Resolving the structure of inner ear ribbon synapses with STED microscopy.

Review Article

Synapse DOI 10.1002/syn.21812

Synapse Review Article – Special issue: The Super-Resolution Revolution in Neuroscience Editor: Henrique von Gersdorff Revision 2 Resolving the structure of inner ear ribbon synapses with STED microscopy

Mark A. Rutherford, PhD Assistant Professor Central Institute for the Deaf Department of Otolaryngology, Campus Box 8115 Washington University School of Medicine 660 South Euclid Ave. St. Louis, MO 63110 Tel.: 314-747-7160 [email protected]

Key words: super-resolution light microscopy, stimulated emission depletion, cochlea, hair cell, spiral ganglion neuron, CtBP2/Ribeye, bassoon, CaV1.3, AMPA receptor, stimulus-secretion coupling

Graphical Abstract: Unlimited by diffraction, 2-color STED microscopy at inner hair cell afferent synapses in the cochlea resolves details of molecular organization within synapses (scale bar 200 nm). The vesicle-handling protein bassoon (magenta) and the voltage-gated Ca2+ channels controlling vesicle release (green; CaV1.3) are intimately aligned at each synapse.

Conflict of Interest statement: The author declares no conflict of interest.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/syn.21812 © 2015 Wiley Periodicals, Inc. Received: Sep 28, 2014; Revised: Feb 03, 2015; Accepted: Feb 08, 2015 This article is protected by copyright. All rights reserved.

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Resolving the structure of inner ear ribbon synapses with STED microscopy Mark A. Rutherford Assistant Professor, Dept. of Otolaryngology, Washington University School of Medicine Abstract Synapses are diverse in form and function; however, the mechanisms underlying this diversity are poorly understood. To illuminate structure/function relationships, robust analysis of molecular composition and morphology is needed. The molecular-anatomical components of synapses – vesicles, clusters of voltage-gated ion channels in presynaptic densities, arrays of transmitter receptors in postsynaptic densities – are only tens to hundreds of nanometers in size. Measuring the topographies of synaptic proteins requires nanoscale resolution of their molecularly-specific labels. Super-resolution light microscopy has emerged to meet this need. Achieving 50 nm resolution in thick tissue, we employed stimulated emission depletion (STED) microscopy to image the functionally and molecularly unique ribbon-type synapses in the inner ear that connect mechano-sensory inner hair cells to cochlear nerve fibers. Synaptic ribbons, bassoon protein, voltage-gated Ca2+ channels, and glutamate receptors are inhomogeneous in their spatial distributions within synapses; the protein clusters assume variations of shapes typical for each protein specifically at cochlear afferent synapses. Heterogeneity of substructure among these synapses may contribute to functional differences among auditory nerve fibers. The morphology of synaptic voltage-gated Ca2+ channels matures over development in a way that depends upon bassoon protein, which aggregates in similar form. Functional properties of synaptic transmission appear to depend on voltage-gated Ca2+ channel cluster morphology and position relative to synaptic vesicles. Super-resolution light microscopy is a group of techniques that complement electron microscopy and conventional light microscopy. Although technical hurdles remain, we are beginning to resolve the details of molecular nanoanatomy that relate mechanistically to synaptic function. Introduction Neural circuits sense our environment, process information, and direct motion. They consist of neurons connected by synapses, submicron in size, which are thought to be the smallest functional processing units of the nervous system. Chemical synapses are supramolecular nanomachines that mediate signaling between cells. They share common features like presynaptic Ca2+-evoked release of neurotransmitter followed by binding to postsynaptic receptors. However, molecular compositions and morphologies may differ by synapse type. Even among synapses of one type, plasticity and heterogeneity of synaptic component substructure are thought to modulate synapse and circuit function (Holtmaat and Svoboda, 2009; Gundelfinger and Fejtova, 2012; Xu-Friedman and Regehr, 2004; Sterling and Matthews, 2005; Meyer and Moser, 2010).

This article is protected by copyright. All rights reserved.

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For synapses in the inner ear, like those in the brain, microscopy demonstrates correlations between morphology and function (rev. by Wichmann and Moser, 2015). Understanding the functions and dysfunctions of neural circuits in health and disease relies upon knowledge of synaptic molecular anatomy and the differences between synapses. However, such molecularanatomical details are not revealed by conventional light microscopy. Synapses are often said to be specialized, but we are only beginning to resolve precisely how morphological details and molecular differences specialize synapses to perform their functions. To ask increasingly detailed questions about synaptic mechanisms, significant emphasis has been placed on studies that quantitatively correlate structure with the functional properties of synaptic transmission. Investigations of form and function converge at the molecular level, where proteins and lipids that support biophysical chemical reactions assemble into observable anatomical patterns. When synaptic function changes over development, or when experiments that manipulate genetic or environmental variables generate an effect on synaptic function, then it is crucial to identify any correlated changes in synaptic molecular anatomy at the nanoscale (e.g., in the cochlea: Khimich et al., 2005; Frank et al., 2010; Wong et al., 2014). Diffraction-limited microscopy By the end of the 21st century, visualizing biological tissues and cells with diffraction-limited microscopy had been optimized with the conventional confocal laser scanning microscope (Minsky, 1988; Shotton, 1989; Paddock and Eliceiri, 2014). Working with the traditional principles of light microscopy, resolution is limited by diffraction as stated by Abbe (1873). Resolution (d) in the image plane (i.e., the XY or lateral plane) is proportional to the wavelength of light (λ) and inversely proportional to twice the numerical aperture (NA) of the objective lens: dX,Y = λ/2NA. Numerical apertures are generally in the range of 1.0-1.5. Thus, the limit of lateral resolution is approximately one half to one third the wavelength of light used to make the image. Because light travels as a wave, the photons used to excite fluorophores cannot be focused to a smaller spot. For blue light around 480 nm the maximum image resolution theoretically attainable is 160 nm. Smaller features or objects cannot be discerned or distinguished through focused illumination alone (Hell et al., 2004). Axial resolution (i.e., along the Z axis) is considerably worse: dZ = λ/NA2. The point spread function (PSF) is the three-dimensional diffraction and interference pattern of light emitted from a point source (a source smaller than the resolution limit). The twodimensional diffraction PSF in the focal plane, given in units of full width at half-maximum (FWHM), is described by the Airy function (Airy, 1838). A good approximation of the resolution attainable is the FWHM of the dominant, first peak in the PSF. This can be thought of as the fundamental unit of an image or the blur in an image that represents an unresolved object. The spatial extent of the PSF has lower bounds that are diffraction-limited, however, in practice the PSF is typically significantly larger than 160 nm. If the numerical aperture of the lens is less than


