excitation) was used already in the first roomtemperature single fluorophore detection experiments in 1990 to suppress contributions from instant laser light scattering into the detected fluorescence signal.121 Time-gated detection is based on the time-resolved detection of fluorescence signal induced by periodic, pulsed excitation, which for single-photon detection most often is referred to as time-correlated single photon counting (TCSPC).266 From histograms of detected events generated by TCSPC, software-wise filtering of the signal in certain time windows can then be performed. Temporally gated detection is also possible hardware-wise, by directly gating the detectors in the experiments.210 For approaches with multispot (or gridline) STED imaging, parallel detector system in the form of charged coupled devices (CCDs) can be applied for faint single molecule condition imaging. CCDs convert incoming photons into electron charges on a well-arrayed semiconductor chip with high quantum efficiency and low noise. Especially modern electron multiplying (EM) CCDs are applicable, in which a series of connected wells at the end of the chip electronically multiply and boost the signal. Thereby, gain factors of about a factor of a hundred can be obtained, without too much noise being introduced. Furthermore, new cameras with complementary metal oxide semiconductor (CMOS) image sensors have emerged that in several aspects can offer a better alternative than EMCCDs, especially for applications where high-speed detection is required. Both camera systems have been applied for parallelized STED imaging.206,250 On the backside is that CMOS cameras have relatively high readout noise levels and variable hot pixels, which demand calibrations when used for super resolution single molecule imaging.267,268 3.2.6. Calibration and Performance Testing. Several recent review articles on super-resolution STED imaging summarize stepwise necessary items to check during assembly, and common “tricks” that can be applied in order to easily and with a high chance of success build a STED nanoscope.48,88−91,269 Calibrating and checking performance of the system is an iterative process, continuously controlling crucial parameters. Starting from the laser side, several strategies have been developed to optimize and make the photon-generation part of a STED system compact and more userfriendly.231,232,243,270 As pointed out in the laser section above, single mode, polarization maintaining optical fibers are often used to guide the depletion light into the microscope scanner, thereby avoiding free space laser paths which are not so easily coaligned. The length of the fibers is adapted to generate the desired pulse lengths, and their mode profiles can clean up distorted wave-fronts (see Figure 6). With a pulsed STED system applying, for example, Ti:sapphire femtosecond lasers, pulses are prestretched with decimeter long glass rods to avoid nonlinear (white light) generation and to achieve broadening of depletion pulses in the fiber.89 The duration of the depletion pulses needs to be known. Too short pulses may result in severe photobleaching, and if they are too long, the required depletion efficiencies may not be reached because of insufficient laser irradiances within the pulses.89 With a fixed selected fiber length, a pulse duration measurement must basically only be done once in the laboratory (or at the vendors factory), since it does not change dramatically as a function of depletion wavelength and power settings when in operation in Z


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Figure 8. Schematic of work flow to initiate daily STED microscopy, from starting a system, checking the PSF’s for misalignment, after which some quick calibrations are applied for performance checking. The listed times can be shortened for the last two steps, but it is crucial to let the system warm-up to a stable operation temperature (including equilibration with the environment in the operating room) to avoid drift.

PSF(s), without any depletion phase-mask(s) yet entered into the beam path(s). By scanning small scattering gold beads, typically sub-100 nm in diameter, and mounted onto a microscope coverslip, the PSF-profiles can be monitored on a large area PMT. Practically, several gold beads should be checked as their size and shape affects the scattering and image results, which are also wavelength-dependent and polarizationsensitive. In the alignment, including checking wave-fronts, collimation, and polarization, the resulting conventional PSFprofiles must be tuned to be “perfect”, where widths and heights can be benchmarked against eqs 2 and 3. An adjustable pinhole, sharp emission filters, and single photon sensitive detectors, including a counter/timer computer card with some controlling software, are then added to produce a fluorescence confocal microscope. Indeed, a STED microscope is in the end also a very good confocal microscope, and confocal images are often used as low-resolution overviews, onto which nanoscale

STED visualizations can be overlaid (if the light dose tolerated by the cells allows previews of this kind). Moreover, several alignments of a STED imaging system are typically evaluated together with confocal PSFs, by imaging fluorescent beads and fluorescent quantum dots (sized 20−40 nm), or a thin layer of a fluorescent solution on top of a coverslip to set best spatiotemporal overlaps of PSFs and pulses. The fluorescent calibration samples are also suitable for basic spatial and chromatic alignment of the detectors. First, depletion power calibration can be tested to see if the available power from the STED laser sent into the back aperture of the objective lens is adequate or how high one should maximally go in order to fully deplete the fluorescence in the calibration samples, without too much re-excitation occurring. The tuning of the excitation and depletion delay in pulsed STED is further possible to calibrate, as shifting the temporal separation can be monitored by seeing worse or better depletion (i.e., more or less fluorescence being AA


