Review

doi: 10.1111/joim.12278 STED : increased resolution for medical research?

H. Blom & H. Brismar

From the Science for Life Laboratory, Royal Institute of Technology, Stockholm, Sweden

Abstract. Blom H, Brismar H (Royal Institute of microscopy technique, because of its targeted on/ Technology, Stockholm, Sweden). STED micro- off-switching of fluorescence, is not hampered by a scopy: increased resolution for medical research? diffraction-limited resolution barrier. STED (Review). J Intern Med 2014; doi:10.1111/joim. microscopy can therefore provide much sharper 12278. images, permitting nanoscale visualization by sequential of individual-labelled biomole- Optical imaging is crucial for addressing funda- cules, which should allow previous findings to be mental problems in all areas of life science. With reinvestigated and provide novel information. The the use of confocal and two- fluorescence aim of this review is to highlight promising devel- microscopy, complex dynamic structures and opments in and applications of STED microscopy functions in a plethora of tissue and cell types and their impact on unresolved issues in biomed- have been visualized. However, the resolution of ical science. ‘classical’ optical imaging methods is poor due to the diffraction limit and does not allow resolution Keywords: fluorescence, nanoscale imaging, STED, of the cellular microcosmos. On the other hand, the stimulated emission depletion microscopy, super- novel stimulated emission depletion (STED) resolution.

dermatological surface layer investigations (e.g. Challenges in medical optical imaging glucose measurement) and ophthalmological inves- microscopy is a tool with superb tigations of the visual sensory system (e.g. assess- sensitivity that allows individual-labelled biomole- ment of a damaged cornea), to specify two clinical cules to be detected by the absorption of and examples. However, longer wavelength (near-infra- the reemission of fluorescent . In the last red) microscopy has been developed and applied decade, the technique has provided new experi- since the early 1990s to allow a better penetration mental possibilities to decipher several biochemical depth into cellular structures, thus enabling questions related to the heterogeneous cellular imaging of several hundred micrometres into the world [1]. Why is this ‘impressive’ optical tool not highly medium of tissue and in living applied in the routine clinical setting, in the same systems [2]. For example, employing near-infrared way as other imaging methods such as X-ray wavelengths, and as in optical , magnetic resonance imaging, ultra- , allows topographically dee- sonography and positron emission tomography? per imaging in ophthalmology to obtain detailed The main physical disadvantage of this method, images within the retina and in interventional which hampers clinical applications, is light scat- cardiology to aid the diagnosis of coronary artery tering, which generates ‘poor’ penetration depth of disease. human tissue. A diffuse image is often the final result with fluorescence microscopy when trying to The main methods for studying human diseases, to visualize deep within dense cellular material whilst provide effective treatment, are as follows: (i) tissue attempting to enable both excitation light to enter biopsy, followed by optical histology; (ii) cellular and emission light to exit the sample and achieving diagnostics of cultivated infectious diseases, for targeted labelling of the area of interest. Internal example with specific fluorescent probes to high- imaging by microscopy avoids the problem of light infectious agents; and (iii) collection of body optical penetration by placing the tool in the vicinity fluids (blood, saliva, urine or spinal fluid) with of the studied objects as in endoscopy. The only subsequent molecular diagnostics using sensitive opportunities for human optical microscopy are fluorescence detection of well-characterized

ª 2014 The Association for the Publication of the Journal of Internal 1 H. Blom & H. Brismar Review: Applications of STED microscopy

biomarkers [3]. The scientific tool used in the latter resolution (i.e. small d values), short wavelengths case is most often simply a pair of optical lenses and high NA values (NA = n sina) are optimal. and a sensitive detector connected to a computer However, this is technically difficult in optical with appropriate software and a coloured probe microscopy. The typical light gathering capacity of that can be detected at very low concentrations immersion oil objectives is about NA = 1.4, so that when specifically attached to the biomolecules of the lowest resolution is given by d k/3. Typically, interest. In other words, this tool that is found in the shortest optical wavelength that is used is almost every clinical environment is an optical above 340 nm, where glass in normal objective that uses fluorescence (or absorption lenses is transparent. Using quartz lenses instead in the case of histology investigations) for profiling allows ultraviolet wavelengths to be used; however, the expression of a set of cellular biomarkers this may have pronounced phototoxic effects, for already known or suspected to be related to a example in live-cell imaging, due to energetic disease. As an indication of the importance of this photons. The use of longer visible wavelengths to field, this tool also represents the hallmark of preserve live-cell capabilities may only resolve cutting edge intellectual quality; many photo- subcellular features with structural extent or sep- graphs of scientists are taken sitting in front of aration of maximally 200 nm in the focal plane of a an . fluorescence microscope (Fig. 1). Attempting to systematically image and visualize the heteroge- Why has this century-old tool become so important neous cellular world and decipher the bimolecular in biomedical science, as it cannot penetrate even microcosmos is therefore futile. One is basically 1 mm into the human body? The two most impor- blind to the scale mechanistically governing the tant qualities of optical microscopy are first the structure and function of the cell, shown for ultimate sensitivity of individual fluorescent mole- example with classical imaging of the postsynaptic cules (highlighted above) and secondly the resolu- protein topology of the membrane-bound sodium tion power of visible light for live-cell imaging. The pump in neurons [5]. resolution of the above tools ranges from submillimetre to tens of micrometres. New optical opportunities with sharper images The resolution of optical microscopy is 100- to 1000-fold higher, meaning that it may achieve It is worth considering whether the resolution of an impressive resolutions of a few hundred nanome- optical microscope could be tuned to the macro- tres in sharp optical focus. Thus, being able to view molecular scale of cell constituents to better inves- the ‘microcosmos’ of the cellular world and its tigate a biomedical hypothesis. Is this possible topological structure, as well as interactions over given that eqn 1 has been assumed to be insur- time, in several colours and in three dimensions, mountable for at least a century? Fortunately, to enables the generation of images carrying informa- gain a better biological understanding of the cell, tion about biological mechanisms. However, no novel imaging modalities have ‘shattered’ the dif- tool can (or should) be used under conditions in fraction limit of light and revolutionized fluores- which it physically cannot provide accurate infor- cence microscopy in the last two decades. Twenty mation. According to basic optical principles, the years ago, there was an important novel develop- resolution of an optical microscope is limited by the ment in this field: stimulated emission was (theo- wave-like nature of light (i.e. diffraction broaden- retically) employed to turn off the spontaneous ing). Thus, optical microscopy fuses individual- fluorescence process in selected regions of the labelled images of fluorescent molecules into a image [6]. Targeted ‘switching’ was then experi- single blurred image if the structural extent or mentally pursued [7, 8] and developed to separate separation in the sample (d) is closer than the Abbe fluorescently labelled biomolecules in the cellular limit [4]: environment in the range of tens of nanometres, to answer biological questions through nanoscale ¼ k=ð aÞ¼k= ð Þ d 2n sin 2NA 1 imaging [9, 10]. This pioneering far-field super- resolution imaging technique was termed stimu- where n describes the of the lated emission depletion (STED) microscopy, as it specimen, a measures the solid half-angle from was realized that switching (i.e. selected stimulated which light is gathered by an objective, k is the depletion of fluorescence) should produce much wavelength of light used to excite the specimen, sharper images. Today, a custom-built or com- and NA is the numerical aperture. To obtain high mercial STED microscope typically generates a