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optimal or when there is a mismatch between the refractive indexes of the sample and the optics, then resolution is degraded. The combination of microscope and sample comprise an imaging system with resolution described as an ‘effective PSF.’ Confocal laser scanning microscopes optimize the resolution of conventional light microscopy by limiting the points in space where the sample is excited and from where emitted light is detected. The coordinates where fluorophores are excited are minimized through diffractionlimited point illumination. Detection of emitted light from outside of the focal plane, where illumination is more diffuse and the PSF is larger, is reduced through use of a pinhole aperture. The confocal laser scanning microscope achieves thin optical sectioning relative to wide-field microscopy, which illuminates the entire field simultaneously and places no pinhole before the detector. Resolution in the optical (Z) axis is improved by the confocal sectioning, which improves the resolution of three-dimensional reconstructions of sample volumes. Still, confocal resolution is 2-3 times longer axially than laterally. Thus, with optimized optics and sample preparation, the resolution limit of conventional microscopy is ~ 200 nm in the XY plane and greater than 500 nm in Z. The image acquired is the convolution of the PSF with the real source of emitted photons. In principle, it isn’t possible to resolve things smaller than the PSF. When two objects are separated by a distance ≤ d, they will appear as one continuous structure. Likewise, when a single object has morphological details ≤ d, they are not observed. Regardless of a microscope’s resolution, properties of the sample may blur structural details or add to the apparent size of an object. For example, with immunochemistry that uses fluorophores conjugated to antibodies that recognize the antigen of interest, the size of the antibodies adds to the apparent sizes of the labeled structures (Ries et al., 2012). In practice, when the distance between two structures or the morphological details of a structure are smaller than ~ 500 nm, such as synaptic components and other internal structures of biological cells, those features are not resolved with confocal microscopy. Still, conventional confocal resolution is sufficient for many purposes. When tissue structures are relatively large, the PSF can be negligible. When an object is sub-diffraction in size, Gaussian curve fitting can still identify its center of mass for precise localization limited by the signal to noise ratio, not the PSF. If objects are well separated, relative molecular abundance can be compared by analysis of pixel intensities. For its disadvantages (see below), super-resolution microscopy is typically applied only as needed. However, because of their sizes, the protein components within synapses require superresolution for precise measurements (Figure 1). Fortunately, the tools for super-resolution light microscopy research are now commercially available. As scientific focus on structure-function relationships has advanced from the micro- to the nano-domain, the barriers of diffraction-limited light microscopy have been overcome through several approaches (Ji et al., 2008; Huang et al., 2010; Sahl and Moerner, 2013).


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Microscopy at nanometer scale is not new. Electron microscopy (EM) and tomographic reconstructions from serial thin-sections enable the most precise measurements of electrondense structures like synaptic ribbons and t-bars (Wichmann and Sigrist, 2010; Frank et al., 2010; Wong et al., 2013). EM is unsurpassed in structural resolution (~ 0.1 nm), but has the drawback of being relatively low-throughput. Although immuno-EM is the gold standard in protein localization at the ultra-structural level, there is a trade-off between preservation of ultrastructure for EM and preservation of antigenicity for immunochemistry, restricting the number of ‘good’ antibodies for EM protocols. Immuno-EM is also limited for studying synaptic diversity because it generally allows detection of only two antigens at the same synapse. Superresolution light microscopy complements EM by enabling the collection of statistics on protein localizations and abundances at a relatively large number of synapses. These optical techniques exploit properties of photons and fluorophores to overcome Abbe’s law and achieve diffraction-unlimited super-resolution. Sometimes called nanoscopy because they achieve resolution in the range of tens of nanometers, super-resolution light microscopy techniques require collecting and/or processing optical data in new ways. Although the various superresolution techniques developed in the past two decades use different methodologies, they all operate on the principle of restricting the population of excited or detected fluorophores, either in space or in time, to resolve things smaller than diffraction limited spots (reviewed by Hell, 2009; Dani and Huang, 2010; Sigrist et al., 2012; O’Rourke et al., 2012; Willig and Barrantes, 2014). Super-resolution light microscopy of ribbon synapses in the inner ear We now focus on stimulated emission depletion microscopy (STED; Hell and Wichmann, 1994) and the first auditory synapses, in the inner ear, through which all information about sound must pass (reviewed by Moser et al., 2006a). As shown in Figure 1, the presynaptic active zones of hair cells feature a cytoplasmic extension of the presynaptic density called the synaptic ribbon (Smith and Sjostrand, 1961). Like their long and thin ribbon-shaped relatives in the retina, hair cell ribbons are comprised mainly of the protein CtBP2/Ribeye (Schmitz et al., 2000). EM has shown that ribbons of hair cells, also called synaptic bars, assemble in ellipsoidal or tubular shapes that can appear to have either a structured, solid interior or a hollow core (Liberman, 1980; Lenzi et al., 1999; reviewed by Moser et al., 2006b). Synaptic ribbons are often regarded as conveyor belts that deliver tethered, 40 nm diameter synaptic vesicles to the presynaptic membrane (reviewed by Lenzi and von Gersdorff, 2001), although other functions have been proposed (Vollrath and Spiwoks-Becker, 1996; Parsons and Sterling, 2003; Jackman et al., 2009). Vesicles morphologically associated with synaptic ribbons constitute functional pools of vesicles that rapidly mediate synaptic transmission upon stimulation via exocytosis (von Gersdorff et al., 1998; Moser and Beutner, 2000; Lenzi et al., 2002; Khimich et al., 2005; Rutherford and Roberts, 2006; Li et al., 2009; Kantardzhieva et al., 2013). Most ribbons of mouse cochlear inner hair cells have short and long axes in the ranges of 100-125 and 200-250


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nm, respectively (EM sections: Meyer et al., 2009). When object dimensions are similar to the lateral resolution, as is for ribbons of mouse cochlear inner hair cells in confocal imaging (both ~ 200 nm), then the objects appear roundish. With laser scanning confocal microscopy, ribbons appear to be ≤ 500 nm in XY, as can be seen in Figures 2 and 4. For ribbons and other similarlysized structures, the presence of one long and one short axis is often just noticeable, depending upon its three-dimensional orientation. When spatial resolution is 200 nm, then the Nyquist criterion for the sampling interval is 100 nm. In conventional light microcsopy, pixel sizes of 50 100 nm2 are sufficient to gather the available information regardless of the smallness of the objects being imaged. As shown in the diffraction-limited images of Figure 2, super-resolution fluorescence imaging is required to resolve the synaptic structures shown in Figure 1. STED is one technique applicable to biological preparations. Diffraction is not broken but it no longer limits resolution. Said to be diffraction-unlimited, STED effectively reduces the size of the PSF by shaping the light that is emitted by the sample (Klar et al., 2000). Like confocal microscopy, a laser beam (λ = 400-700 nm) is passed through the lens to create a focused spot of excitation extending ~ 0.5 λ laterally and > λ axially. After light stimulates a fluorophore, there is a brief window of time over which photons are emitted. During this window (from ~ 10 picoseconds to ~ 4 nanoseconds) the redshifted STED beam depletes excitation of fluorophores by transitioning them to the ground state through the physical process of stimulated emission. The interaction of the STED beam with the sample must begin immediately after excitation and should continue through the end of this fluorescence lifetime. The STED de-excitation pattern has the shape of a bagel or a donut with a hole of zero power in the center (Figure 3), centered on the excitation focal spot. Fluorophores in the periphery of the excitation spot are not allowed to fluoresce. The effective PSF is the excitation pattern subtracted by the depletion pattern or, rather, the area from where fluorescence is allowed to emit. Thus light is detected, or not detected, from only a very small volume in the center of the donut. This tuning of the spatial extent of emission has been called PSF engineering. Resolution is limited by the effective PSF, which is sub-diffraction in size. Resolution can be assessed using fluorescent nanobeads. Figure 4 directly compares the image of 40 nm beads in conventional confocal versus STED mode in panels e and f. With increased resolution, additional information can be extracted from the sample by acquiring smaller pixels. The region of interest is scanned in XY and Z, like in confocal laser scanning microscopy, and emitted light is detected through a confocal pinhole aperture. Thus, precise location information comes from knowing the coordinates of the donut hole. Finer structures and smaller spaces between objects are made visible because STED enables more precise measurements of the voxels in which bright things are not located (Figure 3b, right). The size of the effective PSF depends upon the power of the STED laser and the shape of the depletion pattern, the latter of which is formed by a phase plate in the path of the STED beam.