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to expand on resolving possibilities in microscopy.272 As even, for example, individual fluorescently labeled macromolecular protein assemblies are resolved on the nanoscale with STED microscopy, the mission in sample preparation is to produce a very specifically labeled structure, with high contrast, strong signal, and with preserved overall native biology, while at the same time not inducing too much of unspecific background labeling. Sample protocols used previously for immuno-labeling or live-cell transfection fluorescence microscopy can often be applied largely unchanged. A variety of samples, ranging from single cultured flat cells, tissue slices to whole model animal systems (e.g., C. Elegans or D. melanogaster), may thus be investigated also by STED microscopy. The better resolution offered with STED can in principle come to its full right only if the sample is good enough for visualizing the nanoscale. Thus, highest care is required in the preparation, as the resolving and localization power of STED microscopy will often identify artifacts. Technically, as discussed in the sections above, the compatibility between samples and microscope objective is an important issue. Presently, almost all STED imaging systems apply high-numerical oil objectives (e.g., 100x/1.4 NA), which has a working distance of tens of micrometers (300 nm in diameter) are typically required. With diffraction-limited optical resolution, RICS can only distinguish larger regions of interest and requires low fluorophore concentrations in the nanomolar range. To overcome these latter aspects, RICS has recently been combined with STED microscopy.344 Thereby, spatiotemporal information on molecular diffusion in model membranes and live cells can be

5. APPLICATIONS OF STED IMAGING It is fair to say that the field of SRM is still young, and the potential capabilities of STED imaging and other SRM approaches still remain to be fully explored.153 However, it is evident that SRM is quickly bridging the gap between EM, for decades the prevailing reference technique for imaging of the finest details of subcellular structures, and CLSM and other diffraction-limited fluorescence microscopy techniques, offering limited resolution but flexible and easily accessible approaches for localization and identification of subcellular structures. The major uses of SRM this far have naturally focused on applications, which specifically require biological information stemming from changes in structures smaller than 200 nm (i.e., beyond the resolution limit of conventional, diffraction-limited optical imaging), and where it together with detailed morphometric characterization is of importance to detect and identify specific biomolecules with high sensitivity and specificity. Besides structural and morphological characterization combined with biomolecular detection/identification, SRM techniques are also quickly expanding toward interaction mapping, multiple target detection, and live imaging.277 In these latter application categories, SRM methods benefit from distinctive methodological advantages compared to EM, in particular more flexible sample staining with much higher labeling efficiencies, faster and simpler readouts, and far more gentle sample preparation procedures. Naturally, the areas within which SRM methods will find their major applications will be determined by a combination of the biological information asked for and the limitations of current microscopy modalities used, in particular of EM and diffraction-limited fluorescence microscopy, motivating using SRM instead (i.e., can the requested information be specifically obtained by SRM, or maybe by simpler or more adequate means?). In addition, increasing awareness of the capabilities of SRM, together with discoveries of previously unknown features of the nanoscale organization of proteins in cells, will also guide users to address entirely new questions not possible to answer by other methods. Although STED microscopy can be regarded as the first SRM method to be launched, with the theoretical foundation published over 20 years ago1 and experimentally validated a few years later,2,92 it is still a rather young research method. Since its conception, the first major steps in its development have in large part been taken at the Nanobiophotonics department headed by Nobel Laurate Stefan Hell in Göttingen, Germany, or in close collaboration with his research group. With subsequent spreading of competence to other research groups, for example, via former members of the Göttingen group moving to other academic institutes, or to companies commercializing STED imaging, and with commercially available instrumentation being installed in larger and larger numbers, it is basically only now that an era of everyday use of STED imaging is emerging. Advances in optical fluorescence microscopy typically follow a route of improvements, starting with single-color two-dimensional imaging, multicolor twodimensional imaging, three-dimensional imaging, and finally live-cell applications (isolated tissue, whole organism animal, and last human live imaging). STED microscopy has achieved all these steps (except in vivo imaging in humans) and has been used to visualize previously unseen details with superb AH


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resolution in many areas of life science (see, for example, refs 48, 89−91, 269, 345, and 346 for reviews). However, it is only during the past decade that the method has been further developed and experimentally explored for more advanced life science imaging projects. Particular benefits of STED imaging versus other SRM techniques have been discussed above, and it has recently been pointed out, the potential capabilities of different SRM techniques are still not fully explored.153 There is an active ongoing technique development, and it is difficult even for experts to evaluate the best choice of methodology for a certain application. Different SRM options may also offer complementary information, rather suggesting parallel use of techniques.48 However, generally speaking, in comparison to coordinate stochastic single molecule localization techniques, STED as a coordinate-targeted approach typically offers faster acquisition rates, requires no image processing, and has a simpler way to control fluorophores. The sample preparation is more flexible with a broader range of dyes available than, for example, in PALM/STORM (especially compared to activator based versions carrying two different fluorophores) and with no imaging buffers required in the samples (as for dSTORM). Because of the confocal nature of the imaging system, imaging of thicker samples and combinations with highly time-resolved fluorescence fluctuation spectroscopy are typically more straightforward. It would lead too far to cover all different areas in which STED imaging is likely to play an important role or areas in which it already has proven its capacity. Below, we have rather concentrated on three different application areas of STED imaging, fundamentally different in character, which illustrate how particular benefits of STED imaging can be taken advantage of and which have interesting potential for further development.