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400 nm 300 nm 200 nm

Fig. 1 The sharpness of the diffraction-limited focus only allows entities to be resolved and separated simultaneously if they are at a distance of at least the width of the generated optical blur, that is, d in eqn 1. The physical separation of labelled fluorescent molecules thus needs to be considerably >200 nm to be resolved using conventional microscopy at visible wavelengths. maximum resolution of 20–50 nm, which has To further understand STED microscopy and its allowed the nanoscale topology of the cellular intrinsic physical principles, it is necessary to microcosmos to be imaged. understand Einstein’s quantum theory [11]. Essentially, optical fluorescence microscopy What is the underlying mechanism of this fluores- involves three processes: absorption, spontaneous cence microscopy technique and how does it gen- emission and stimulated emission. These pro- erate sharp images? Targeted switching allows cesses and the effect they may have are shown fluorescence to occur only within a nanoscale area, schematically in Fig. 2c. Initially, when a fluores- thus separating and resolving neighbouring fluor- cent probe molecule absorbs light (Fig. 2c, blue escently labelled molecules (Fig. 2). Thus, super- photon), the electronic organization of the absorb- resolution imaging requires only the addition of a ing probe is changed by exciting an electron from (red-shifted) STED to a scanning confocal the ground state to a higher energy level. Within a fluorescence microscope with the beam profile relatively short time (typically a few nanoseconds), modified to produce no light in some area(s) of the absorbed energy can then be spontaneously the focus. Most often, the STED profile is optically reemitted as fluorescence (Fig. 2c, green photon) sculpted to generate a ‘doughnut-shaped’ light relaxing the fluorescent probe to its ground state. distribution in the focal plane (i.e. a ring of high Because of internal energy losses in the probe laser intensity surrounding an area of zero inten- molecule, due, for example, to vibrational or rota- sity). Finally, overlapping the STED profile onto a tional movements, the emitted fluorescence has Gaussian-shaped excitation laser beam profile less energy than the absorbed photon. This differ- allows targeted switching. Fluorescent molecules ence is utilized in fluorescence microscopy to are thus either switched off by stimulated emission separate spectrally excitation light from spontane- in the outer areas (induced via the STED beam) or ous emission (i.e. separate the background from allowed to generate a detectable fluorescence sig- the fluorescent signal). It is also possible to actively nal from the few central molecules that are opti- force the excited fluorescent molecule to reemit the cally ‘un-switched’. Scanning the coaligned beams absorbed energy. After the molecule has been over the fluorescent sample and detecting sequen- excited to a higher state and internally relaxed, tially each nanoscale pixel builds up a super- interaction with another photon (Fig. 2c, red pho- resolution image; no data processing is necessary ton) can stimulate the excited molecule back to the as the raw detection of nanoscale-separated posi- ground state. Excess energy can then be removed tions is induced via a purely physical (optical via an exact copy of the stimulating light (Fig. 2c, switching by stimulated emission) process. Sepa- additional red photon), which can again be sepa- rating individual fluorescent molecules and rated spectrally from the fluorescence or excitation sequentially building up the targeted imaged posi- light. This latter duplication process is stimulated tions via the switching profile are thus the novel emission, which in STED microscopy basically principle underlying the high resolution of STED operates as an off switch for fluorescence, instan- microscopy. taneously removing absorbed energy in selected

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(a) (c) Excitation STED Emission

Excitation

STED doughnut (t) t1 t2 t3

(d)

Emission

Phase plate

Excitation STED

(b) (e) – STED + STED

Depleted emission

Standard resolution STED effective resolution

Fig. 2 (a) Laser light is used to excite (blue) fluorescence emission (green) in the area of focus. A sculptured STED focus (red) is used to selectively turn off emission in the outer rim allowing better separation of fluorescent entities. (b) Increased depletion power (red) reduces the common area of emission and generates a sharper effective focus with nanoscale resolution power. (c) A fluorescent molecule (black circle) following absorption of excitation light (blue photon) spontaneously emits fluorescence (green photon) after a short time (usually a few nanoseconds) to return to its ground state. Addition of STED light (red photon) can rapidly stimulate the excited molecule to emit the absorbed energy (additional red photon) and return to the ground state, thus turning off fluorescence (green). (d) Schematic diagram of an optical STED microscope with a phase plate sculpturing the depletion beam (red) into a ‘doughnut-shape’ in the focal plane, overlapped with the excitation beam (blue), and the resulting effective detected fluorescence emission (green). (e) Standard fluorescence microscopy image of a mixture of 40 nm fluorescence beads (red and green colour coded) and the super-resolved STED image allowing nanoscale separation and visualization. parts of the focal volume. Furthermore, the wave- excitation of fluorescent probe molecules. This length of the stimulated emission depletion beam is selection also simplifies spectral separation of the selected to be sufficiently long (red-shifted com- signal and the background. Increasing the inten- pared to the excitation beam) to avoid secondary sity of the STED beam to saturate the depletion

