2.1.

Concepts of Super-Resolution Microscopy

Over the past 20 years, tremendous progress has been made in the development of imaging techniques that provide diffraction unlimited resolution. A number of such super-resolution imaging techniques have been developed that make use of the ability to switch fluorophores on and off (photoswitching/photoconversion). Although based on the same principle of photoswitching/photoconversion, the different techniques rely on different implementations to attain subdiffraction resolution. These concepts can be divided roughly into two categories according to the main principle used to exploit photoswitching of fluorophores to achieve super-resolution: one employing the modulation of the point spread function (PSF), whereas the other uses a stochastic approach. The most prominent example for the first approach is stimulated emission depletion (STED) microscopy, which was awarded with the Nobel Prize in 2014. The diffraction pattern of an illuminated target, obtained by a microscope, exhibits a specific intensity distribution, the so-called PSF. The full width at half maximum (FWHM) of this PSF [see Fig. 1(a) for illustration] describes the microscope’s resolution. Therefore, minimizing the PSF of a microscope by depleting a subset of excited fluorophores using stimulated emission describes one way of obtaining subdiffraction resolution.

Fig. 1

(a) STED microscopy, principle of the technique. The excitation beam (blue) and the doughnut-shaped STED beam (orange) overlap so the excited fluorophores in the periphery of the excitation beam are depleted. Thus, only fluorophores in the center, where the STED beam power is zero, are allowed to fluoresce. This leads to a fluorescent spot with an FWHM of 66 nm (green).² (b) Comparison of confocal (left) and STED resolution (right). The mitochondrial protein Tom20 (red) and the F1F0 ATP synthase (green) were imaged, using antibody labeling. Two-color STED microscopy is able to resolve single Tom20 clusters, whereas conventional confocal microscopy is not.³ Scale bar: 200 nm. Figures were reprinted with permission from Refs. 2 and 3. © 2007, Elsevier.

The second principle used for super-resolution imaging is based on the ability to only activate a subset of fluorophores stochastically. Sequentially collecting the emission of only a sparse set of activated fluorophores makes it possible to localize those fluorophores with high precision. This approach is employed in super-resolution imaging techniques, such as stochastic optical reconstruction microscopy (STORM) and photoactivated light microscopy (PALM).

2.2.

Stimulated Emission Depletion Microscopy

The principle of STED microscopy was published in 1994 by Hell and Wichmann.⁴ They were the first to describe the principle of STED as a tool to break the diffraction barrier. As mentioned earlier, STED microscopy is based on the modulation of the microscope’s PSF relying on the principle of reversible saturable optical transition.⁵ This means the switching of a fluorophore between two different states (here, the excited and de-excited states) can be optically induced. In STED microscopy, the switching from an excited state to the nonfluorescent de-excited state is induced by stimulated emission. In general, an electron in the ground energy state $S_0$ can be excited by the absorption of a photon to the first excited electronic state $S_1$. The electron in the higher energy state can relax back into the ground state by emitting a photon, thus emitting fluorescence. This spontaneous emission of photons can be “depleted” by stimulated emission. Upon encountering a photon of a specific energy level, the electron is forced back into the ground energy state by emitting a photon of the same wavelength as the photon encountering the electron in the first place. Due to this forced emission of a photon, the electron cannot undergo spontaneous emission of a photon. As the photon forced to be emitted is of the same wavelength as the photon used for the STED effect, the wavelength can be chosen in such a way that it can be filtered out from the spontaneous fluorescence one wants to detect. In an STED microscope, this principle is realized by using two lasers that are aligned so they overlap. One of those is the excitation laser, used to excite the respective fluorophores as in a normal confocal microscope. The second laser beam is the STED beam, which is phase modulated to a doughnut shape and set to a longer wavelength than the excitation laser to deplete the fluorophores in the periphery of the excitation beam. This is done by stimulating the fluorophores to emit photons at this longer wavelength outside of the detection range of the microscope setup.^5,6 This reduces the spot where fluorophores are allowed to fluoresce to the center of the overlapping excitation and STED beams, where the power of the STED laser is zero [Fig. 1(a)]. By increasing the power of the STED laser, the concentric area of depletion is increased, thus the fluorescent spot in the center is decreased. Thereby, a resolution can be achieved that is much higher than the resolution limit of conventional microscopes. The average resolution of an STED microscope, measured by the FWHM, by now is $\sim 60$ to 70 nm, although a resolution of $<20\mathrm{nm}$ has been demonstrated as well.^7,8 With a two-color STED setup, it is possible to image samples labeled with two different fluorophores, acquiring images in two separate channels with STED resolution.^3,9 Using this setup, the authors of a colocalization study were able to resolve nanoclusters of the mitochondrial membrane protein Tom20 in comparison to the $F_1{F}_0$ ATP synthase [Fig. 1(b)].³ Another important tool in super-resolution imaging is provided by live-cell STED imaging. This was used for example by Nägerl et al.¹⁰ to monitor changes in the morphology of dendritic spines. Various studies have also shown that live-cell STED imaging can be combined with a two-color setup.^9,11 Live-cell imaging has been improved further by achieving video-rate temporal resolution, which allowed to record synaptic vesicle movements in hippocampal neurons.¹² Another STED-based application details the investigation of diffusion and interaction dynamics of fluorescently labeled molecules by combining STED with fluorescence correlation spectroscopy.¹³ All of the examples above clearly show that STED microscopy is an important tool to investigate versatile questions with a technique that allows resolving single protein clusters.