dynamics is crucial in order to better understand how the brain functions. While EM can provide images of the ultrastructure of synapses with unparalleled resolution in 2D as well as 3D, the possibilities for molecule-specific labeling are somewhat limited. This is partly due to restrictions caused by fixation steps blocking labeling of affinity molecules as discussed above. Moreover, as the sample size is often small in EM (e.g., tens of nanometer thin plastic embedded slices), only a minute amount of affinity molecules may contribute to information in the final image. Statistical quantifications of amount can thus see huge variations as the ultrastructure may also suffer from unspecific labeling. Optical super-resolution modalities thus have a complementing possibility to extract information from the nanoscale in neuroscience even with its lower resolution. Imaging of live cells and species is furthermore not possible with EM. All synaptic phenomena, including the trafficking and release of vesicles, are governed by small protein complexes with highly interdependent components, and it was relatively early on realized that the development of fluorescence SRM could open new perspectives in neuroscience. As a consequence, many technological advances in STED imaging have been developed in the context of neuroscience applications. With the recent development of STED and other forms of SRM, details of synapses are no longer too small to be resolved by optical microscopy, and imaging can be performed without some of the above-mentioned shortcomings of EM. Video-rate imaging of synaptic vesicle (diameter 40−80 nm) trafficking in live neurons,247 as well as topological imaging of presynaptic protein architecture has been reported.348−351 However, sample preparation “artifacts” must still be considered when labeling the nanoscale for STED imaging as discussed for example in the context of synaptic vesicle visualization.352 The minute size and dense packing of neuronal proteins has brought up the issue of label sizes and have sparked a development of a new generation of smaller affinity molecules, further discussed in ref 277. Figure 10 shows an example of how STED imaging, combined with biochemical analyses might bring orthogonal results (i.e., imaging data points toward that the two labeled proteins are not “interacting” on the same place, whereas Western blotting is showing a large association). This discrepancy may however be resolved, considering the size of the antibody complex used for labeling, which may shift “interaction coordinates” in space. A simple simulation can resolve the discrepancy as shown in the lower graphs (Figure 10). In the field of neuroscience, there is a manifold of examples of how the nanoscale resolution offered by STED imaging has contributed to our understanding of synaptic function, and neuronal function in general, shedding new light on previously inaccessible phenomena. In dendritic spines (the excitatory contact points between neurons), topological findings from super-resolved images have allowed several neuronal proteins to be quantitatively mapped on the postsynaptic side.353−356 In these examples, the ion-channel AMPA, the G-protein coupled Dopamine 1 receptor, as well as the membrane-bound sodium−potassium pump and the cytosolic DARPP-32 substrate have been studied, all in all providing a better understanding of the fast and slow postsynaptic regulation in dendritic spines. Several hundred other proteins and their synaptic distributions are also possible to investigate and disentangle. Along this line, STED imaging has for example been applied to image the protein synaptotagmin, known to be involved in membrane fusion of individual synaptic vesicles,

5.1. STED Imaging in Neuroscience

Neuroscience is a scientific field in which nanoscale resolution provided by STED microscopy has made it possible to investigate previously inaccessible areas. The cellular constituents of the brain, such as within neurons and especially their synaptic machinery, are an attractive realm for nanoscale imaging, as the physical size of pre- and postsynaptic areas can be smaller than a few hundred nanometers. Such structures are thus too small to be uniquely resolved by diffraction-limited optical microscopy. In spite of this, much of the understanding of, for example, synapses, recognized as fundamental units of neuronal computation, have historically come from light microscopy studies using one-photon as well as two-photon imaging.347 Understanding the molecular mechanisms by which neurons process and integrate synaptic inputs, as well as how these mechanisms are modified by activity, is a central challenge in neuroscience. Of particular interest are neuronal mechanisms that may be responsible for regulating signal localization and controlling the spatiotemporal regulation of biological functions in the brain. The accurate determination of the spatial distribution of a protein inside a cell is often intimately related to its function. It is widely accepted that precise subcellular locations of proteins critically affect their functional role. In the case of postsynaptic proteins in neurons, clustered directly opposite to the presynaptic active zone, their activation and response may be fast. In contrast, when the same proteins are distributed in the extra-synaptic membrane far away from the active zone, they may activate and respond differently, due to, for example, a lower amount and slower action of distributed neurotransmitters. Visualizing this localization and interaction AI


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and this highlights the usefulness of STED microscopy as a means of studying a myriad of protein complexes related to neuroscience. Dynamic morphology of dendritic spines and their inner protein distributions have also been visualized deep into living tissue and even into the brain of living animals.210,363−366 This has been made achievable by STED imaging, when combined with either two-photon excitation or index-matching glycerol objectives. The activity-dependent synaptic changes observed in these studies (often referred to as plasticity) are considered to be core mechanisms in the brain for memory storage and learning. Moreover, even when going from super-resolution imaging of live cells, with minimal perturbations by sample preparation, to studies of protein distributions in fixed neural cells, structures have been visualized which have never been seen with EM. In pioneering experiments applying STORM,367 actin, spectrin, and associated proteins were found to form a periodic cytoskeletal structure in axons. This previously unseen phenomenon has also been resolved by STED imaging, has been extended to dendritic structures, and has also been found in live cultures and tissue.71,368,369 Figure 11 shows live neurons imaged with STED nanoscopy, visualizing subdiffraction spaced periodic structures of actin. This previously unknown and presently hot topic of neuron architecture is spurring further studies. It indicates that STED imaging together with other SRM techniques are likely to disentangle also other presently unknown and unanticipated structural features in neural cells at subdiffraction length scales. Studies on how these structural features are related to functional connections to other neuronal proteins and their dynamics is now being pursued.