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profile finally confines the fluorescence focal spot In the last decade, complementing methods in several-fold, in all directions [10]. In other words, super-resolution microscopy have also pursued STED allows nanoscale imaging by ensuring that switching to generate sharper fluorescent images. narrowly spaced fluorescent probe molecules are Methods for localization of stochastically switched switched to different states; they are either stimu- individual molecules [e.g. PALM, photoactivated lated not to fluoresce (off) or left undepleted and localization microscopy (PALM) and stochastic allowed to fluoresce (on). It is noteworthy that if the optical reconstruction microscopy (STORM)] or duplication process occurs simultaneously for spatially nonuniform illumination and targeted many molecules placed within a cavity (e.g. (nonlinear) switching [e.g. structured illumination between two mirrors bouncing light back and microscopy (SIM)] have been developed. The multi- forth), stimulated emission will amplify light as it ple choices available are similar in many respects, oscillates and produce a bright laser (i.e. light but distinct in others (data generation, speed, amplification by stimulated emission radiation). multiplexing, resolution and analysis), which Thus, the same fundamental stimulation process allows the most suitable technique to be selected used to create a bright laser beam has advanced for the particular research purpose [15–17]. The the route to diffraction-unlimited imaging in STED main characteristics of STED are (i) the possibility microscopy, by selectively switching fluorescent of nanoscale (i.e. reduced back- molecules off and on, which directly generates a ground in thicker samples), (ii) fast imaging rates raw nanoscale image [12]. (i.e. tracking of dynamic processes with a low level of artifacts due to imaging drift), (iii) use of stan- Fluorescence is the common signal in STED dard fluorescent proteins or suitable bright dyes microscopy, but its probability of occurring can and (iv) direct generation of a raw super-resolution be controlled with the stimulated emission deple- image (with optional mathematical processing to tion beam. To effectively deplete or quench the enhance image quality before data analysis) [18]. fluorescent state, the rate of stimulated emission depletion must outperform the spontaneous decay In the following sections, we will review the tech- rate. Mathematically, a modified Abbe equation nological and methodical developments in STED expressing the resolution improvement can be microscopy in recent years. Thereafter, we will approximated as: discuss considerations of the technique. Finally, we will consider the application possibilities of 1=2 d ¼ k=2NA ð1 þ 1Þ ð2Þ STED microscopy. All sections can be read inde- pendently and in no particular order. where ς is the saturation factor expressing the ratio of the applied (maximum) STED intensity to Technological developments in STED microscopy the saturation intensity, ς = Imax/Is. The satura- tion intensity (corresponding to the value of half A typical STED microscope uses a pair of coaligned the emission signal) is dependent on the orienta- laser beams, one for excitation and a second (high tional and rotational behaviour of the fluorescent power) beam for the depletion of fluorescence probe, as well as on the wavelength, temporal (Fig. 2d). The spatial shape of the depletion beam structure and polarization of the STED light [13]. is such that it produces an annular pattern, with a Thus, with increasing intensity of stimulated node of zero intensity in the middle that allows emission depletion, increasingly smaller fluores- fluorescence to be switched off in the periphery of cence focal widths can be generated, which in turn the focal volume. Combining lateral and axial allows smaller and more narrowly spaced fluores- depletion patterns allows nanoscale resolution in cent objects to be visualized. With sufficiently three dimensions, where the ‘doughnut-shaped’ intense depletion intensities, the fluorescent focal profile enhances lateral resolution (X,Y) and an spot can thus be scaled down to molecular sizes in axial depletion profile (see Fig. 3) improves the Z- a STED microscope with saturation factors above resolution. As targeted switching using STED 100 and maximum focal widths of below 20 nm as microscopy requires light to selectively ‘turn off’ reported using common fluorescent dyes at the molecules sequentially, developments in laser single-molecule level [14]. The achievable resolu- technology have been linked to a simpler, user- tion is thus increased by 5- to 15-fold with STED friendly and turnkey STED approach. Historically, microscopy, compared to conventional light an expensive and somewhat complex pulsed laser microscopy. system has been used for stimulated emission

ª 2014 The Association for the Publication of the Journal of Internal Medicine 5 Journal of Internal Medicine H. Blom & H. Brismar Review: Applications of STED microscopy

depletion microscopy [7]. Applications to ‘simplify’ way to simplify the STED approach and conve- the method by introduction of continuous-wave niently provide multicolour imaging [21–24]. Fur- (CW)-STED have generated resolution down to thermore, even live-cell imaging of Caenorhabditis 30 nm laterally (approximately 100 nm axially) elegance labelled with enhanced green fluores- using scientific . Thus, adding a bright CW cence protein (eGFP) has been achieved with a laser for STED can convert a regular scanning similar compact and flexible laser system [25]. As confocal fluorescence microscope into a three- the biological cell is a densely packed microcosm, dimensional super-resolution system [19], appli- imaging at the nanoscale in three dimensions is cable even for nanoscale imaging of organelles in often necessary to be able to reveal its internal living cells [20]. A summary of common STED components and unravel the mechanistic pro- modalities is shown in Table 1. cesses. Several approaches to spatially sculpture in three dimensions, the distribution of the STED In parallel to the technological approach of apply- light with a central minimum (see Figs 2a and 3), ing nonpulsed lasers as in CW-STED, the imple- allow nanoscale imaging for biological as well as mentation of spectrally broad laser systems (e.g. material science applications [22, 26]. Moreover, white light laser sources or stimulated Raman more technologically complex STED systems using scattering light sources), where excitation and dual-opposing high-numerical objectives to gener- depletion pulses are generated from the same light ate interfering focal patterns, which suppress the source, has been shown to be a straightforward spontaneous fluorescence process isotropically, have been applied for biological nanoscale imaging inside cells (maximum resolution approximately 30 9 30 9 30 nm) [27, 28].

The improved image resolution in stimulated emis- sion depletion microscopy is encoded spatial by the STED beam which optically targets fluorescent molecules to be switched off or remain on. As the STED beam has a spatially varying strength around its focal minimum, the (stimulated and spontaneous) lifetime of neighbouring fluorophores will vary. This selective information can be applied to further improve the nanoscale localization of individual-labelled molecules by filtering the detected fluorescence signal in time. Theoretical and experimental applications of this time-gated Fig. 3 A conventional focal volume of fluorescence (green) approach have been applied in STED microscopy to has an axial extent of several hundred nanometres. By provide higher targeted resolution [29, 30] (i.e. applying an axial STED profile (red) and increasing its temporal filtering to detect fluorescence signals laser power, selective depletion of the fluorescence effec- occurring only from the centre of the nondepleted tively sharpens the focal depth (and additional lateral focus; see Fig. 2b,c). Given the often challenging resolution improvement can be achieved) to about 100 nm. and laborious preparation of biological samples for

Table 1 STED modalities

Modality Laser type Probes Excitation and depletion CW-STED CW excitation Blue/green dyes Blue/green excitation CW depletion Yellow (red) fluorescent proteins Orange/red depletion Gated CW-STED Pulsed excitation Blue/green dyes Blue/green excitation CW depletion Yellow/red fluorescent proteins Orange/red depletion Pulsed (NIR) STED Pulsed excitation Red/far-red dyes Green/orange excitation Pulsed depletion Red/far-red depletion

CW, continuous wave; STED, stimulated emission depletion; NIR, near-infrared.

