Dendritic spines are actin-rich protrusions on neurons that are critical for neurotransmission, as they are sites for the majority of excitatory postsynapses ^{1– 3} . Abnormal spines are found in a wide range of neuropsychiatric, neurodegenerative, and neurodevelopmental disorders ^{4– 6} , further highlighting the importance of these structures in cognition. Spines typically consist of a thin neck and a bulbous head, which is 0.5 to 1 μm in diameter. Therefore, analyzing spine and synapse organization in detail was previously difficult owing to their small sizes, which are near the diffraction limit for conventional light microscopy ^{7, 8} . The advent of super resolution imaging has revolutionized the study of spines and synapses. Whereas conventional light microscopy has an effective limit of resolution at ~200 nm due to the diffraction of light, super resolution fluorescence microscopy can bypass this limit, increasing the resolving power to tens of nanometers. In terms of resolving power, super resolution microscopy is limited by the brightness and photostability of the probes used ^{9, 10} ; the principles underlying super resolution microscopy have been discussed in detail in previous reviews ^{7, 9, 11} . This enhanced resolving power enables more detailed examination of protein mobility in living cells. The live-cell application of super resolution microscopy is what currently sets it apart from electron microscopy, which can achieve a somewhat higher resolution (picometer) but is not compatible with live-cell imaging and requires stringent fixation conditions ⁹ . Because super resolution microscopy is compatible with live-cell imaging, dynamic changes in spine and synapse morphology can be readily observed ^{12– 14} . Particularly exciting is the possibility of imaging the very early stages of spine formation and subsequent maturation, which has not been possible to study with conventional light microscopy. Additionally, the enhanced resolving power of super resolution microscopy permits a more precise analysis of protein localization and the organization of protein nanodomains within individual spines and synapses ^{15, 16} . This type of microscopy will be critical for detailing the organization and dynamics of the hundreds of proteins that are packed together in submicron structures, such as dendritic spines. Consequently, super resolution microscopy will enhance our knowledge of dendritic spine and synapse architecture to possibly reveal nanoscale abnormalities in diseased states and lend further insight into the mechanisms underlying neurodevelopmental disorders.

New probes created in the last few years, as discussed below, have made super resolution microscopy even more conducive to visualizing neurons, especially fine neuronal structures (i.e. dendritic spines), because these probes are optimized for live-cell imaging. Super resolution studies are primarily performed using small molecule fluorophores and photoactivatable and photoswitchable fluorescent proteins ^{17– 19} . Although small molecule fluorophores are less bulky, brighter, and more photostable compared to fluorescent protein tags, they can fail to bind to their intended targets and/or bind to undesired targets. However, by fusing a protein of interest directly to a fluorescent tag (i.e. green fluorescent protein [GFP]), this limitation can be overcome, but these fluorescently tagged proteins tend to be bulky and display weaker photostability and brightness than small molecule fluorophores. Over the past few years, researchers have focused on developing probes for super resolution microscopy that overcome the limitations of traditional fluorescent proteins and synthetic fluorophores. For example, quantum dots (QDs) ²⁰ , which are semiconductor nanoparticles, and nanobodies ²¹ , which are composed of the smallest fragment of an antibody that will still bind to antigens, have also been used to label proteins for super resolution microscopy. The advantage of QDs is that they are highly photostable and therefore amenable to live-cell, single-molecule fluorescence microscopy. The major shortcoming for using QDs to visualize small neuronal structures is their size. QDs have an average diameter of 15–35 nm ²² , making them difficult to utilize in spatially confined areas such as the synaptic cleft ²³ . To address this problem, Cai et al. developed small QDs (sQDs), which are about 7 nm in size and can easily label proteins in neuronal synapses ²² . As a proof of concept, Wang et al. used sQDs conjugated to a GFP nanobody to label the exogenously expressed α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluR2, which is fused to pH-sensitive GFP (pHluorin), to track the lateral diffusion of AMPARs in synapses ²⁴ . As an alternative approach to traditional fluorescent proteins, Viswanathan et al. created “spaghetti monster” fluorescent proteins (smFPs) ²⁵ that contain multiple copies of commonly used tags such as Myc, FLAG, or HA on their surface. These tags create additional antibody binding sites on smFPs, which leads to high antibody labeling density, making these probes brighter than traditional fluorescence proteins or individual antibodies and nanobodies. Probe brightness is a critical determinant of spatial resolution in single-molecule super resolution microscopy. To test the ability of smFPs to reveal submicron structures, smFPs were expressed as a filler to visualize a major class of spines in CA3 neurons, called “thorny excrescence” spines. These spines have small protrusions from their spine neck, which are difficult to label with conventional fluorescent proteins and dyes. Even at low expression levels, smFPs labeled these protrusions significantly better than enhanced GFP (EGFP) or lucifer yellow ²⁵ . Furthermore, the epitope tags on smFPs allow for strong labeling of proteins for which suitable, specific antibodies and nanobodies are not available. Finally, smFPs are also especially attractive for investigating the early stages of spine and synapse formation because their brightness makes them well suited to imaging proteins that are present at low levels.