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The phase plate and STED beam can be configured to generate a depletion pattern predominantly laterally or axially, dramatically improving resolution in XY or Z, respectively (Figure 3, STEDX,Y). To simultaneously increase resolution in the focal plane and along the optical axis, the techniques termed 3D STED and isoSTED combine two de-excitation patterns to create spherical effective PSFs with diameters of about 40 nm (Harke et al., 2008b; Schmidt et al., 2008; Hell et al., 2009). In principle, the resolution of STED is not limited, depending only on laser power (Harke et al., 2008a). The STED pulse must be bright enough to make sure that enough photons are available to deplete excited fluorophores before they emit photons, over a period of 1 nanosecond. As the STED power increases, the volume in which stimulated emission is saturated increases, as it closes in around the center of the excitation spot resulting in greater resolution (Figure 3c-d). Therefore, in STED microscopy the ability to discern a dark spot from a neighboring bright spot depends upon the spatial profile of saturation of stimulated emission (Klar et al., 2000). Stefan Hell and colleagues thus extended the Abbe equation from d = λ/2NA to d = λ/(2NA*√(1+(I/IS))), where I is the STED intensity and IS is the ‘saturation intensity.’ The larger the ratio I/IS >> 1, the smaller the effective PSF (Hell et al., 2004). Note that resolution still scales with λ; diffraction is not broken but it no longer limits resolution. In principle, the resolution of STED is unlimited, but in practice it is limited by the intensity of light that can be tolerated by organic fluorescent dyes (Hell, 2005). STED-resolved structural correlates of stimulus-secretion coupling Voltage-gated Ca2+ channels play a central role in stimulus-secretion coupling at synapses. In hair cells the predominant voltage-gated Ca2+ channel, CaV1.3, is required for exocytosis of synaptic vesicles as well as for regenerative potentials during development (Brandt et al., 2003). Confocal microscopy showed that these channels localize with synaptic ribbons (Brandt et al., 2005). As illustrated in Figure 2, spots of CaV1.3 are nearly diffraction-limited. Therefore, their true shape is not observable with conventional microscopy and, like ribbons, they appear more or less round depending upon their three-dimensional orientation relative to the image plane. Earlier studies using freeze-fracture scanning EM observed rows of densely-packed intramembranous particles underneath hair cell ribbons (Saito and Hama, 1984; Roberts et al., 1990), and speculated that some of these particles were ion channels. However, the lack of molecular markers in the EM and the blurr of overlapping synaptic proteins in conventional confocal images left this issue unresolved until recently. As shown in Figure 4, STED microscopy revealed that clusters of CaV1.3 channels formed elongated stripes alongside ribbons acquired in confocal mode (Frank et al., 2010), consistent with the position and shape of the rows of particles seen directly underneath presynaptic ribbons in EM. The synaptically ubiquitous cytomatrix protein bassoon has many binding partners and is thought to be involved in the handling of synaptic vesicles in hair cells (reviewed in Rutherford


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and Pangršič, 2012). At ribbon synapses bassoon has the additional job of anchoring the ribbon to the synaptic membrane. When bassoon is disrupted ribbons float away, synaptic exocytosis is reduced, and sound encoding is compromised (Khimich et al., 2005; Buran et al., 2010; Frank et al., 2010; Jing et al., 2013). Bassoon directly binds with the predominant ribbon protein CtBP2/Ribeye (tom Dieck et al., 2005). Thus it was unclear if bassoon protein should be found throughout the ribbon or localized strictly to the spaces between synaptic ribbons and CaV1.3, where the ribbon is anchored and where the majority of docked vesicles reside adjacently. Due to the spatial overlap in confocal mode of fluorescent signals coming from the ribbon, CaV1.3 channels, and postsynaptic AMPA-type glutamate receptors (see Figures 2-4), improved resolution was required in order to determine the precise position of bassoon relative to other members of the active zone. As shown in Figure 5, 2-color STED microscopy revealed an intimate association between CaV1.3 and bassoon (Wong et al., 2014), inconsistent with localization of bassoon throughout the ribbon (see Vincent et al., 2014; their figure 6). Because CaV1.3 is thought to be restricted to the plasma membrane underneath the ribbon, the close association with bassoon strongly suggested that bassoon localization was restricted to the base of the ribbon, as confirmed by pre-embedding immuno-gold labelling of bassoon in EM (Wong et al., 2014). The number and precise localization of voltage-gated Ca2+ channels relative to synaptic vesicles and other active zone proteins like bassoon has a direct effect on the properties of exocytosis and synaptic transmission (Matveev et al., 2011; Kim et al., 2013). Similar to other synapses, the intrinsic Ca2+-dependence of exocytosis in cochlear inner hair cells is a non-linear function of Ca2+ concentration (Beutner et al., 2001). However, when changing the amount of synaptic Ca2+ influx in a physiological way, by changing the membrane potential and therefore the number of open channels, the magnitude of exocytosis scales linearly with Ca2+ influx (Brandt et al., 2005). It has been hypothesized that this linearity results from a nanoscale coupling between voltagegated Ca2+ channels and vesicles, such that the opening of a single channel would be sufficient to saturate the vesicular Ca2+ sensor for exocytosis such that the non-linear intrinsic dependence of the molecular sensor is not apparent in the relationship between Ca2+ influx and exocytosis. In such a Ca2+ “nanodomain” regime exocytosis scales with the number of open channels, so long as vesicle supply and active zone clearance are not rate limiting (Haucke et al., 2011). Cochlear inner hair cell ribbon synapses of the mouse display this property of nanoscale stimulus-secretion coupling only after the first two weeks of postnatal development, around the onset of hearing, when CaV1.3 channels are organized almost exclusively into elongated stripes alongside bassoon underneath ribbons (Frank et al., 2010; Wong et al., 2014). Before hearing onset, on postnatal day 10 (p10), only some CaV1.3 channels are located with ribbons; many are extrasynaptic, located far from ribbons, as shown with confocal microscopy (Wong et al., 2014). Moreover, STED microscopy revealed that CaV1.3 channels did not form