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super-resolution microscopy, there is a need to system being studied [45]. Even the physical simplify the technical aspects of STED and make properties of the fluorescent probe and its linker the method more robust to broaden its accessibility must be considered for optimal measurement and to the research community. This has been the goal data extraction [46]. Thus, as always in fluores- of several developments regarding the focal profile cence microscopy, the microenvironment of the used for targeted switching [31, 32]. As the system probe may influence the outcome of the measure- essentially contains only one extra optical compo- ment, and vice versa the way of labelling may affect nent, it is possible that this microscope could be the achievable outcomes. For example, not all implemented as a nanoscopic endoscope, thus epitopes are available because steric hindrance simplifying in vivo imaging at high resolution. blocks ‘true’ labelling using antibodies; in addition, application of fluorescent proteins being transfect- To further allow better penetration depth into ed into the biological system may severely perturb cellular structures, several groups have also expression levels, and proteins might be guided to recently extended STED microscopy to two-photon incorrect cellular positions. In the (new) eyes of a excitation, using different optical systems and nanoscopic imager, all the considered questions modalities [33–36]. This has allowed deeper imag- asked above are thus entangled and need consid- ing into tissues, such as brain slices [37–39]. eration to allow seeing the truth. Switching from the surface-based high-numerical oil objectives (index matched to the coverslip) Thus size is important when it comes to super- commonly used for STED to more biocompatible resolution; not only the labelling density needs to glycerol-based optics (index matched to the spec- be high to achieve (or claim) a resolution of tens of imens) has also advanced the possibility of pene- nanometre, the ‘disturbance’ on the nanoscale trating deeper into living tissue and allowed must also allow higher densities to be deduced so nanoscale images even within the brain of living that true biological topology can be accurately animals [40]. For dynamic nanoscale investigation dissected. Advances in methods for site-specific of fluorescently labelled biomolecules, STED targeting of small-molecule probes to cellular pro- microscopy has, after initial proof-of-concept stud- teins are thus important for nanoscale imaging ies [41], also been applied to study membrane- [47–50] as shown by live-cell compatible imaging in anchored lipids in cellular systems using STED HeLa cells and fibroblasts. Approaches to make combined with fluorescence correlation spectros- smaller ‘click chemistry’ linkers have also been copy FCS [42, 43]. This single-molecule analysis shown to be suitable for STED imaging [51], with approach complements video-rate imaging (using a combinations of SNAP-tag fusion proteins allowing small region of interest) achieved by fast scanning resolution of the cytoskeleton structure in living technology that has been applied, for example, to mammalian cells. Moreover, switching to imaging dynamically follow labelled synaptic vesicles in of thicker samples places strong demands on axons [44]. optically matched samples. Advances in imaging matching material applicable for fixed groups of cells or tissue slices have allowed nanoscale imag- Methodical developments to improve STED imaging ing with STED with performance maintained when What are (i) the best probes, (ii) the best way of optically penetrating the sample [52–54]. measuring, (iii) the maximally achievable resolu- tion and (iv) the most suitable analytical approach Pessimistically speaking, is there no easy gain in to extract data to investigate nanoscale biomedical using super-resolution STED microscopy? Opti- hypotheses? The answers to these questions mistically, the STED system is a basic fluorescence depend on the biological system being studied; microscope with one laser for excitation and the how well the fluorescent sample is prepared and same or another for targeted off-switching; STED the extent to which the nanoscale being imaged can microscopy might thus be used to generate un- be correlated with the biomedical function of blurred images of any fluorescent sample. Visual- interest. This suggests that several inherent (mea- ization of such unblurred nanoscale images might, surement) problems may be present when attempt- however, generate evidence that does not support ing to visualize the nanoscale. currently accepted hypotheses. Figure 2e shows an example of nanosized beads (mixture of red and The size of a bulky fluorescent probe such as an green; diameter 40 nm) first imaged by conven- antibody–dye complex might perturb the molecular tional confocal fluorescence microscopy, which is

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performed with the STED beam turned off. When most commonly used are clones of eGFP, yellow interpreting such an image (Fig. 2e, left), it is often fluorescent protein (YFPs) and some red fluores- erroneously assumed that ‘yellow’ overlap reveals cent proteins [7–61], which have been applied, for nanoscale colocalization in the sample. Knowing example, to image the dynamics of the endoplas- that the confocal microscope, in the best case, can mic reticulum in living mammalian cells and the resolve entities separated by around 200 nm, morphology of filled dendritic spines in hippocam- essentially no separation below this scale is possi- pal neurons. Both pulsed and CW depletion lasers ble. Switching on the STED beam in the micro- allow resolution of <50 nm in living cells. There- scope allows visualizing of the nanoscale and fore, studies that are currently performed in cells dissecting individual nanobeads being separated, using confocal may be carried out in pointing towards that with increased resolution, no future with substantially improved spatial resolu- colocalization at all is present in the sample. This tion using STED microscopy. loss of information is, however, overcome by the now available much more complex description of Bright and photostable dye molecules have been the topological distribution of the nanobeads used since the introduction of this technique to (assuming this could have a physiological mean- visualize the nanoscale in cell biology; more than ing). The insight available from super-resolution 20 are suitable for nanoscale imaging (see www. thus demands methodical development of suitable nanoscopy.de). Additional developments to gener- analytical approaches for information extraction ate dedicated reporters with superior performance [55]. With knowledge on super-resolution STED as well as excellent biocompability are ongoing [62, demands and possibilities, rethinking of cell 63]. This is expanding STED microscopy to all biological mechanisms might also be necessary, areas covered by ‘classical’ optical imaging, thus prompted by the biomedical imaged nanoscale allowing it to be combined with any microscopy being resolved or shown to be perturbed [56]. In modality, fluorescent probes or fluorescence analy- the case of colocalization, STED microscopy has a sis techniques. New ‘exotic’ reporters consisting of topological benefit (as well as the improved resolu- so-called Farbenzentra in diamond are also being tion) as the sculptured profile defines the origin of considered as fluorescent probes. Trapped atomic the signals. In multicolour imaging, where some vacancies inside the rock solid crystal act as misalignment of the two excitation foci becomes fluorescent emitters that can be optically switched ‘realigned’ by the central targeting zero of the by STED [64]. Manufacturing advances are pres- depletion beam, it is beneficial to apply STED ently being pursued (such as the development in microscopy for ‘true’ colocalization [24]. the last decade of quantum dots) to produce biomodified nanocrystals with single vacancies for Improving the resolution of a fluorescent micro- biological nanoscale imaging. A summary of the scope in all spatial directions allows increasingly common advantages and disadvantages of STED better resolution of the nanoscale. However, if the microscopy is shown in Table 2. preparation of the biological sample (and its label- ling density) is not simultaneously improved (i.e. 2- Considerations in STED microscopy fold to keep the required resolution in a bottom-up perspective), resolved images may not be limited by The performance of STED microscopy is dependent the performance of the STED microscope but by on several associated factors. First, the perfect the poor sample preparation. As in any fluorescent minimum of the STED profile needs consideration microscope, the quality of the image and its con- as residual depletion intensity will dim the signal in trast and resolution are ultimately determined by the nanoscale image, and, in the worst case, it may the fluorescent reporter molecule; this is especially not be visible above the background. Secondly, the the case in STED where the intrinsic states of the spatial overlap of the excitation and depletion molecules are selectively targeted in the optical beams is important as this selects centrally located switching process. Demands on fluorescent repor- fluorescent molecules to generate the sequentially ter molecules are high (photostable, bright etc.), detected signal by the targeted switching principle. but with a plethora of suitable fluorescent report- For ‘perfect’ depletion performance, the overlap ers, advances in STED applications are shown with should not be off more than 10–20 nm and this almost any type of probe. It has been shown several should be stable over time so as not to deplete the times that genetically expressed fluorescent pro- signal focus [13]. It is thus crucial to minimize drift teins are suitable probes for STED microscopy. The between the two laser beams; this has been the