2.3.

Stochastic Optical Reconstruction Microscopy and Photoactivated Light Microscopy

STORM and PALM also rely on the switching or conversion of fluorophores from the on-state to the off-state and vice versa. Their technical implementations are based on the stochastic activation of a small, randomly positioned subset of fluorophores at a given time. Iterative imaging of that subset and activation of a new subset of excited fluorophores lead to the accumulation of a series of images all containing the positions of the different fluorophores [the principle is shown in Fig. 2(a)]. The centers of those sparse, diffraction-limited spots in one image can be calculated by fitting to a Gaussian function. Thus, the position of each fluorophore can be determined with high precision, due to the lack of nearby interfering fluorophores. From the collected data, an overall image can be reconstructed. With this imaging technique, the resolution is mainly dependent on the density of the labeling and the number of photons one is able to collect from one fluorophore. Thus, with a high quantum yield and a low background noise, the localization precision can be very high, therefore, the resolution is not limited. Usually, a resolution (localization precision) of $\sim 20\mathrm{nm}$ is achieved with these imaging techniques.

Fig. 2

(a) STORM and PALM, principle of STORM and PALM imaging. Present fluorophores in a sample are either in a dark state from the beginning or are turned off by a pulse (red arrow). Iterative activation of a subset of fluorophores and imaging of that subset lead to the accumulation of images, from which the precise locations of the molecules can be calculated. An overall image of all localized molecules can be constructed from this series of single images.¹⁴ (b) Comparison of conventional and 3-D STORM images of clathrin coated pits (magenta) and transferrin (green). (c) and (d) Zoomed-in clathrin coated pits indicated in (b), showing the $xy$ projections (left panels) or $xz$ projections with (middle panels) and without (right panels) the transferrin label.¹⁵ Scale bars: (b) 500 nm and (c) and (d) 100 nm. Scheme in (a) was reprinted with permission from Ref. 14. © 2006, Macmillan Publishers Ltd. Figures (b) to (d) were reprinted with permission from Ref. 15. © 2011, Macmillan Publishers Ltd.

Both techniques, STORM¹⁴ and PALM,¹⁶ were described first in 2006 by separate research groups, although they both use the same main principle. Initially, STORM imaging was developed for antibody-based labeling with inorganic dyes, whereas PALM imaging was developed for labeling with fluorescent proteins.