Actin is the main cytoskeletal element in dendritic spines and underlies spine morphology and plasticity. Despite its importance, until recently, super resolution live-cell imaging of actin remodeling in spines was limited to the use of low-affinity actin probes, such as ABP-tdEosFP ²⁶ or exogenous expression of actin fused to a fluorescent protein ¹⁹ . To address this, Lukinavičius et al. developed probes for live-cell imaging of actin and tubulin using a silicon-rhodamine derivative conjugated to ligands that bind to these cytoskeletal elements ²⁷ . The high specificity, enhanced fluorescence, and low phototoxicity of these probes make them invaluable for super resolution imaging of cytoskeletal remodeling in dendritic spines.

Collectively, the creation of these new probes has made super resolution microscopy even more amenable to studying small structures in neurons, such as dendritic spines and synapses, with unprecedented detail compared to conventional light microscopy. Although these probes overcome some of the weaknesses of older probes, newer probes are still needed that have all the characteristics of an ideal probe for imaging dendritic spines, including high specificity, brightness, and photostability, as well as small size.

Although the proper development of dendritic spines and synapses is critical for normal cognitive function, their small size has limited the acquisition of detailed images of their nanoscopic substructures via conventional light microscopy. Super resolution microscopy overcomes the diffraction barrier, which allows for the imaging of these structures. While electron microscopy can achieve even higher resolution, it is limited because it cannot currently be performed in living cells. In contrast, super resolution microscopy is amenable to live-cell imaging. Moreover, super resolution microscopy will be critical for visualizing interactions between actin and actin-binding proteins during the early stages of dendritic spine formation and their subsequent maturation. The recent development of probes that are smaller, brighter, more specific, and more conducive to live-cell imaging are turning super resolution microscopy into a vital new tool to better understand dendritic spine morphology, organization, function, and plasticity. Indeed, super resolution microscopy has already revealed fascinating and important new information about both the gross anatomical structure of spines and the protein nanodomain composition as well as actin remodeling within them in both healthy tissue and in diseased states. Super resolution microscopy will be invaluable to many applications in neuroscience, but it specifically offers the potential to examine spine and synapse development at a level of detail in living cells which was previously not possible but is necessary to understand the underlying mechanisms that regulate this process.

Super resolution microscopy can now be used to address a number of intriguing questions about the development of dendritic spines. For example, when are synaptic nanodomains established during spine formation, and how do they affect filopodia and spine morphology? Do all synaptic proteins enter a forming spine simultaneously, or are they recruited sequentially? Which protein nanodomains assemble independently, and which domains require synaptic scaffolding proteins to assemble appropriate nanodomains? How does nanodomain composition correlate with overall spine morphology? For instance, are the properties of nanodomains in a developing filopodium the same as what is seen in mature synapses, or are there immature stages, where nanodomains show different properties in immature synapses? The answers to these and other interesting questions would lend insight into novel functions for synaptic proteins. It will be critical to not only assess the normal development of dendritic spines but also evaluate how spine formation and maturation are perturbed in neurological disorders, such as Alzheimer’s disease, schizophrenia, and FXS. Super resolution imaging has the potential to reveal the mechanisms that underlie these abnormalities and allow for the generation of new treatments for these disorders.