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Table 2 Advantages and disadvantages of STED modalities maximum absorption of haemoglobin in blood cells or chlorophyll in plant cells). On the other hand, it Parameter Advantages | Disadvantages is also possible to select an optimal fluorescent Fluorescent dye Bright, more photostable probe (a dye molecule or fluorescent protein) for a (immunolabelling) compared to fluorescent proteins STED laser as the spectrum of the probe and the below | Cell-penetration protocol laser need to be simultaneously ‘tuned. In essence, needed the STED wavelength needs to be far-red-shifted to Fluorescent protein Less bright, less photostable avoid reexciting the probe. As the microenviron- ment of the fluorescent probe might shift the (transfection) compared to dye molecules above excitation and emission spectra (sometimes even | Genetically expressible in cells abolishing the fluorescence in unfavourable condi- Cell cultures (not Thin structures, lower tions), the tuning of the STED laser must often be confluent) background | Less physiological reinvestigated before the final nanoscale imaging Tissue (<30 lm) Thick structure, higher can be optimally obtained. Different optimal con- ditions might thus be found in different biological background | More physiological systems and with different probes. As STED Resolution Setup dependent: typically XY: 40 microscopy relies on switching the fluorescent –50 nm; Z: ~100 nm probes (i.e. manipulating which photophysical Imaging speed ROI dependent: typically seconds; state is populated), this is not an unforeseen effect, Video-rate possible (~20 fps) but it may be beneficial or may be a quantum Image processing Not mandatory (raw image); mechanical problem that needs to be resolved. Practically, the STED depletion wavelength oper- optional to enhance contrast ates equally well within a range of 20–30 nm, thus finding suitable working conditions is not difficult, STED, stimulated emission depletion; ROI, region of interest; fps, frames per second. especially using tunable lasers or with knowledge of suitable fluorescent probes acquired in the last decade. driving force for implementation of some of the Fourthly, the question of whether high laser inten- reported easy STED approaches [31, 32]. Sample sity can affect the sample should be addressed. The drift must also be minimized to image true nano- only possible ‘drawback’ of including an additional scale topology, even though STED is a fast scan- STED laser in fluorescence microscopy is a small ning approach that is able to generate a nanoscale decrease in the detected signal, due to residual image of a whole cell within a few seconds. How- intensity in the centre of the annular depletion ever, movements of tens of nanometres over the pattern, and a slight increase in photobleaching of acquired image might then be deceiving when the fluorescent molecules. The latter is not a new conclusions are being drawn. Stable optical sys- finding within the field of fluorescence microscopy tems with feedback loops to prevent (severe) drift and has so far not prevented the novel STED are thus always needed for the application of technique from showing very prominent resolution super-resolution microscopy. The climate of the enhancement of 5- to 15-fold in each spatial room, the presence of any vibration and the selec- dimension. Selection of photostable fluorescent tion of the optimum coverslip thickness and probe molecules and/or adaptation of their mic- immersion mixture can all severely affect the roenvironments (e.g. addition of antifading agents) possibility of achieving maximal resolution. minimizes photobleaching in STED microscopy Thirdly, assuming all environmental and physical [46]. Addition of a red-shifted (depletion) laser parameters are well controlled, the interplay may sometimes also prevent photobleaching and between the STED depletion intensity and the increase the fluorescence signal as this laser fluorescent probe in the sample is an important depopulates additional electronic energy levels of consideration. It is also crucial to selecting the the fluorescent probe molecules [65, 66]. This ‘best’ powerful STED laser that matches the high- process is not entirely beneficial in STED micros- est possible depletion wavelength, without induc- copy as the repopulation of the fluorescent state ing any reexcitation, photodestruction or sample adds a signal which is often not depleted. This damage (e.g. induced by dirty mounting medium, means that there may be an additional or residual use of lung cells from a smoker, or reaching the fluorescence over a diffraction-limited volume,

ª 2014 The Association for the Publication of the Journal of Internal Medicine 9 Journal of Internal Medicine H. Blom & H. Brismar Review: Applications of STED microscopy

which will weaken the degree of resolution as biological material commonly has an autofluo- enhancement that can be achieved. rescent background in the blue/green part of the spectrum, selection of red/far-red fluorescent Fifthly, the selection of pulsed versus CW modes probes, and suitable longer wavelength STED should be consider: which is the best technique microscopy, might be beneficial to achieve higher and which will produce the best resolution? In signal-to-noise ratio (i.e. better contrast of the simplistic terms, most pulsed STED microscopes nanoscale image). The practical conclusion is use high power near-infrared light for depletion, therefore that an infinite degree of resolution whereas the wavelength for the depletion using CW without a detectable specific signal will (unfortu- lasers is in the visible range. Already, a tuning nately) provide a dull image. towards shorter wavelengths allows a higher reso- lution, as eqn 2 is scalable in regard to this Having considered the additional characteristics of parameter. However, the way depletion is achieved the STED technique compared to confocal fluores- must also be considered and is sensed by the cence microscopy (i.e. the STED laser and its saturation factor (ς) in the latter equation. The intrinsic profile), the final two important consider- effectiveness of the fluorescence depletion differs in ations are as follows: (i) how samples are prepared pulsed and CW-based STED microscopy. In the and (ii) which biological issues can be addressed. case of a pulsed STED system, the intensity of the As the STED microscope uses fluorescence as a depletion beam is concentrated during one pulse, signal generator for producing a nanoscale totally depleting the fluorescence immediately after image, sample protocols used previously for im- an excitation pulse. In the CW approach, the STED munolabelling or live-cell transfection fluores- intensity is distributed equally over time and thus cence microscopy can be applied with largely an excited molecule only senses a fraction of the unchanged. However, they need to be superbly depletion intensity. CW mode STED thus needs an prepared as the super-resolution power of the average of about 3- to 4-fold increased laser power STED microscope will identify the slightest artefact for the same resolution enhancement [19]. How- [18]. The following conditions are recommended ever, a benefit of using CW excitation is that the for optimum sample preparation to achieve the fluorescence signal can be increased by a similar highest STED resolution: use of high-precision factor due to the continuous excitation, which 170 lm coverslips (No. 1.5H) matching the objec- makes fast imaging possible [20]. In addition, fast tives carrier correction; use of index-matched scanning modulation decreases photobleaching of mounting medium that does not absorb any STED the fluorescent probe molecules by not allowing the light; addition of antifading reagents suitable for probe to enter reactive sensitive states such as the the fluorescent probes; and stable temperature triplet or any radical states. As the probe in general and degree of vibration. The major goal is to be only switches between the ground and excited able to visualize the topology of interest with a states (i.e. absorbing and emitting photons), the resolution of 20–50 nm in a crowded cellular effective fluorescence per unit time is actually environment in three dimensions with several increased. Similar signal enhancement can be colours and a fast time scale in dynamic live-cell gained with a pulsed STED system, in which a settings. However, as stated above, the effective- lower repetition rate of excitation and stimulated ness of the STED microscopy usually depends on emission depletion maintains the fluorescent probe the system being studied, the question being molecule in the absorption–emission cycle [67]. In asked, how well the fluorescent sample is prepared other words, before fluorescent molecules ‘sense’ and how well the image can be correlated with the an additional excitation and depletion event (CW or function under investigation. Thus, increasingly pulsed), they can relax to the ground state and higher resolution is sought. However, as the res- thereby avoid potential ‘photoexecution’. A combi- olution increases, the sample also needs to be nation with pulsed excitation and CW depletion improved. Labelling strategies to identify the basically merges the two laser protocols and allows nanoscale landscape and density of the biomole- additional flexibility of signal gating and thus cule of interest demand suitable small probe temporally filters out a higher spatial resolution carriers such as directly conjugated antibodies [30]. In addition to filtering the signal, the repeti- (or fragments thereof), fluorescent proteins, apta- tion rate of the excitation may also be tuned to mers or nanobodies [45, 57, 68]. Such methodical allow the fluorescent probes to relax (i.e. cool) endeavours are highly related to the future devel- between imaging events. It is also worth noting that opment of super-resolution microscopy, including