One example of STORM-based investigations demonstrates the possibility to visualize the organization of pre- and postsynaptic proteins in mouse brain tissue with a localization precision of 14 nm.¹⁷ Furthermore, an improvement in localization precision along the $z$-axis (with regard to conventional and even STED imaging) has been shown by the application of three-dimensional (3-D)-STORM, achieving an axial resolution of 25 to 30 nm.^18,15 Combining 3-D STORM with a dual-color system by employing a second photoswitchable dye and a second laser, Jones et al.¹⁵ were able to localize transfer in the middle of clathrin coated pits. The comparison to conventional microscopy shows the immense improvement in the precision of colocalization along all three axes [Fig. 2(b)].

Two years after the emergence of STORM and PALM, in 2008, photoactivated localization microscopy was demonstrated to be suitable for live-cell imaging with an initially slow temporal resolution of 25 s per frame.¹⁹ Later, the use of STORM and PALM live-cell imaging with video-rate temporal resolutions as well has also been achieved.²⁰

Taken together, all of the examples described above show that STORM and PALM present versatile tools to investigate cellular processes with a resolution in the nanometer range, even exceeding the high resolution of STED microscopy.

2.4.

Limitations of Super-Resolution Microscopy in the Temporal Domain

STORM, PALM, and STED microscopy prove how immensely imaging techniques have been improved in the past years by breaking the diffraction barrier and thus providing subdiffraction resolution. The improvement in resolution and the possibility of multicolor and live imaging render those techniques suitable for a wide range of applications, although each exhibits a different set of advantages and limitations, making them suitable for distinct applications. One advantage of STED microscopy is the possibility to image relatively thick samples, as it is built on the principle of confocal microscopy. This is not the case for STORM/PALM microscopy, where imaging is limited to relatively thin samples unless it is combined with light sheet microscopy. While STORM/PALM imaging provides information on every molecule and thus allows for further analysis, such as counting of molecules, this amount of detailed information on each molecule is not provided in STED imaging. Nevertheless, by applying further data-processing methods, one can extract information about the number and distribution of molecules from STED images, as recently performed by Ta et al.²¹ One major limitation of STORM/PALM microscopy is the need for deconvolution of the raw data acquired by those imaging techniques, which limits the speed of STORM/PALM in contrast to STED imaging. STORM/PALM also face the limitation of background and noise signals, and the images have to be subjected to the careful correction of drift and chromatic aberration, and finally, the overall image has to be constructed. This amount of data processing not only makes the image production a slow process but is also a potential source of artifacts.

In addition to individual limitations of each technique, STED, STORM, and PALM further exhibit limitations that are shared by each of the described imaging techniques. The choice of dyes is limited by the need for extremely photostable and photoswitchable/photoactivatable fluorophores. This again leads to a limited spectrum of fluorophores that can be used in a multicolor experiment. Furthermore, live-cell imaging, especially for most STORM and PALM setups, still exhibits a poor temporal resolution regarding the investigation of fast cellular processes. Frame rates of about 20 to 30 s are not suitable to resolve processes such as synaptic vesicle release. Faster frame rates have been accomplished, but often at the expense of maintaining physiological conditions during long-term imaging. The utilization of faster cameras and appropriate localization algorithms is currently greatly improving temporal resolution, but possible long-term effects in terms of photo-toxicity on the samples might occur. Furthermore, super-resolution microscopy techniques are not able to monitor long-term changes in the cell, which is especially important for the investigation of cell activity, i.e., protein turnover. Here, photobleaching is one of the biggest challenges that limits long-term imaging to a few tens of minutes at most.¹⁹ Both STED and STORM/PALM operate with relatively high laser intensities, not only inducing photodamage but also increasing the irreversible bleaching of fluorophores in fixed cells as well as in live cells. In STED microscopy, the high intensity of the STED laser leads to rapid bleaching even of highly photostable dyes, such as Atto647. In STORM and PALM, slightly lower laser intensities are used, but those are still enough to bleach fluorescent proteins or dyes after continued imaging. Thus, the pool of available fluorescent molecules that can be activated will decrease, which in turn leads to a reduced signal-to-noise ratio. As the resolution in all these techniques depends on the number of photons that the fluorophore emits before bleaching, photobleaching will result in a drastic decrease in resolution. Nevertheless, many examples from recent years show that super-resolution microscopy techniques are important tools in the investigation of biological questions, closing the gap between conventional light microscopy and electron microscopy.