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elongated stripes in immature, p10 mice. Rather, they existed in round spot-like clusters (Figure 6A). Similarly, in bassoon-deficient inner hair cells of mature mice, in which most active zones lack ribbons, synaptic CaV1.3 channels existed only in spot-like clusters even at the few remaining ribbon-occupied synapses (Figure 6b). The difference between one elongated CaV1.3 stripe at a wild-type synapse versus two or three CaV1.3 spots at a bassoon-deficient synapse (Figure 6) was not observable in confocal because the distance between spots is similar to the PSF. Different from immature wild-type inner hair cells, stimulus-secretion coupling was linear at bassoon-deficient synapses of mature mice (Frank et al., 2010; Jing et al., 2013), consistent with normal nanodomain coupling between remaining Ca2+ channels and docked vesicles. However, a reduction in number of CaV1.3 channels and vesicles did result in a reduction of release-ready vesicles and abnormal encoding of auditory signals. Finding that elongated stripes of CaV1.3 are not required for nanodomain stimulus-secretion coupling in the adult bassoon-deficient mice suggested that spots of CaV1.3 don’t readily explain looser functional coupling in immature mice. Combined with the finding of similar intrinsic biochemical coupling one week before and after hearing onset, this suggests that looser stimulus-secretion coupling in immature mice is likely due to the extrasynaptic localization of CaV1.3 during postnatal development (Wong et al., 2014). In another recent study, STED microscopy showed that unlike the elongated CaV1.3 stripes in mature cochlear inner hair cells, the CaV1.3 channels at ribbon synapses of mature type I vestibular hair cells form smaller, spot-like shapes (Vincent et al., 2014). However, unlike immature cochlear inner hair cells which displayed CaV1.3 spots and loose stimulus secretion coupling (Wong et al., 2014), type I vestibular hair cells displayed spots and tight, nanodomain-like coupling (Vincent et al., 2014). In comparison with cochlear inner hair cells, CaV1.3 channels in vestibular type I hair cells were more tightly organized with ribbons and stimulus-secretion coupling was tighter in physiological tests. This suggests that for a given pool of vesicles, round CaV1.3 channel clusters may minimize the distances between channel and vesicle. Moreover, elongated stripelike CaV1.3 channel clusters may contribute to the increased dynamic range observed for exocytosis in cochlear inner hair cells (Vincent et al., 2014). These investigations of CaV1.3 channel cluster morphology at hair cell ribbon synapses are improving our understanding of the relationships between molecular anatomy and synaptic function in the inner ear. It appears that CaV1.3 cluster shapes are cell-type specific and have direct influence on the encoding of auditory and vestibular stimuli. Moreover, within the inner hair cells, these afferent synapses exhibit heterogeneity in the machineries of stimulus-secretion coupling (compare synapses in Figure 5). This presynaptic heterogeneity is likely to contribute to the diversity of soundresponse properties found among fibers of the auditory nerve, which each receive their sole excitation from a single synapse (Liberman, 1980). However, the mechanistic relationships between presynaptic morphological heterogeneity and diversity of encoding are not presently understood.


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AMPA receptors distributed inhomogeneously in the postsynaptic membrane After exocytosis from the inner hair cell, neurotransmitter binds to AMPA-type glutamate receptors on the postsynaptic bouton of an auditory nerve fiber (also called spiral ganglion neuron, see Figure 2A). The single postsynaptic density on each spiral ganglion neuron postsynaptic bouton is approximately one micron across in EM (Merchan-Perez and Liberman, 1996). Further investigation using antibodies to AMPA receptors with STED microscopy revealed a clear ring-like shape of the AMPA receptor array on each bouton (Meyer et al., 2009). This postsynaptic ring-like shape surrounds the release face of the presynaptic active zone, demarcated by the ribbon, as shown in Figure 7. The ring-like shape of the AMPA receptor array persists in the presence of bassoon disruption and concomitant presynaptic disorganization (Jing et al., 2013). The size of an AMPA receptor array is one determinant of the size of excitatory postsynaptic potentials. Fluorescence line profiles across the ring-like AMPA receptor arrays measured with STED microscopy showed that they are on average ~ 900 nm in diameter with fluorescence dropping to about one-half the peak intensity in the center (Meyer et al., 2009). The significance of smaller AMPA receptor density in the center of the array is not clear. One peculiar property of synaptic transmission at synapses between inner hair cells and spiral ganglion neurons is the large heterogeneity of amplitudes of excitatory postsynaptic potentials (Glowatzki and Fuchs, 2002). A recent study investigated the effect of variability in presynaptic release position within the active zone, given the size and shape of the AMPA receptor clusters observed in STED. Modeling suggested that the large ring-like array embracing the presynaptic release face has the effect of making the amplitude of excitatory postsynaptic currents quite independent of presynaptic release location (Chapochnikov et al., 2014). This finding suggests that rather than variation of release position, other factors such as variation in quantal content or a dynamic fusion pore determine the variability in size and shape of excitatory postsynaptic potentials. Practical considerations for interest in STED Is super-resolution microscopy necessary and, if so, which type? For some purposes, superresolution is not required. When the morphological details of interest are known to be at least twice as large as the conventional confocal PSF, for example groups of entire cells, then application of super-resolution could lead to unwarranted spatial oversampling. Similarly, when objects are well-separated from each other, then conventional microscopy may provide sufficient resolution. For example, unlike many chemical synapses in the brain, hair cell ribbon synapses are relatively large and generally well-separated spatially, such that they can be analyzed in isolation with confocal microscopy (nearest neighbor distance: 0.5 – 5 µm; Meyer et al., 2009). However, information on the true size and shape are lacking. Without resolving synaptic substructure, centers of mass and pixel intensities can be measured for comparisons of