Figure 10. STED imaging of synaptic proteins. (Top) STED imaging of immuno-fluorescently labeled cultured striatal neurons, where the sodium−potassium pump (NKA − isoform α3, green|Alexa594) and Dopamine 1 receptor (D1R, red|Atto647N) are located postsynaptically in dendritic spines (dashed line). The overlay indicate that there is a minute spatial co-occurrence of the sodium−potassium pump (NKA) and the Dopamine 1 receptor (D1R), which is in contradiction to both conventional imaging results and biochemical studies.398 (Middle) Immunoprecipitation experiment where the striatum from a rat brain was homogenized, and the purified striatal lysate thereafter incubated with antibodies and eluded onto a gel (SDS-PAGE for Western Blotting). An association between D1R and NKA is clearly seen (i.e., a strong dark band). The negative control against an IgG antibody shows very little unspecific association. (Bottom) Simulated imaging results assuming two proteins at the same place (i.e., green + red = yellow) labeled with a primary−secondary antibody complex (assumed 10 nm + 10 nm in size; in random directions). A typical confocal (PSFfwhm = 200 nm) and STED microscopy (PSFfwhm = 40 nm) image are shown to highlight that diffraction limited imaging is not well-suited to investigate colocalization on the nanoscale, and that immuno-labeling can skew results.

5.2. Plasma Membrane Organization and Dynamics

The plasma membrane (PM) is an interface between the internal cytoplasm of the cell and its outside environment and houses many important cellular functions, including selective transport of molecules and ions, signaling, sensing, and propagation of action potentials. In general, compartmentalization of specific cellular functions, through spatial localization, increases regulation efficiency. Similarly, in the PM there are specialized cellular regions with specific components enriched or specifically recruited, such as the basal and the apical membrane in polarized cells, the immunological synapse in interacting leukocytes, or the multimolecular membranecytoskeleton assemblies of focal adhesions and podosomes.370 Two well-known concepts to describe PM compartmentalization are the lipid rafts371 [i.e., small (10−200 nm) transient membrane compartments with enriched contents of cholesterol and sphingolipids] and membrane cytoskeleton “fence-picket” models,372 with PM diffusion barriers based on an underlying meshwork of filamentous proteins. However, despite the central role of the PM, with PM compartmentalization as a major functional modulator, it has remained a highly enigmatic cellular structure.373 Although the microscopic structure is getting clearer, the organization at the nanometer level, the relevance of lipid raft or fence-picket models, and how the detailed spatiotemporal organization of different proteins and lipids within the membranes is related to the biological functions of the PM is still not fully understood.153 With the rapid development of STED imaging and super-resolution optical microscopy in general, new tools are now emerging, providing improved spatiotemporal resolution, specificity, and sensitivity to capture interactions of lipids and proteins directly in living cells. For the characterization of the mere interaction

showing that the protein remains concentrated in small clusters after exocytosis, instead of being dispersed across the plasma membrane.230 STED microscopy has also been used to visualize other membrane fusion proteins and their interactions (e.g., those between the SNARE motif and syntaxin clusters at plasma membrane sites where secretory granules and caveolae fuse).357,358 The distribution of the Bruchpilot protein centered at synaptic zones in Drosophila neuromuscular junction has also been revealed.359,360 These studies provide examples of how STED imaging can reveal details of how the assembly and organization of presynaptic protein complexes contribute to the efficacy of vesicle release and shed new light on how vesicular proteins are recycled after exocytosis. Furthermore, with macromolecular resolution, STED imaging has been able to resolve the punctuated structure of intermediate filaments in neurons361 and the organization of the amyloid precursor protein in neuroblastoma.362 Such images have previously been accessible only by EM, with its limitations regarding, for example, sample preparation and labeling, as discussed above, AJ