3.1.

Secondary Ion Mass Spectrometry Imaging

A complementary technique, both to autoradiography and MALDI, is secondary ion mass spectrometry (SIMS), an imaging technique for acquiring atomic and molecular ion maps.²⁶ SIMS with high mass and lateral resolution capabilities and parts per million or parts per billion detection limit is widely applied in biology. In SIMS analysis (Fig. 3), a focused primary ion beam is used to sputter material from a sample and produce secondary ion species, which are analyzed with a mass spectrometer and detected as a mass spectrum.²⁷

Fig. 3

Schematic representation of the sputtering process in SIMS. Primary ion beam (monoatomic or cluster ion species of metals, gases, or carbon) is scanned over the sample surface and sputters away secondary species (neutrals, electrons, and ions). Only charged species ($\sim 1\%$) will be extracted to the mass spectrometer and analyzed.²⁵

SIMS analysis may be performed with two different mass analyzers: time-of-flight (ToF)²⁸ and magnetic sector.²⁹ The ToF-SIMS technique allows recording of a mass spectrum at each pixel for known and unknown ion species and thus represents a label-free imaging technique. However, its lateral resolution of $\sim 200\mathrm{nm}$ and lower secondary ion yields during high-spatial resolution acquisition are limiting factors for the application for subcellular imaging.

3.2.

NanoSIMS Technique for High-Resolution Imaging

Nowadays, a promising technique for high-resolution chemical imaging has been magnetic sector SIMS, which provides higher secondary ion yields in comparison to ToF-SIMS. Commercial versions of magnetic sector SIMS in existence today are Cameca NanoSIMS 50 or 50L instruments (Fig. 4).

Fig. 4

Scheme of NanoSIMS 50L showing the main parts of the instrument. The primary ion beam consisting of ${\mathrm{Cs}}^{+}$ or ${\mathrm{O}}^{-}$ species is focused onto the sample. Coaxial ion optics allows reduction of the primary ion beam spot size and therefore improving the lateral resolution. Secondary ion species are separated in the dual-focusing sector-field mass analyzer and collected in parallel with five (NanoSIMS 50) or seven detectors (NanoSIMS 50L) in the multicollection chamber. EM, electron multiplier and FC, Faraday cup. Original schematics by courtesy of CAMECA.

NanoSIMS is a dynamic SIMS instrument with the ability to analyze most of the elements in the periodic table. Continuous primary ion bombardment allows breakage of molecular bonds in the sample while continuously generating usually monoatomic and diatomic secondary ions, therefore, molecules of interest must have a specific element or have to be isotopically labeled. The unique feature of this instrument is the presence of coaxial ion optics, which enables copropagation of primary and secondary ions. This design allows ion optics to be close and perpendicular to the sample, thus reducing the primary ion beam spot size and increasing the lateral resolution and the secondary ion collection efficiency (transmission). Due to the coaxial design of optics, the primary and secondary ion beams should be opposite polarities. Primary ion sources commercially available are either cesium sources, which generate negative secondary ions and can be focused down to 30- to 50-nm beam diameter, or duoplasmatron sources for ${\mathrm{O}}^{-}$ primary ion probes with 40-nm diameter (newest model of the source), which generates positive secondary ions. The secondary ions are separated according to their mass to charge ratio ($m/z$) in the dual-focusing sector mass analyzer first with an electrostatic filter and further with a magnetic sector. Double focusing capability of the mass spectrometer allows the NanoSIMS instrument imaging with high mass resolution. Unlike ToF-SIMS, NanoSIMS can detect only up to seven species simultaneously at electron multiplier or Faraday cup detectors.^27,30