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position and molecular abundance. However, when the fluorescently-labeled structures have spacing or sizes smaller than the conventional confocal PSF, then true size and shape are not observed (Figures 2-4) and super-resolution is required. When the morphologies of labeled protein structures are not known, then super-resolution microscopy can be explorative. Generally, when super-resolution is needed to resolve morphology, the appearance of subdiffraction structures in conventional confocal microscopy will suggest the presence of unresolved substructure (Figure 2). STED has some potential detriments that should be considered. As it scans, the powerful STED beam can bleach fluorophores laterally and axially around the focal spot before they come into focus. Working with difficult or sparse antigens, immunoreactivity that might be acceptable in conventional fluorescence microscopy can bleach significantly. Thus, depending upon the sample, it may not be practical to screen samples or to search for the region of interest in STED mode prior to image acquisition. STED modules are commercially available as additions to laser scanning confocal microscopes. In this case, regions of interest can be identified in epifluorescence or conventional confocal mode. This is especially helpful in tissues where the synapses are spatially restricted to small regions of the tissue, such as in the endorgans of the inner ear. Moreover, a single STED beam and phase plate increase lateral or axial resolution only. Due to the problem of bleaching and the confocal-like resolution in Z (Figures 4-7), rigorous 3D reconstruction of antibody-labelled synaptic structures in the organ of Corti has so far not been realized with STED. New developments like pulsed excitation and time-gated detection may help achieve this because they allow improved resolution with lower STED laser power (Vicidomini et al., 2011). The STED laser must be powerful because the molecular transition that must be acted upon, fluorescence decay, is a very rapid process (order of ~ 1 nanosecond). Alternative STED-like approaches, also in the category of Reversible Saturable Optical Fluorescence Transitions (RESOLFT) microscopy techniques, can be used with much lower light levels because the switchable fluorescent proteins or organic fluorophores used have much slower molecular transitions (Hofmann et al., 2005; Bossi et al., 2006). STED microscopy has certain advantages and disadvantages relative to other super-resolution techniques. As a laser scanning technique generating raster plots, STED microscopy is familiar due to its similarity with confocal microscopy. The confocal aperture makes it possible to use STED microscopy deep in relatively thick samples without significant contamination of the signal from out of focus light above and below the focal plane. STED microscopy has been used to image synapses 15 microns below the cover slip in the organ of Corti (Figures 4-7). In contrast, localization based microscopy (LBM) super-resolution techniques like STORM (stochastic optical reconstruction microscopy) and PALM (photoactivated localization microscopy) are widefield methods that generally rely on TIRF (total internal reflection


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fluorescence) illumination to limit the thickness of the excited optical section and thereby preclude contamination by light from out of focus fluorophores. In consequence, the depth of the focal plane is limited to less than one micron below the cover slip. Thus, STED may be preferred for fluorescently thick samples. When imaging deep in a sample, resolution and brightness can be limited by spherical aberrations created by a mismatch in refractive index between the tissue mounting medium and the immersion oil. This problem is nearly the same in STED and in conventional confocal. The difference is that the problem is more prevalent when the resolution is higher. However, the mismatch can be largely avoided through use of alternative embedding media (Staudt et al., 2007). STED is sufficiently fast for live nanoscopic imaging of synaptic vesicle movement in cultured hippocampal neurons and for imaging changes in spine morphology in the living mouse brain (Westphal et al., 2008; Berning et al., 2012). In LBM single-molecule detection techniques, rather than making an image by the ensemble of PSFs via photon collecting, images are constructed pointillistically as all-or-none events of localization via offline calculations. The location of each single-molecule emitter is calculated but the PSF is not visualized in the processed image, which is constructed from a sequence of raw images. In comparison with STORM/PALM and other stochastic point-source detection methods, STED has the advantage of being fast and deterministic, not requiring post processing. The raster plot is generated live as in conventional confocal microscopy. For its speed relative to STORM/PALM, STED may be more suited to live imaging of cells (Nägerl et al., 2008; Pellett et al., 2011; Takasaki et al., 2013). LBM methods like STORM/PALM offer the advantage of high resolution in Z (50 nm) and XY (20 nm) at low light levels, albeit over a more limited focal range with TIRF illumination (< 1 µm). For STED in fixed tissue, one advantage is that using traditional immunohistochemistry with secondary antibodies requires only one modification, that the conjugated fluorophore be “STEDable” (i.e., resistant to bleaching and efficiently depleted by the STED beam). A list of fluorescent dyes used in STED microscopy is available online from the Department of Nanobiophotonics at the Max Plank Institute for Biophysical Chemistry in Göttingen, Germany (http://nanobiophotonics.mpibpc.mpg.de/old/dyes/). STED works on multiple laser lines, enabling colocalization experiments. Measurements of colocalization can be limited by misalignment of the beams. However, one STED line may be used to sequentially deplete two fluorophores excited by two excitation lines, as in Figure 5 (Göttfert et al., 2013; Wong et al., 2014). This approach ensures that the PSFs are aligned even with slight misalignment of the two red excitation focal spots of 590 and 647 nm light. Currently published research may be limited to two simultaneous channels. However, adding another STED laser line is in principle technically straightforward. In this scenario the additional STED beam, used with 488 excitation, could be at 590 nm and would overlap the excitation spectrum of the red dye at 590 nm.


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Imaging of the red dyes first should prevent the additional STED laser from bleaching the red dye(s) before they are imaged. Conclusion In summary, synaptic structures previously observed only in EM can now be observed with light microscopy at the nanoscale. At the synapses in the inner ear, between sensory hair cells and 8th nerve neurons, STED microscopy has been successfully employed in thick tissue to achieve resolution of ~ 50 nm in XY with the molecular specificity of immunohistochemistry. One and two-color STED images have illustrated the shapes and relative positions of presynaptic ribbons, voltage-gated Ca2+ channels, bassoon protein, and postsynaptic AMPA receptors. The shape of clusters of presynaptic CaV1.3 channels relative to the ribbon and bassoon may be a determinant of the properties of stimulus-secretion coupling in hair cells. The size and shape of the AMPA receptor arrays at synapses on inner hair cells in part determine the properties of synaptic transmission. Over other super-resolution techniques STED offers the advantages of fast acquisition speed, no post-processing, and deep tissue penetration. Certain technical limitations must be overcome to make further improvements. At present, STED microscopy in general is limited to two colors and requires relatively high laser power that can result in bleaching, depending upon the sample. So far, STED in the inner ear has been limited to increasing resolution in XY, but not Z, precluding three-dimensional super-resolution reconstruction of synaptic components. Perhaps gated 3D- or iso-STED will simultaneously reduce bleaching and allow for improved Z resolution. Acknowledgments This work was supported by research funds provided to M.A.R. by the Dept. of Otolaryngology at Washington University in St. Louis, School of Medicine. Illustrations of STED microscopy in the organ of Corti were sourced from previously published figures and data sets. STED microscopy was performed in collaboration with the groups of Tobias Moser (Inner Ear Lab, Dept. of Otolaryngology, University of Göttingen Medical Center) and Stefan Hell (Dept. of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry) in Göttingen, Germany.


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Figure legends:

Figure 1. Molecular anatomy of the hair cell ribbon synapse derived from electron microscopy and super-resolution light microscopy. (a) Transmission electron micrograph of the synapse between a cochlear inner hair cell and a spiral ganglion neuron. The main structural elements visible are the synaptic ribbon and its halo of vesicles, the anchor between the ribbon and the plasma membrane, and the presynaptic and postsynaptic densities. (b) Color overlay on the same micrograph shows synaptic vesicles (gold) and our interpretation of the locations of the structural components labeled in (a), molecularly-identified with confocal and super-resolution microscopy: presynaptic ribbon (antiCtBP2/Ribeye, red); presynaptic cytomatrix protein bassoon (anti-Sap7f, magenta); presynaptic voltagegated Ca2+ channels (anti-CaV1.3, green); and AMPA-type glutamate receptors (anti-GluA2/3, blue). (c) Tomographic reconstruction from electron microscopy is a 3D rendering of the synapse, structurally segmented and colored (see panel label), showing the ribbon surrounded by vesicles (upper, side view) as well as membrane-tethered vesicles docked underneath the ribbon and aligned with the presynaptic membrane density (lower, top or en face view). a-b are adapted from Meyer et al., 2009; c is reproduced from Frank et al., 2010.