10 ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine H. Blom & H. Brismar Review: Applications of STED microscopy

approaches better applicable to analysing dis- Figure 4 shows an early outlook of what STED cretely resolved nanoscale images. could achieve and now actually, only a few years later, basically have achieved in neurobiology, where it is now possible to visualize the protein Possible applications of STED microscopy topology in the synaptic area [5, 55, 73, 74]. To Advances in optical fluorescence microscopy most further advance the understanding of molecular often follow the route of improvements in single- (protein) localization and dynamics, STED has colour two-dimensional imaging, multicolour two- been applied to image, for example, the protein dimensional imaging, three-dimensional imaging synaptotagmin in individual synaptic vesicles and finally live-cell applications (isolated tissue, showing that it remains concentrated in small whole organism animal and lastly human live clusters after exocytosis instead of being dispersed imaging). STED microscopy has achieved all these across the plasma membrane [75]. Additionally, steps (except imaging in humans) and been used to STED has been used to visualize the interaction visualize previously unseen details with superb between the SNARE motif and syntaxin clusters at resolution in many areas of life science. To visual- plasma membrane sites where secretory granules ize further details beyond the 250–400 nm resolu- and caveolae fuse [76, 77]. The distribution of the tion afforded by one-photon confocal or two-photon Bruchpilot protein centred at synaptic zones in fluorescence microscopy, it can be beneficial to use Drosophila neuromuscular junction has also been STED microscopy (as well as complementary revealed [78]. Furthermore, with macromolecular super-resolution techniques). One example of a resolution, STED has been able to resolve the scientific field in which nanoscale resolution pro- protein patterns on endosomes, the punctuated vided by STED has been used to investigate previ- structure of intermediate filaments in neurons and ously inaccessible areas is neuroscience. The nuclear protein speckles in mammalian cells [79]. cellular constituents of the brain such as neurons The organization of the amyloid precursor protein and the synaptic machinery are attractive for in neuroblastoma and the nicotinic acetylcholine nanoscale imaging as the physical size of pre- receptor in ovary cells has also been imaged by and postsynaptic areas can be considerably less STED [80, 81]. Such images have previously been than a few hundred nanometres. Video-rate imag- accessible only by electron microscopy, highlight- ing of synaptic vesicle (diameter 40–80 nm) traf- ing the usefulness of STED as a means of studying ficking in live neurons [44] as well as topological a myriad of protein complexes. imaging of protein architecture has been reported [69–72]. The accurate determination of the spatial Many fundamental functions of the human body distribution of a protein inside a cell is often are also regulated by receptors that respond intimately related to its function. In dendritic uniquely to specific ligands. In other words, recep- spines (the excitatory contact points between neu- tors in the cell membrane or in the cytoplasm form rons), topological findings of super-resolved images the signalling interface of the cell and play a crucial have allowed additional neuronal proteins to be integrative function in cellular communication and quantitatively mapped [5, 55, 73]. Dynamic mor- regulation; the importance of the discovery of these phology of dendritic spines and their inner protein receptors has been recognized by several Nobel distributions have also been visualized [59], even Prizes [82]. A malfunction of a G protein-coupled in living tissue and in the brain of living animals receptor or its signalling chain is often the cause of [39, 40], as mentioned above. Such activity-depen- a wide range of mood disorders and related dis- dent synaptic changes (often termed plasticity) are eases such as Parkinsonism, schizophrenia, atten- considered to be essential in the brain for memory tion deficit hyperactivity disorder, anxiety and storage and learning. depression. Therefore, substances that selectively bind to and alter the function of receptors and Understanding the molecular mechanisms by thereby influence behavioural disorders are attrac- which neurons process and integrate synaptic tive targets for the development of new pharmaco- inputs, as well as how these mechanisms are logical strategies. Application of STED microscopy modified by activity, is a central challenge in has already identified mechanisms of transport, neuroscience. Of particular interest are neuronal localization and interaction of receptors in neurons mechanisms that may be responsible for regulating on the nanoscale; in addition, use of this technique signal localization and controlling the spatiotem- in the near future is likely to lead to further poral regulation of biological functions in the brain. elucidation of the nature of the physiological

ª 2014 The Association for the Publication of the Journal of Internal Medicine 11 Journal of Internal Medicine H. Blom & H. Brismar Review: Applications of STED microscopy

(a) (b) (c) Fig. 4 (Top) (a) Schematic rep- resentation of a presynaptic site with synaptic vesicles and the postsynaptic side in an excitatory synapse, drawn to scale from an electron micro- graph. (b–f) Simulated images with STED resolution of 10, 20, 40, 70 and 150 nm. At present, (d) (e) (f) resolution of around 40 nm is commonly achieved in STED neuronal studies. Reprinted with the permission from the licensed publisher Elsevier Ltd. (Bottom) STED microscope images taken from hippocam- pal primary cultures transfect- ed with eGFP (blue) to outline cellular borders and subse- quently stained with antibodies against the presynaptic scaf- fold protein Bassoon (red) and the postsynaptic protein Homer (green). Scale bar indicates 1 lm. The image has been post- processed using deconvolution software (Huygens, SVI, Hilver- sum, The Netherlands).

function, regulation and signalling roles of these order of magnitude larger than typical interaction- receptors [55, 73]. relevant interprotein distances. Thus, macromolec- ular protein–protein interactions cannot be The super-resolution STED method holds great positively identified at present, using confocal or promise for the investigation of dimensions on the two-photon microscopy. With the STED technique, scale of tens of nanometres, as all the above however, the necessary resolution can be reached, examples show. Moreover, use of dual-colour imag- and therefore, this type of positive identification ing allows nanoscale colocalization studies [83, 84], may be possible (see Fig. 2e). which provides the possibility of investigating (transient) cooccurrence of protein nanoassem- To improve the resolution of STED imaging even blies. In a recent study using STED microscopy, in further, increased laser depletion intensity and/or conjunction with quantitative colocalization analy- smaller fluorescent probe complexes might be sis, nanoscale analysis was applied to investigate useful. In live-cell STED, fluorescent proteins with the complex distribution of membrane proteins in masses of tens of kilodalton are applied [57, 58, mitochondria [85]. As noted above, one of the 60], whereas in immunofluorescence applications, biggest disadvantages of colocalization studies antibodies with larger masses (sizes) are used [9]. using ‘classical’ fluorescence imaging techniques The much larger size of the antibodies (10–12 nm) is that the smallest achievable resolution is an compared to the fluorescent proteins (3–4 nm)