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time-resolution of STED imaging is not limited in the same way. Frame rates can be increased by diminishing the field of view or possibly by parallelization. Moreover, with STED-FCS (ref 344, see above), combining the high spatial resolution of STED with statistical analysis of photon fluctuations at high time resolution by FCS, characterization of even faster lipid and protein dynamics in the PM at the nanoscale is possible (see Figure 12). Thereby, membrane dynamics, including, for example, anomalous lipid diffusion behavior can be characterized at the time scale of microseconds and at spatial scales down to tens of nanometers. This has allowed the observation of anomalous, hindered diffusion of fluorescent lipid analogues in the PM of live mammalian cells, and characterization of its underlying mechanisms.303,335,374 With the spatial resolution of STED-FCS, the hindered diffusion could be attributed to trapping of the lipids to slowly moving entities (presumably proteins) for durations of 1 to 10 ms, and within areas smaller than 20 nm in diameter. With diffraction-limited FCS measurements, these transient events tend to be averaged over the approximately 2 orders of magnitude larger (>300 nm in diameter) observation areas employed and are thus not directly observable. By scanning-STED-FCS (ref 340, see above and Figure 13), rapidly recording FCS data along a scanned line or circle, depletion of fluorescent molecules in the membrane region of the excitation/STED beams due to photobleaching can be minimized, and the membrane dynamics at the nanoscale can be mapped not only in one spot but also along trajectories which are several micrometers long. ScanningSTED-FCS experiments have shown that diffusion speeds and trapping characteristics of lipid analogues can strongly vary both in space and time across the PM of mammalian cells.340 Interestingly, STED-FCS measurements in live cell PMs have this far not indicated the presence of lipid phase separation driven domains (lipid rafts), even though cytoskeleton and cholesterol dependent diffusion of lipid analogies have been observed.153 This may on the one hand be a matter of short transient lifetimes or small sizes of the rafts, calling for yet higher acquisition speeds, sensitivities, and spatial/temporal resolutions. However, apart from the instrumental performance, the local mobility of fluorescent lipid analogues or membrane proteins in the PM may not fully reflect the actual weak and transient interactions between lipids and proteins in the PM. Alternatively, fluorescence labeling may introduce perturbations, making it difficult to characterize subtle, localized, and transient interactions in the PM in a correct manner. The further fulfillment of the promising potential of STED and SRM to reveal the fundamental principles of PM organization at the very details is thus not only a matter of instrument performance. Maybe even more important is the development of new fluorescent probes, minimally perturbing and small in size, compatible with STED, with high photostability and brightness, but also with further improved lipid labeling compatibility and with distinct phase partitioning properties.

Figure 11. (Top) STED image of live hippocampal primary neurons (grown for 6 days in vitro). Prior to live cell imaging, neurons were labeled with the cell penetrating SiR-actin dye to label the cytoskeleton structures. Central images show close-up of the selected dendritic segment (white rectangle in upper image) visualized by STED and a preceding confocal microscopy image (depletion beam turned OFF). (Bottom) Line profile along the dendrite (white dashed line) showing periodic cytoskeletal actin structures with a period of ∼180 nm. Images were acquired with a Leica SP8 STED 3× microscope using 635 nm excitation and 775 nm depletion with a 2D vortex plate.

between biomolecules in a membrane, diffraction-unlimited optical imaging techniques do not necessarily offer more distinct measures than other approaches, based on, for example, FRET, FCS, and fluorescence anisotropy using diffractionlimited optical readouts. However, combined with fluorescencebased techniques for molecular dynamics characterization, such as single-particle tracking (SPT)329 and fluorescence correlation spectroscopy (FCS),262,321,322 diffraction-unlimited imaging have the potential to reveal currently concealed mechanisms for PM function and compartmentalization, not within reach by other approaches. By sptPALM, with a subset of fluorophores remaining active over many image frames, the diffusion of thousands of individual molecules in the PM can be followed with nanoscale spatial resolution (ref 334, see above). A limitation with sptPALM, however, is that direct interaction measurements are difficult, since only a very small fraction of the emitters is stochastically activated at a given moment.46 Moreover, the switching kinetics of the fluorophores, together with the frame rate of the camera, limits the time resolution to hundreds of milliseconds.268 As a raster-scanning method, the

5.3. Diagnostics and Understanding Disease Mechanisms at the Nanoscale

In many diseases, underlying mechanisms take place at a subcellular level, well ahead of any evident clinical signs of disease. For several different disease categories, an increased understanding of the disease mechanisms, at a subcellular, nanoscopic level, can thus have a large impact on diagnostics and the development of new possible treatment strategies. In this context, combining high-spatial resolution with molecular AK


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Figure 12. STED-FCS; (Top) STED allows the focus to be scaled down in size with increasing depletion power. Single molecules moving around in the plasma membrane will, because of this, transverse smaller and smaller observation spots (i.e., focal diameter, d, going from 250 nm → 30 nm). Analyzing average fluorescence transit times (i.e., the dwell time in the focus, tD) and average concentrations (i.e., the inverse of the fluctuation amplitude being proportional to number of molecules, Nm) by fluctuation correlation analysis allows one to study molecular mobilities down to the nanoscale. The graph schematically shows a correlation curve, recorded in a diffraction-limited focus (black) and the same system investigated within a scaled-down STED focus (gray), where the first has a longer dwell (passage) time and more molecules in observation spot. (Bottom) The plasma membrane has an actin-rich meshwork in its very vicinity, which will affect spatiotemporal molecular mobilities. To study this, STED-FCS can be applied to disclose different diffusion modes in the system while rescaling the observation spot diameter. For free diffusion (i.e., Brownian motion), the apparent diffusion coefficient [D(d): d2/tD] will remain constant as the transit time scales with the area. When the diffusion is transiently trapped (i.e., transient interaction with immobilized or slow moving binding partners), a stop in the diffusion path will result in a continuous decrease of the apparent diffusion coefficient with smaller d, since the transit time is increasingly dominated by the trapping times. As the plasma membrane is typically seen as a meshwork with differently sized domains, STED-FCS allows probing their existence. A transient incorporation into a domain or complex in which diffusion is still possible but slowed down would result in a decrease of the apparent diffusion coefficient with smaller d. However, when reaching the domain scale, D(d) will level off again showing “free” diffusion. Moreover, even hop diffusion modes can be studied with STEDFCS, showing an increase in the apparent diffusion coefficient as a function of observation spot diameter. Freely moving molecules may cross from one fence-like structure to the next (bottom left), thus searching out (i.e., hopping out from) their environment faster in the scaled-down observation spot investigations.