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Figure 2. Ribbon synapses at the limits of confocal resolution demonstrate the need for superresolution. (a) Confocal projection of a synaptic triple-label in the organ of Corti whole-mount preparation from an adult mouse cochlea. Three inner hair cells, outlined with nuclei (dotted lines and circles), are immunolabeled for ribbons with anti-CtBP2/Ribeye (red), voltage-gated Ca2+ channels in the presynaptic membrane with anti-CaV1.3 (green), and AMPA-type ionotropic glutamate receptors in the postsynaptic membrane with anti-GluA2 (blue). Five synapses from the bottom of one inner hair cell (IHC) are enlarged below, showing the merged image (red box) and the separate channels in gray scale. Each synapse has a different 3D orientation relative to the XY plane. One spiral ganglion neuron (SGN) is drawn contacting the presynaptic IHC via its postsynaptic bouton. The resolving capability of the microscope is approximately 220 nm in XY and 700 nm in Z. (b) The appearance of the synapse depends upon its orientation relative to the image plane. The pre- and post-synaptic elements may be separated along the vertical (Y) axis as shown in side view or cross section. (b’) Or, the pre- and postsynaptic elements may be separated along the optical (Z) axis as shown in the top view or en face section. Either way, structures are closer to each other than the PSF of the microscope. Therefore, they appear to overlap somewhat as in (c-f). (c-f) Cross section and (c’-f’) en face views of two hair cell ribbon synapses from (a). Pixels are 50 nm. (c, c’): raw data; (d, d’): oversampled and smoothed; (e, e’): deconvolved with the PSF; (f, f’): deconvolved, oversampled, and smoothed. Oversampling and smoothing are cosmetic and don’t improve resolution. Deconvolution with the Richardson-Lucy algorithm of Deconvolution Lab in ImageJ uses assumptions based upon PSF theory to change the image. Deconvolution reduces the contribution of defocused light to the image and reduces the apparent sizes of fluorescent objects. The labeled structures are close in size to diffraction-limited spots, making them too small to resolve their shapes. When viewed en face, the membrane-delimited clusters of Ca2+ channels and glutamate receptors often appear more elongated and inhomogeneously distributed, suggestive of unresolved substructure.


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Figure 3. Principles of super-resolution using the STED method. (a) Black circles represent five fluorescently-tagged structural elements evenly spaced at imaging coordinates along the X-axis. Excitation light is focused to a diffraction-limited spot (blue), shown in the XZ and XY planes (upper and lower) for X-axis pixel position b. All structural elements within the blue excitation PSF are simultaneously excited (green) to emit light that is quantified and assigned to the pixel corresponding to X-axis position b. In confocal microscopy (left), three structural elements become bright at each X-axis coordinate a-c, while two of the elements remain dark. In STED microscopy (right) the same excitation light (blue) is followed by the STED laser beam (red) that is focused into the shape of a donut or bagel, having a hole of zero intensity in the center. Where it overlaps the space of excitation (purple), the STED beam prevents excited fluorophores from fluorescing, thereby reducing the size of the PSF to the nonoverlapping space in the center of the field of view. In this example, one structural element becomes bright in each pixel. Note that the green circles indicate the locations of emitting fluorophores accounted at X-axis position b, not the emitted light; the PSF for emitted light is no better than the PSF for excitation. (b) The structural element at position b has been removed. In confocal, two elements become bright in each pixel, but they cannot be discerned from each other and the structural gap is not resolved. In STED, the size of the PSF (blue space of non-overlap) is similar to the size of each structural unit. The gap is resolved at X-axis position b because all fluorophores fall outside the effective PSF. (c) To resolve labeled structures, the microscope must be able to detect the regions in space where fluorophores are absent. On right, the hypothetical structural elements and thus the gap are smaller. (d) The PSF of the STED microscope can be tuned, depending on the intensity of the STED laser. On left, the same STED beam as depicted in panels (a) and (b) results in a PSF that is unable to detect the gap in the array of smaller structural elements. On right, a more intense STED laser results in a smaller region of non-overlap ( blue), enabling resolution of the gap.


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Figure 4. Voltage-gated Ca2+ channel (CaV1.3) clusters in confocal and STED microscopy. (a) In confocal, clusters of Ca2+channels appear as elongated spots with minimum dimensions limited by the diffraction of light. (b) The same field of view in STED mode. (a1-a6 and b1-b6) 600 x 600 nm zoom views of Ca2+ channel clusters in (a) and (b) are a direct comparison of the same clusters viewed in conventional confocal microscopy versus STED mode. The effective PSF of the STED microscope was approximately 80 nm, therefore the minimum dimensions appear to be about 80 nm. The fluorophore used for STED was Atto647N-conjugated to a secondary antibody (Atto-Tec). (c) Conventional confocal image of ribbons (red) and Ca2+ channel clusters (green). (d) Ca2+ channel clusters in STED mode (green) with ribbons overlaid in conventional confocal mode (red). (e) 40 nm fluorescent beads measured in confocal. (f) The same field of view in STED mode. The beads approximate point sources of light, illustrating the difference in resolution between conventional confocal and STED mode. In STED, the diffraction-limited PSF is not detected so the beads appear less blurry. Their images do not overlap because the spatial frequency of the optical transfer function is increased due to the smaller effective PSF.


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Figure 5. Two-color STED microscopy demonstrates morphological coupling of bassoon with voltagegated Ca2+ channels. The same STED laser was pulsed through a vortex phase plate to deplete two fluorophores activated sequentially at different wavelengths in an interleaved pattern (Wong et al., 2014). Voltage-gated Ca2+ channels (CaV1.3, green) control exocytosis of synaptic vesicles. The cytomatrix protein bassoon (magenta) handles vesicles and organizes the presynaptic active zone. Each subpanel (740 x 740 nm) contains one synapse. The resolution of the microscope was approximately 50 nm in XY and the pixels are 20 nm. The displayed images are not oversampled but they are smoothed with a 2D Gaussian in ImageJ (sigma = 1). The resolution in Z is approximately 700 nm and each image is a maximum intensity projection of optical sections containing the entire synapse volume. CaV1.3 forms an elongated stipe-like shape most noticeable en face (upper and left panels). Some synapses have two stripes or more complex shapes (lower panels). Bassoon labeling is relatively less continuous and tends to parallel or surround but not directly overlap with CaV1.3. Fluorophores: Atto590- and Atto647Nconjugated secondary antibodies (Atto-Tec).