12 ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine H. Blom & H. Brismar Review: Applications of STED microscopy

slightly widens the range of physical size of the the internal nucleosome in cardiomyocytes to cellular object that can be investigated. This may, improve knowledge of the chromatin structure in part, prevent the most accurate image localiza- [92]. The centriole, a key component of the cellular tion that present STED setups typically allow from control and division machinery, has also been being reached [14] and might require selection of investigated with STED to provide previously unob- smaller probe molecules. However, the photosta- tainable details [93]. bility and photoemission of the dye molecules used in immunochemistry are higher than fluorescent The primary cilium, the organelle that serves as a proteins rendering better imaging contrast. Fur- signalling centre of the cell, has been imaged by thermore, the achievable resolution for STED STED, revealing a differential protein topology [94]. microscopy is influenced by the movement of the In bacteriology, for example, STED microscopy has fluorescent probe. Therefore, sophisticated label- been applied to determine which factors control ling strategies with smaller fluorescent labels are cytoplasmic translocation and activation of lytic being developed and applied [48–51]. At present, proteins in Streptococcus pneumoniae, as only a the typical STED fluorescent probe molecule is a small fraction was found to be attached to the small dye (~1 nm); it does not need to be reduced in extracellular cell wall [95]. Furthermore, using size until the required resolution starts to reach the super-resolution STED fluorescence microscopy, nanometre range [64]. Of note, coupling of fluores- nanoscale visualization of the viral envelope on the cent probes to a biologically interesting molecule surface of individual HIV-1 particles has revealed a always introduces additional freedom of move- correlation between surface clustering and the ment, which may alter the resolution in STED efficiency of viral entry [96]. microscopy due to jitter. However, the nanoscale resolution of STED actually allows probing influ- During the last decade, it has become clear from ences from this dynamics [46] making it possible to findings of the human genome project that a large potentially use this ‘disturbance’ to further under- fraction of all genes encode plasma membrane stand the nanoscale. proteins. To understand the molecular mecha- nisms of proteins complexes, it is of fundamental In addition to the studies discussed above, STED importance to understand their structure (topol- microscopy has been used to characterize the ogy) and assembly dynamics [97, 98]. Of note, protein distribution of synapses at different tono- many membrane proteins are only active in protein topic regions of the cochlea [86]. STED microscopy complexes, so-called molecular machines, and it is could be applied for investigation of such physio- not possible to determine their subcellular assem- logical function in the ear [87], as well as in the eye bly topology using confocal or two-photon fluores- [88] and internally via endoscopic developments cence microscopy. On the other hand, STED [89], to generate and enhance understanding of microscopy may be used to resolve several mem- these and other biological systems in the human brane proteins and their cooccurrence [5, 55]. It is body. For example, understanding of the physio- also noteworthy that the total number of human logical function of pancreatic beta-cells from the proteins is (only) about 20 000, and their localiza- islets of Langerhans could be enhanced consider- tion is currently being imaged by ‘classical’ fluo- ably by the use of STED microscopy [90]. In rescence microscopy [99]. The application of STED particular, the elucidation of insulin granule exo- microscopy to this project would enable unprece- cytosis would be readily achievable, as has already dented detail of the localization of essentially all been shown with granules in natural killer (NK) these proteins, which should further increase cells and the immune synapse [91]. The immune understanding of the cellular protein microcosmos. synapse is involved in the transfer of information across the mediated junction formed between T Moreover, the super-resolution properties of STED cells and/or NK cells and foreign agents (such as have been identified as essential in the field of viruses). Diffraction-limited fluorescence imaging translational medicine, to resolve cellular func- has been applied to investigate this connection tions (with additional complementary imaging between NK cells and target cells. Using STED techniques) ‘from molecule to man’. The latest microscopy, the immune synaptic junction has initiative is part of a project by the European been even further resolved, which has led to a Research Infrastructure for Imaging Technologies better understanding of its structure and function in Biological and Biomedical Sciences, in which [91]. Furthermore, STED has been used to study super-resolution flagship centres will provide open

ª 2014 The Association for the Publication of the Journal of Internal Medicine 13 Journal of Internal Medicine H. Blom & H. Brismar Review: Applications of STED microscopy

access to service and training in nanoscale imaging potential for visualizing cellular biology, including for European scientists [100, 101]. Additionally, in details of cellular and even macromolecular struc- a recently completed European collaborative pro- ture, has been made compatible with live-cell ject, STED nanoscopy was used to monitor the imaging in several spectral channels and has been molecular distributions within tumour-specific applied to measure fast nanoscale dynamics even cells for detection of breast and prostate cancer on the single-molecule level; in the meantime, [102]. The nanoscopic resolution power of STED STED was also selected as the life science method was used to image morphological markers that of the year in 2008 [12]. After more than 100 years correlate with malignant transformation and clin- of being limited in terms of resolution, fluorescence ical tumour aggressiveness. The profound reorga- microscopy has thus entered a totally new para- nization of the cytoskeleton of cells, driven by a digm. In summary, nanoscale imaging of protein deregulation of signal transduction pathways as a assemblies allows initial quantification of topolog- response to changes in the extracellular environ- ical distributions (i.e. the precise location of an ment, suggests the possibility of visualizing a assembly and its coordinated connections to other correlation with tumourigenesis using STED assemblies) and estimations of the amount of [103]. Additionally, the nanoscale topology of pro- proteins (i.e. the number of protein assemblies tein expression in blood platelets was imaged with and their individual intensities thus possibly STED microscopy, providing a means for clinical determining protein count) [5, 24, 55, 73, 75]. As cancer diagnostics [104]. the resolution of STED microscopy is technically limited by the hollow depletion beam minima and What advances in STED microscopy can be the surrounding peak intensity for off-switching expected in the next 20 years? It is clear that with (controlled by the photokinetics of the fluorescent the technology being made commercially available probe molecules being manipulated), further more life scientists will gain access to STED advances are likely to be in the field of chemistry. microscopy [18]. This will mean rethinking how Suitable and flexible laser sources are needed to imaging of the nanoscale can be pursued along allow optical manipulation of the fluorescent mol- with understanding the associated problems and ecules, switching them to different states to possibilities [56]. In addition, most scientific fields sequentially separate individual entities. This sug- in which confocal fluorescence microscopy is cur- gests that optical engineering and physics holds rently used to view the intracellular environment the key to further developments. It is necessary to (tissue, animals, etc.) will be able to benefit from develop data analysis methods that approach the STED microscopy by 2020. There will certainly be noncontinuous super-resolution of separated and further developments in technology (e.g. optics, distinct nanoclusters. Thus, information theorists lasers, detectors), fluorescent probes and probe and computer scientist can examine a myriad of carriers (e.g. dyes, nanobodies, aptamers), but as possible hypotheses to provide accurate informa- always predicting the future with accuracy is tion from STED imaging. However, as this is a difficult. The greatest advantage of STED imaging, biomedical review, any advancement in this field is as noted above, is that it operates at the scale of the basically ‘owned’ by the production of the perfect key players and building blocks of the cell. This biological sample (if the question being asked is makes it possible to localize macromolecular assemblies, colocalize assembly complexes to reveal possible connections, and investigate Table 3 Applications of STED dynamics to understand mobility, interactions and densities via spectroscopic (STED-FCS) analy- Scientific area References ses in living cells with nanoscale resolution. A Neuroscience [5–40, 44, 53–55, 59, 69–78, 81] summary of STED applications is provided in Cell biology [9, 19, 20, 25, 27, 28, 42, 43, 51, Table 3. 57, 58, 60, 85, 86, 92–94, 101– 104] Concluding remarks Immunology, [91, 95, 96] There have been 20 years of advances since the bacteriology and initial development of this novel super-resolution virology STED microscopy technique [6]. During this time, this nanoscale imaging tool has shown tremendous STED, stimulated emission depletion.