myocytes, and the changes in underlying membrane structures cannot be resolved by CLSM nor be investigated by EM. Although cardiomyocytes may not be amenable for diagnostic sampling, these STED imaging studies have revealed early stage, nanoscale mechanisms of heart failure. Knowledge about these mechanisms may ultimately provide new ways to interfere therapeutically in the disease process. Alzheimer’s disease (AD) is the main cause of dementia in the elderly population and is neuro-pathologically characterized by aggregates of amyloid-beta peptides (Aβ) and Tau proteins (i.e., proteins stabilizing and regulating cytoskeletal dynamics in neural cells, which in AD tend to self-aggregate). For early detection of AD, detection of large plaques of Aβ by positron emission tomography (PET) and Aβ-specific PET tracers is an important manifestation (see ref 379 for a review) but needs to be complemented with, for example, detection of Tau protein aggregation for a definite diagnosis. Recently, several studies

specificity, sensitivity, and multichannel detection capabilities, STED imaging and other SRM techniques can play an important role. Recent STED imaging studies concerning the pathophysiology of heart failure, Alzheimer’s disease, and cancer provide examples of the first steps toward exploiting this clinical diagnostic potential. The pathophysiology of heart failure is known to take place over vastly different spatial scales, and STED imaging has recently been used to resolve its finer details, revealing membrane remodeling in cardiac myocytes from rat375 and from a postmyocardial infarction mouse model.376−378 The membrane remodeling can be correlated to heterogenic intracellular Ca2+ release, which in turn can be an underlying mechanism of arythmia susceptibility, tissue degeneration, and contractile dysfunction (i.e., clinical manifestations of heart failure observed at a later stage). Notably, detailed investigations of nanodomains of Ca2+ release units in live cardiac AL


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Figure 13. Application of scanning STED-FCS. (Top) Applying scalable STED focal volumes allows one to probe interaction dynamics on many spatiotemporal scales. By labeling different kinds of plasma membrane lipids (e.g., Sphingolipids or Phosphoglycerolipids) with a fluorophore and studying their diffusion characteristics, information about interaction dynamics in the plasma membrane can potentially be visualized. (Bottom) By combining scanning FCS with STED microscopy (scanning STED-FCS or sSTED-FCS), mobility maps along plasma membrane paths can be deduced. These maps contain spatiotemporal information about the heterogeneity of lipid dynamics and may reveal, for example, long interaction times (i.e., transient trapping) or free diffusion on the nanoscale, both events not possible to reveal with diffraction-limited microscopy.340

and the spatial distribution patterns of relevant proteins can be studied in the context of more or less intact cells. To further understand the mechanisms of AD, STED imaging of pyramidal neurons has also been combined with whole-cell patch-clamp recordings, and computational modeling showing that neuronal hyper-excitability and structural degeneration of dendrites are crucially linked.384 This possibly constitutes novel pathological mechanisms underlying neuronal network dysfunction in AD and is of potential relevance also for other neurodegenerative diseases. Along similar lines, STED microscopy has also revealed defects in presynaptic terminal structure and function in Drosophila models of the neurodegenerative disease amyotrophic lateral sclerosis (ALS).385 Like for the referenced AD studies above, STED imaging can reveal nanoscale defects which may precede and contribute to the disease progression, in this case, the motor neuron degeneration characteristics of ALS. In the context of neurodegenerative diseases related to aberrant aggregation of proteins, STED imaging could also analyze the formation in neuronal cells of fibrillary species of the mutant huntingtin, a disease-related protein in Huntington’s disease.386 For many diseases, diagnostic sampling of cells or tissue is not advisable, because of the fragility or vital character of the organs of interest (e.g., brain or heart) or because the sampling itself can accelerate the disease progress by, for example, dissemination. This also sets limits for the possible benefits of STED imaging for clinical diagnostics. On the other hand, this limitation may also open an important role for STED imaging diagnostics. By SRM, not only the cellular content (up- or down-regulation) of disease-related biomarker proteins can be

indicate that STED imaging and other super-resolution imaging techniques can provide such complementary information. A procedure for ex-vivo testing of AD has been proposed,380 where cerebrospinal fluid (CSF) from patients is incubated with amyloid proteins of interest (e.g., Aβ, Tau, or other proteins). The presence of seeds (indicative of disease) in the CSF would then be probed by super-resolution imaging of elongation of the added monomer proteins/peptides, with the frequency of elongated fibrils being correlated to the number of seeds present in the CSF and therefore to the stage of disease. In a related study,381 analysis of the structural organization of Aβ and Tau in CSF from patients with AD, mild cognitive impairment (MCI), and controls was investigated by STED microscopy. From the number of aggregates in the CSF samples, their imaged sizes and intensities, the CSF samples from the AD patients could to a large extent be distinguished, providing additional support for SRM as a possible tool for AD diagnostics. It may be noted that the above studies380,381 do not actually use the information content of the spatial distribution patterns of the aggregates. This means that complementary information about aggregate number, size, and shape could possibly also be obtained by spectroscopic means, with no imaging required. For example, combining FCS with Förster resonance energy transfer (FRET), initial oligomerization of Aβ can be followed with very high sensitivity.382 STED-imaging has also been demonstrated to resolve AD-specific Taufilaments and oligomers in autopsied 50 μm thick brain sections from AD patients.383 This demonstrates that STED imaging is highly amenable for studies of human brain tissue from biobanks. Thereby, nanoscale disease mechanisms of AD AM