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Figure 6. Morphology of voltage-gated Ca2+ channels at inner hair cell active zones. (a) In immature inner hair cells, before the onset of hearing, voltage-gated Ca2+ channels (CaV1.3, STED) appear only as small round spots as shown at postnatal day 10 (p10). By one week after the developmental onset of hearing, CaV1.3 clusters have matured into elongated stripes as shown at p19. The developmental change of CaV1.3 cluster morphology is correlated with a maturation of the coupling between Ca2+ influx and exocytosis. Each subpanel is 740 x 740 nm. Adapted from Wong et al., 2014. (b) CaV1.3 (green, STED) appears only as small round spots in bassoon-disrupted mature inner hair cells. The failure to form elongated clusters is a form of synaptopathy associated with functional hearing deficits in bassoondisrupted mice. Upper panels, bassoon-deficient; lower panels, wild-type. Ribbons (red) in confocal mode. Adapted from Frank et al., 2010.


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Figure 7. AMPA receptors have a ring-like morphology at inner hair cell – spiral ganglion neuron synapses. (a) Glutamate receptors on postsynaptic spiral ganglion neurons are labelled with antiGluA2/3 (green, STED) and presynaptic ribbons are labelled with anti-CtBP2/Ribeye (red, confocal). The patch of glutamate receptors in each postsynaptic density has a ring-like appearance when viewed en face. The inside of the ring roughly approximates the perimeter of the shadow of the ribbon projected onto the synaptic membrane. A lower density of receptors is apparent directly underneath the ribbon. (b) Fluorescence line scans from STED en face views of glutamate receptor patches from the apical (blue) or midcochlear (red) regions; individual traces and averages overlaid in bold. Line profiles across AMPA receptor arrays, aligned at the center, showed that the density in the center of the array dips to approximately one-half of the side-peaks. For comparison, gray bars represent counts of immunogold particles as a function of distance from synapse center in rat inner hair cells (Matsubara et al., 1996). a.u., arbitrary units. Adapted from Meyer et al., 2009.


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Figure 1. Molecular anatomy of the hair cell ribbon synapse derived from electron microscopy and superresolution light microscopy. (a) Transmission electron micrograph of the synapse between a cochlear inner hair cell and a spiral ganglion neuron. The main structural elements visible are the synaptic ribbon and its halo of vesicles, the anchor between the ribbon and the plasma membrane, and the presynaptic and postsynaptic densities. (b) Color overlay on the same micrograph shows synaptic vesicles (gold) and our interpretation of the locations of the structural components labeled in (a), molecularly-identified with confocal and super-resolution microscopy: presynaptic ribbon (anti-CtBP2/Ribeye, red); presynaptic cytomatrix protein bassoon (anti-Sap7f, magenta); presynaptic voltage-gated Ca2+ channels (anti-CaV1.3, green); and AMPA-type glutamate receptors (anti-GluA2/3, blue). (c) Tomographic reconstruction from electron microscopy is a 3D rendering of the synapse, structurally segmented and colored (see panel label), showing the ribbon surrounded by vesicles (upper, side view) as well as membrane-tethered vesicles docked underneath the ribbon and aligned with the presynaptic membrane density (lower, top or en face view). a-b are adapted from Meyer et al., 2009; c is reproduced from Frank et al., 2010. 169x58mm (300 x 300 DPI)


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Figure 2. Ribbon synapses at the limits of confocal resolution demonstrate the need for super-resolution. (a) Confocal projection of a synaptic triple-label in the organ of Corti whole-mount preparation from an adult mouse cochlea. Three inner hair cells, outlined with nuclei (dotted lines and circles), are immunolabeled for ribbons with anti-CtBP2/Ribeye (red), voltage-gated Ca2+ channels in the presynaptic membrane with anti-CaV1.3 (green), and AMPA-type ionotropic glutamate receptors in the postsynaptic membrane with anti-GluA2 (blue). Five synapses from the bottom of one inner hair cell (IHC) are enlarged below, showing the merged image (red box) and the separate channels in gray scale. Each synapse has a different 3D orientation relative to the XY plane. One spiral ganglion neuron (SGN) is drawn contacting the presynaptic IHC via its postsynaptic bouton. The resolving capability of the microscope is approximately 220 nm in XY and 700 nm in Z. (b) The appearance of the synapse depends upon its orientation relative to the image plane. The pre- and post-synaptic elements may be separated along the vertical (Y) axis as shown in side view or cross section. (b’) Or, the pre- and postsynaptic elements may be separated along the optical (Z) axis as shown in the top view or en face section. Either way, structures are closer to each other than the PSF of the microscope. Therefore, they appear to overlap somewhat as in (c-f). (c-f) Cross section and (c’-f’) en face views of two hair cell ribbon synapses from (a). Pixels are 50 nm. (c, c’): raw data; (d, d’): oversampled and smoothed; (e, e’): deconvolved with the PSF; (f, f’): deconvolved, oversampled, and smoothed. Oversampling and smoothing are cosmetic and don’t improve resolution. Deconvolution with the Richardson-Lucy algorithm of Deconvolution Lab in ImageJ uses assumptions based upon PSF theory to change the image. Deconvolution reduces the contribution of defocused light to the image and reduces the apparent sizes of fluorescent objects. The labeled structures are close in size to diffraction-limited spots, making them too small to resolve their shapes. When viewed en face, the membrane-delimited clusters of Ca2+ channels and glutamate receptors often appear more elongated and inhomogeneously distributed, suggestive of unresolved substructure. 177x102mm (300 x 300 DPI)


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Figure 3. Figure 3. Principles of super-resolution using the STED method. (a) Black circles represent five fluorescently-tagged structural elements evenly spaced at imaging coordinates along the X-axis. Excitation light is focused to a diffraction-limited spot (blue), shown in the XZ and XY planes (upper and lower) for Xaxis pixel position b. All structural elements within the blue excitation PSF are simultaneously excited (green) to emit light that is quantified and assigned to the pixel corresponding to X-axis position b. In confocal microscopy (left), three structural elements become bright at each X-axis coordinate a-c, while two of the elements remain dark. In STED microscopy (right) the same excitation light (blue) is followed by the STED laser beam (red) that is focused into the shape of a donut or bagel, having a hole of zero intensity in the center. Where it overlaps the space of excitation (purple), the STED beam prevents excited fluorophores from fluorescing, thereby reducing the size of the PSF to the non-overlapping space in the center of the field of view. In this example, one structural element becomes bright in each pixel. Note that the green circles indicate the locations of emitting fluorophores accounted at X-axis position b, not the emitted light; the PSF for emitted light is no better than the PSF for excitation. (b) The structural element at position b has been removed. In confocal, two elements become bright in each pixel, but they cannot be discerned from each other and the structural gap is not resolved. In STED, the size of the PSF (blue space of non-overlap) is similar to the size of each structural unit. The gap is resolved at X-axis position b because all fluorophores fall outside the effective PSF. (c) To resolve labeled structures, the microscope must be able to detect the regions in space where fluorophores are absent. On right, the hypothetical structural elements and thus the gap are smaller. (d) The PSF of the STED microscope can be tuned, depending on the intensity of the STED laser. On left, the same STED beam as depicted in panels (a) and (b) results in a PSF that is unable to detect the gap in the array of smaller structural elements. On right, a more intense STED laser results in a