14 ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine H. Blom & H. Brismar Review: Applications of STED microscopy

(a)

(b)

Fig. 5 STED microscopy of intramorphological processes in dendritic spines in organotypical hippocampal slice cultures from mice (imaging depth 10–80 lm). Comparison between control and chemically stimulated neurons, transfected with actin-binding Lifeact-YFP, reveals dynamic morphological changes in the confined spine structures. Reprinted with permission from the licensed publisher Elsevier Ltd. suitable), if one is to select only one field of nanoscale imaging using STED (or other super- improvement. Fruitful collaboration between dif- resolution techniques). In other words, nanoscale ferent scientific areas is likely to advance the field imaging will build on the knowledge gained during as a whole. the last decades, but allow previous findings to be resolved with greater clarity. In particular, analysis How accessible is this novel STED technique? At of (complex) distributions of proteins via colocal- present, there are only a few research facilities with ization will in the future benefit, and indeed to either custom-built or commercial STED systems; some extent have already benefited, from the however, this number is likely to continue growing nanoscale resolution of STED microscopy [85]. It in the near future, stimulated by the drive to better will thus be possible to ‘correct’ conclusion drawn visualize complex biological structures and from diffraction-limited confocal imaging (see dynamic functions in a plethora of cell and tissue Fig. 2e); this could lead to the development of types on the nanoscale, possibly even in living better models and increased understanding of organisms. This advance in technology (interplay structural and functional interactions. For exam- between advances in chemistry, physics, biology, ple, neuronal investigations of controlled and etc.) must also be accompanied by a greater stimulated situations (Fig. 5), using super-resolu- number of skilled scientist to be able to fully tion fluorescence STED microscopy in living sys- exploit this technique for biomedical research. A tems, have dramatically increased understanding complete understanding of molecular functions of how regulation can be influenced on the mor- within cells is only achievable if all interactions phological nanoscale. Such resolved finding may are considered and visualized. Therefore, all inves- allow a solid handle to pinpoint differences in the tigations using confocal or two-photon fluores- biomedical hypothesis of normal and abnormal cence microscopy must be reexamined with (sick) situations. With increased understanding of

ª 2014 The Association for the Publication of the Journal of Internal Medicine 15 Journal of Internal Medicine H. Blom & H. Brismar Review: Applications of STED microscopy

the cellular nanomachinery – how it works, how it the possibility of making the ‘macroworld’ of our is regulated and the signals involved – the devel- whole bodies healthier. STED microscopy has in opment of novel and more effective pharmacolog- this process begun to allow nanoscale resolution of ical strategies can be expected, promoted in part by cellular biology and its connections to disease higher-resolution STED imaging. processes.

STED is in fact one of a number of fluorescence Conflict of interest statement switching techniques that allows the nanoscale to be viewed with optical microscopy. Switching in The authors have no conflict of interest to declare. STED is, as discussed above, accomplished by turning the fluorescence off via stimulated emis- Acknowledgements sion depletion. It is also possible to switch fluores- cence off by inducing conformational changes in This work was supported by funds from the Swed- fluorescent probe molecules, or ‘photoswitching’ ish Research Council (VR-2013-6041). We thank them into a different (triplet or radical) state. Dr. Daniel Ronnlund€ at the Royal Institute of Applications of several switching techniques, Technology, Stockholm, Sweden and Dr. Lars including the initial STED technique, have Kastrup at the Max Planck Institute for Biophysical emerged in recent years and shown that the Chemistry in Gottingen,€ Germany for assistance diffraction limit of fluorescence microscopy is with the acquired STED images shown in Figs 2e becoming dated [10]. This ‘switch’ has provided and 3. We also thank Werner Zuschratter at the much sharper focus, which has led to initiation of Combinatorial NeuroImaging Core Facility at Leib- the exposure of the cellular microcosmos. The niz Institute for Neurobiology, Magdeburg, Ger- generalized modality for targeted super-resolution many for kindly providing the STED microscope imaging, including STED, has been termed revers- image shown in Fig. 4 (lower part). ible saturable optical fluorescence transition (RE- SOLFT); such techniques must be driven by light and aim to separate and resolve (sequentially) References neighbouring molecules. Recently, specially engi- neered fluorescent proteins (optically switched 1 Xian T, Li N, Fang X. Single-molecule fluorescence imaging between long-lived conformational states) have in living cells. Annu Rev Phys Chem 2013; 64: 459–80. allowed targeted live-cell imaging under low-light 2 Yuste R. Fluorescence microscopy today. Nat Methods 2005; 2: 902–4. levels of photoswitching, and this has technologi- 3 Blokzijl A, Friedman M, Ponten F, Landegren U. Profiling cally been extended to highly parallel large field of protein expression and interaction: proximity ligation as a views [105, 106]. Further advances in targeted tool for personalized medicine. J Int Med 2010; 268: 232–45. nanoscale optical switching seem possible. 4 Abbe E. Beitrage€ zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv Mikros Anat 1873; In conclusion, from the perspective of cellular 9: 413–68. € biology and biomedicine, a fundamental goal is to 5 Blom H, Ronnlund D, Scott L et al. Spatial distribution of Na+,K+-ATPase in dendritic spines dissected by nanoscale understand the mechanisms operating in living superresolution STED microscopy. BMC Neurosci 2011; 12: cells by investigating interactions that elicit and 16. direct molecular events. To date, the sheer com- 6 Hell SW, Wichmann J. Breaking the diffraction resolution plexity of cellular systems has meant that most limit by stimulated emission: stimulated emission depletion optical experimental investigations have focused microscopy. Opt Lett 1994; 19: 780–2. on single systems using relatively crude bulk 7 Klar TA, Jakobs S, Dyba M, Egner A, Hell SW. Fluorescence microscopy with diffraction resolution limit broken by stim- ensemble-average measurements. Thus, to gain a ulated emission. Proc Natl Acad Sci USA 2000; 97: 8206–10. better experimental understanding of cellular func- 8 Weiss S. Shattering the diffraction limit of light: a revolution tions, the use of more powerful and precise nano- in fluorescence microscopy? Proc Natl Acad Sci USA 2000; scopic techniques is required, allowing (at a 97: 8747–9. bottom-up molecular level) the resolution of all 9 Dyba M, Jakobs S, Hell SW. Immunofluorescence stimulated functions and connections in different biological emission depletion microscopy. Nat Biotechnol 2003; 21: 1303–4. systems. It is hoped that an experimentally 10 Hell SW. Far-field optical nanoscopy. Science 2007; 316: resolved cellular microcosmos would allow better 1153–8. understanding of the molecular world, and via 11 Einstein A. Emission and absorption of radiation according to preclinical and clinical research entangle this into the quantum theory. Verh Dtsch Phys Ges 1916; 18: 318–23.

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ª 2014 The Association for the Publication of the Journal of Internal Medicine 17 Journal of Internal Medicine H. Blom & H. Brismar Review: Applications of STED microscopy

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ª 2014 The Association for the Publication of the Journal of Internal Medicine 19 Journal of Internal Medicine