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analyzed, as traditionally used for diagnostics, but characterization of highly resolved, spatial distribution patterns of certain proteins within individual, intact sampled cells can provide additional diagnostic information. With increased information content per sampled cell, the sample sizes needed for conclusive diagnoses can possibly be reduced and thereby obtained with more gentle and patient-friendly sampling with far less severe side-effects. This can possibly also open for sampling from previously nonadvisable locations. This diagnostic strategy may be generally applied but is probably particularly relevant for cancer diagnostics. Early stage cancer diagnoses, achieved when lesions still are very small, and before the onset of metastasis, can dramatically increase patient survival. However, current cancer diagnostic practices often require sample amounts not possible to collect at a very early stage or amounts too large to sample without risks of severe sampling-induced side effects. Fine needle aspiration (FNA) cytology and core needle biopsy (CNB) offer less invasive sampling modalities than, for example, surgical excision. However, also for FNA and CNB there is a trade-off between sampling-related side effects and diagnostic reliability.387−389 FNA is patient friendly, minimally invasive, rapid and cost efficient but yields small amounts of sample with cells taken out of their tissue context. By CNB, tissue samples are obtained, and typically higher diagnostic sensitivity and specificity can be reached. However, CNB also display higher rates of complications, including hematoma, infections, and higher risks of cancer cell seeding along the needle tracts. To better combine diagnostic reliability with minimized sampling-related side effects, STED imaging has been tested as a way to reach a necessary level of diagnostic information, also from smaller FNA samples with a limited number of cells.390 Following the general strategy outlined above, and given that biomechanical properties in cancer cells have been found to strongly correlate with invasiveness,391,392 distribution patterns of cytoskeletal proteins were analyzed with nanoscale resolution in individual, normal, and genetically modified malignant fibroblast cells. In the study,390 it could be shown that STED microscopy can uniquely resolve and quantitatively analyze differences in the nanoscale spatial distribution of cell adhesions and of filaments of the cytoskeletal protein vimentin, underlying malignant development and metastatic competence of these cells. This shows that characterization and analyses of subcellular distribution of these and similar proteins by STED imaging can provide novel means to identify metastasizing cells, possibly adding the extra information needed for early cancer diagnostics based on very limited amounts of cells, as provided in FNA samples. Notably, the proteins analyzed by STED imaging do not necessarily have to be specific biomarker proteins, specifically up- or down-regulated as a sign of disease. In principle, STED imaging of any protein which distinctly changes its spatial distribution patterns in the sampled cells upon disease can be considered. As a further confirmation, STED imaging of vimentin fibers in fibroblast cells have shown more entangled fibers with increased widths also in other oncogene-expressing cells, compared with control cells. Moreover, from complementary investigations by single cell atomic force microscopy, the changes in the spatial distribution patterns of vimentin observed by STED imaging could be directly coupled to changes in the cellular stiffness.393 Cells from FNA samples can be imaged in a similar fashion as the cultured cells used in the study referred to above (Figure 14). Along similar principles, it has also been shown that STED

Figure 14. STED image (lower part) and corresponding confocal image (upper part, acquired with the STED laser off) of an FNA sampled cell from a breast cancer patient. Imaged are the spatial distribution patterns of the Insulin-like Growth Factor 1Receptor (IGF-1R) and HER1. HER1 belongs to the epidermal growth factor receptor (EGFR) family, and amplification or overexpression of its gene is believed to play an important role in the pathogenesis and progression of breast cancer. IGF-1R is a receptor protein found in the cell surface membranes of human cells. IGF-1R is implicated in several cancers, not the least breast, prostate, and lung cancers. Both membrane proteins HER1 and IGF1R can interact with a broad range of different molecules and also form complexes with each other in the membranes. After FNA sampling, cells were fixated with PFA, permeabilized with Triton-X, labeled with primary antibodies + dyelabeled secondary antibodies (IGF-1R) or with a specific dye-labeled affibody (HER1), and then mounted in Mowiol. Dyes used: Atto594 (IGF-1R) and Atto647N (HER1). It can be noted that STED imaging opens possibilities to analyze protein spatial distribution features, not accessible with diffraction-limited resolution (i.e., confocal imaging), forming a basis for additional, single cell information and with possible use for cancer diagnostics.

super-resolution microscopy is capable of revealing nanoscale protein distributions in paraffin-embedded human rectal cancer tissues, which have been stored for decades in biorepositories.394 STED imaging has also been applied to explore storage and release mechanisms of angiogenesis-regulating proteins in platelets.395 These mechanisms are believed to be involved in the role platelets play in early malignancies, selectively providing proteins needed by the tumor to stimulate local formation of blood vessels and promote its growth (Figure 15). Protein distribution patterns dissected by STED have been shown to give fingerprints of platelets from healthy donors, distinguishing what different distinct activations the platelets have been subject to395 and allowing up to four different proteins to be imaged at a time.319 Hypothetically, platelet fingerprints indicative of platelet activations coupled to malignant disease could then also be distinguished. If so, STED imaging of distribution patterns of selected angiogenesisregulating, or other relevant in platelets can find a diagnostic AN