The NanoSIMS imaging technique has proved to be useful in a wide range of research fields, such as material science,³¹ biological geochemistry and cosmochemistry,³⁰ plant research,³² environmental microbiology,³³ cell biology,³⁴ and others. An outstanding spatial resolution has allowed it to be applied in biological studies, specifically, imaging cells and their compartments.³⁵ In such studies, a ${\mathrm{Cs}}^{+}$ primary ion beam of 16-keV energy is usually applied, and stable isotopes, such as ${}^2\mathrm{H}$, ${}^{13}\mathrm{C}$, ${}^{15}\mathrm{N}$, and ${}^{18}\mathrm{O}$ are used for labeling of target molecules. Owing to excellent mass resolution with high transmission, NanoSIMS is capable of resolving ion species, such as ${}^{13}\mathrm{C}{{}^{12}\mathrm{C}}^{-}$ and ${{}^{12}\mathrm{C}}_2^{-}$, and even isobars with the same nominal mass, such as ${}^{12}\mathrm{C}{{}^{15}\mathrm{N}}^{-}$ and ${}^{12}\mathrm{C}{}^{14}\mathrm{N}{}^1\mathrm{H}$. It operates in an ultrahigh vacuum environment and thus necessitates adequate preparation of water-rich samples. Therefore, the preparation of biological specimens requires attention due to the possible migration or complete loss of soluble compounds during fixation and embedding phases.³⁰

NanoSIMS has proven to be capable of imaging proteins by pulsing isotopically labeled amino acids³⁶ or nucleotides³⁵ into the cell and imaging cellular turnover. However, understanding metabolism at the subcellular level, gene, and protein expression, the study of protein complexes and single organelles are still limited as this technique cannot provide chemical maps of specific proteins.^34,36 Attempts to improve the specificity of the protein imaging have been done by Angelo et al.,³⁷ where NanoSIMS was used for imaging cells labeled with metal conjugated antibodies. Primary antibodies were coupled with up to 100 isotopically pure lanthanides, and positive secondary ions of metals were sputtered away with an oxygen primary ion beam. The chemical distribution of different lanthanide elements corresponded to a specific antibody and therefore to a specific epitope. Although this methodology allows single-step-specific protein analysis without the need to correlate with other imaging technique(s), it still suffers from several pitfalls, such as difficulty in targeting some epitopes with antibodies,³⁷ and reduction of accurate imaging as epitopes should be labeled at high density and metal-labeled antibodies react with only a small portion of epitopes and thus incorporate rather poorly into the biological sample.³⁸

As for super-resolution optics, NanoSIMS is limited in terms of both contrast and resolution. The former depends on the number of ions that can be determined from the particular structure. For example, a gold particle of $\sim 50$ to 100 nm in diameter will provide excellent contrast,³⁶ but small particles, such as the 5-nm ones used commonly in electron microscopy, are extremely difficult to detect. At the same time, the image contrast depends on the difference in the abundances of the isotopes measured between the structure of interest and the surrounding material. For example, ${}^{13}\mathrm{C}$ has been used as a common isotope for labeling proteins in biological samples. ${}^{13}\mathrm{C}$ is present at an abundance of $\sim 1.05\%$ of all C atoms in all biological structures and thus provides a strong background signal, which reduces the contrast. ${}^{15}\mathrm{N}$ should be used whenever possible for protein labeling, since it is normally far less abundant ($\sim 0.36\%$) and thus provides better contrast.

The image resolution in the lateral ($X-Y$) plane depends mainly on the primary beam thickness. A beam of $\sim 50\mathrm{nm}$ results in a comparable resolution (35 to 50 nm), since it produces isotopes only from this small area. The axial resolution has been debated, albeit it seems to be identical to the amount of material ablated by the primary beam during scanning. In biological samples, this has been determined to be in the order of 10 to 20 nm.³⁶ The main limitation in this type of experiment is the sample drift, which should be minimized as it otherwise distorts both the lateral and the axial measurements.