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smaller region of non-overlap (blue), enabling resolution of the gap. 119x119mm (300 x 300 DPI)


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Figure 4. Voltage-gated Ca2+ channel (CaV1.3) clusters in confocal and STED microscopy. (a) In confocal, clusters of Ca2+channels appear as elongated spots with minimum dimensions limited by the diffraction of light. (b) The same field of view in STED mode. (a1-a6 and b1-b6) 600 x 600 nm zoom views of Ca2+ channel clusters in (a) and (b) are a direct comparison of the same clusters viewed in conventional confocal microscopy versus STED mode. The effective PSF of the STED microscope was approximately 80 nm, therefore the minimum dimensions appear to be about 80 nm. The fluorophore used for STED was Atto647N-conjugated to a secondary antibody (Atto-Tec). (c) Conventional confocal image of ribbons (red) and Ca2+ channel clusters (green). (d) Ca2+ channel clusters in STED mode (green) with ribbons overlaid in conventional confocal mode (red). (e) 40 nm fluorescent beads measured in confocal. (f) The same field of view in STED mode. The beads approximate point sources of light, illustrating the difference in resolution between conventional confocal and STED mode. In STED, the diffraction-limited PSF is not detected so the beads appear less blurry. Their images do not overlap because the spatial frequency of the optical transfer function is increased due to the smaller effective PSF. 177x178mm (300 x 300 DPI)


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Figure 5. Two-color STED microscopy demonstrates morphological coupling of bassoon with voltage-gated Ca2+ channels. The same STED laser was pulsed through a vortex phase plate to deplete two fluorophores activated sequentially at different wavelengths in an interleaved pattern (Wong et al., 2014). Voltage-gated Ca2+ channels (CaV1.3, green) control exocytosis of synaptic vesicles. The cytomatrix protein bassoon (magenta) handles vesicles and organizes the presynaptic active zone. Each subpanel (740 x 740 nm) contains one synapse. The resolution of the microscope was approximately 50 nm in XY and the pixels are 20 nm. The displayed images are not oversampled but they are smoothed with a 2D Gaussian in ImageJ (sigma = 1). The resolution in Z is approximately 700 nm and each image is a maximum intensity projection of optical sections containing the entire synapse volume. CaV1.3 forms an elongated stipe-like shape most noticeable en face (upper and left panels). Some synapses have two stripes or more complex shapes (lower panels). Bassoon labeling is relatively less continuous and tends to parallel or surround but not directly overlap with CaV1.3. Fluorophores: Atto590- and Atto647N-conjugated secondary antibodies (Atto-Tec). 177x177mm (300 x 300 DPI)


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Figure 6. Morphology of voltage-gated Ca2+ channels at inner hair cell active zones. (a) In immature inner hair cells, before the onset of hearing, voltage-gated Ca2+ channels (CaV1.3, STED) appear only as small round spots as shown at postnatal day 10 (p10). By one week after the developmental onset of hearing, CaV1.3 clusters have matured into elongated stripes as shown at p19. The developmental change of CaV1.3 cluster morphology is correlated with a maturation of the coupling between Ca2+ influx and exocytosis. Each subpanel is 740 x 740 nm. Adapted from Wong et al., 2014. (b) CaV1.3 (green, STED) appears only as small round spots in bassoon-disrupted mature inner hair cells. The failure to form elongated clusters is a form of synaptopathy associated with functional hearing deficits in bassoon-disrupted mice. Upper panels, bassoon-deficient; lower panels, wild-type. Ribbons (red) in confocal mode. Adapted from Frank et al., 2010. 155x93mm (300 x 300 DPI)


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Figure 7. AMPA receptors have a ring-like morphology at inner hair cell – spiral ganglion neuron synapses. (a) Glutamate receptors on postsynaptic spiral ganglion neurons are labelled with anti-GluA2/3 (green, STED) and presynaptic ribbons are labelled with anti-CtBP2/Ribeye (red, confocal). The patch of glutamate receptors in each postsynaptic density has a ring-like appearance when viewed en face. The inside of the ring roughly approximates the perimeter of the shadow of the ribbon projected onto the synaptic membrane. A lower density of receptors is apparent directly underneath the ribbon. (b) Fluorescence line scans from STED en face views of glutamate receptor patches from the apical (blue) or midcochlear (red) regions; individual traces and averages overlaid in bold. Line profiles across AMPA receptor arrays, aligned at the center, showed that the density in the center of the array dips to approximately one-half of the sidepeaks. For comparison, gray bars represent counts of immunogold particles as a function of distance from synapse center in rat inner hair cells (Matsubara et al., 1996). a.u., arbitrary units. Adapted from Meyer et al., 2009. 68x93mm (300 x 300 DPI)


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Graphical abstract text: Unlimited by diffraction, 2-color STED microscopy at inner hair cell afferent synapses in the cochlea resolves details of molecular organization within synapses (scale bar 200 nm). The vesicle-handling protein bassoon (magenta) and the voltage-gated Ca2+ channels controlling vesicle release (green; CaV1.3) are intimately aligned at each synapse.


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Unlimited by diffraction, 2-color STED microscopy at inner hair cell afferent synapses in the cochlea resolves details of molecular organization within synapses (scale bar 200 nm). The vesicle-handling protein bassoon (magenta) and the voltage-gated Ca2+ channels controlling vesicle release (green; CaV1.3) are intimately aligned at each synapse. 141x141mm (72 x 72 DPI)


Relating structure and function of inner hair cell ribbon synapses.

Immunoelectron microscopy of the human inner ear.

Ribbon synapses in zebrafish hair cells.

Spectrin Organization at Synapses.

Development of the inner ear.

Superresolving dendritic spine morphology with STED microscopy under holographic photostimulation.

Inner Ear Inflammatory Pseudotumor With Middle Ear Cholesteatoma.

Toxic inner ear lesion following otitis media with effusion: a comparative CT-study regarding the morphology of the inner ear.

Simultaneous dual-color 3D STED microscopy.

Two-Photon Excitation STED Microscopy with Time-Gated Detection.

Gated STED microscopy with time-gated single-photon avalanche diode.

[Formation of the inner ear lymphs. Permeability of inner ear membranes (author's transl)].

The windows of the inner ear.

Resolving Intra- and Inter-Molecular Structure with Non-Contact Atomic Force Microscopy.

Cochlin in autoimmune inner ear disease: is the search for an inner ear autoantigen over?

Metal casts showing the three-dimensional structure of the human inner ear were converted into jewelry.

STED microscopy - towards broadened use and scope of applications.

Tricellular Tight Junctions in the Inner Ear.

Autophagy in the Vertebrate Inner Ear.

Diving-related inner ear injuries.

Sp8 regulates inner ear development.

Diving injuries to the inner ear.

Pressure-regulating mechanisms in the inner ear.

Normative inner ear volumetric measurements.