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exploring its full potential. Stimulated by the drive to better visualize and understand complex biological structures and dynamic functions in a plethora of cell and tissue types on the nanoscale, possibly even in living organisms, we predict STED nanoscopy will contribute to a revolution in fundamental as well as applied life sciences in years to come.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Jerker Widengren: 0000-0003-3200-0374 Notes

The authors declare no competing financial interest. Biographies Hans Blom received his M.Sc. in Engineering Physics from the Uppsala University, Sweden, in 1998 and his Ph.D. in Biophotonics from the Royal Institute of Technology, Stockholm, Sweden, in 2003. The latter was a collaboration with the Medical Physics group at Karolinska Institutet, Stockholm, Sweden. After working for industry as a research engineer at Olympus in Tokyo, Japan, and being a postdoc in Professor Stefan W. Hell’s group at the Max-PlanckInstitute for Biophysical Chemistry in Göttingen, Germany, to develop super-resolution microscopy for single molecule analysis, he returned to Sweden and is currently Facility Head at the national bioscience hub Science for Life laboratory in Stockholm, training and supporting Swedish scientists in need of super-resolution microscopy.

Figure 15. STED images (lower part) and corresponding confocal images (upper part, acquired with the STED laser off) of plateletfactor 4 (PF-4, green) and vascular epidermal growth factor (VEGF, red) in platelets from a healthy donor. These two proteins regulate growth of blood vessels, acting as anti- and pro-angiogenic factors, respectively. The platelets were subject to no stimulation (left) and to stimulation by adenosine diphosphate (ADP). From the highly resolved protein distribution patterns in the STED images, plateletstate specific storage, release, and uptake of proteins can be characterized. This may open for future diagnostic applications (see ref 395 for further details).

Jerker Widengren (M.Sc. Engineering Physics, M.D., Ph.D.) received his Ph.D. at the Karolinska Institute, Stockholm, in 1996. In between periods of clinical work as a physician, he did postdoctoral work at the Max-Planck-Institute for Biophysical Chemistry, Göttingen, in single molecule fluorescence spectroscopy with the group of Professor Claus M. Seidel. Since 2003, he has been a professor and chair of Experimental Biomolecular Physics at the Royal Institute of Technology (KTH), Stockholm. His main research interests are ultrasensitive and ultrahigh resolution fluorescence spectroscopy and imaging for fundamental biomolecular studies and clinical diagnostics. Along these lines, Widengren and his research group are specifically developing STED nanoscopy towards subcellular cancer diagnostics and for fundamental membrane dynamical studies.

role, indicating presence or progress of an ongoing malignant disease and based on plain, peripheral blood samples. The protein distribution patterns in the platelets obtained by STED imaging may also provide clues for how to manipulate the platelet mechanisms of protein storage and release to counteract early tumor development.

6. CONCLUSIONS Not very much more than 20 years have passed since it was first realized that the optical resolution limit set by diffraction can be overcome1 and less than 20 years since the first experimental demonstration of the concept.2 Within this relatively short time-range, STED imaging together with other super-resolved fluorescence microscopy techniques have already been applied on a large scale in major fields of the biological sciences, like cell biology, microbiology, and neurobiology. In this review, we have described how STED imaging originally evolved, how it compares to other optical super-resolution imaging techniques, and what advantages it provides compared to previous golden standards for biological microscopy, such as diffraction-limited optical microscopy and electron microscopy. The triad of prerequisites for an ideal super-resolved image by stimulated emission depletion is as we have tried to highlight: (1) full control of instrumentation (hardware and software), (2) full control of sample preparation, and (3) full control of the fluorophores and their photophysical possibilities. With the prerequisites fulfilled, a highly versatile imaging modality is at hand, and we are only in the beginning of

ACKNOWLEDGMENTS This work was supported by grants from the Swedish Research Council (VR 2013-6041 and VR2011-3045), the EU FP7 (FLUODIAMON 201 837), the Knut and Alice Wallenberg Foundation (KAW 2011.0218), the Swedish foundation for strategic research (RFI14-0091), and an HMT grant from KTH and the Stockholm County Council. The authors would like to express their sincere gratitude to all colleagues at KTH, at MPIBPC, Göttingen, and within the EU project FLUODIAMON for their valuable help and support. The authors highly appreciate help from Johan Tornmalm (KTH) with the modeling presented in Figure 5; David Unnersjö-Jess (KTH) for vortex plate images in Figure 6−8; Jan Bergstrand, Dr. Lei Xu, Dr. Daniel Rönnlund (KTH), and Prof. Gert Auer (Karolinska Institutet, Stockholm) for provision of Figures 14 and 15; and the support with figure design (i.e., Figures 2, 10, 11, 12, and 13) provided by Prof. Christian Eggeling at Radcliff Department of Medicine at University of Oxford, UK, Dr. Volker Westphal at the Max Planck Institute for Biophysical AO


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