1. Super-Resolution Microscopy: From Single Molecules to Supramolecular Assemblies 
Andrew M. Sydor, Kirk J. Czymmek, Elias M. Puchner, Vito Mennella 
Trends in Cell Biology 
Volume 25, Issue 12, Pages (December 2015)
DOI: /j.tcb
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2. Figure 1
Counting Single Molecules and Oligomers Using Super-Resolution Microscopy. (A) One method to count molecules is through photobleaching steps (top). In this methodology, the proteins under investigation are labeled with genetically encoded fluorescent protein tags, which are imaged until they photobleach. The number of resulting stepwise decreases in the fluorescence intensity reveals the stoichiometry of the complex. Alternatively, single-molecule counting can be conducted by sequentially activating and bleaching the fluorescent proteins (bottom). This cycle is repeated until all fluorescent proteins have been activated and imaged such that the number of observed fluorescent peaks reveals the number of molecules in the sample. Abbreviation: GlyR, glycine receptor. Adapted from [22]. (B) Nan et al. [27] observed oligomers of CRAF (Raf-1 proto-oncogene), a serine/threonine kinase involved in regulating cell growth, via quantitative PALM. The authors labeled CRAF with the fluorescent protein PAmCherry1. When imaged using total internal reflection fluorescence (TIRF) microscopy, it is difficult to determine the oligomeric state of the protein. However, the application of PALM greatly improves the resolution and the individual signals from the labeled CRAF can be observed. Clustering algorithms determined that CRAF was mostly dimeric in state but, upon fusion of CRAF to the K-RAS (Kirsten rat sarcoma viral oncogene homolog) 4B motif, higher-order oligomers were observed (scale bars: left box, 5μm; middle and right box, 200nm). Adapted from [27].
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3. Figure 2
Elucidation of the Structure of Protein Assemblies by Super-Resolution Microscopy (SRM). (A) (i) The nuclear pore complex (NPC) is composed of several Y-shaped nucleoporin (Nup) protein complexes which form a ring-like structure responsible for transport in and out of the nucleus. The relative orientation of these protein complexes was unknown before the application of SRM. To examine the spatial orientation of this complex, individual protein components were labeled with GFP (labeling sites are indicated by the colored spots). (ii) The authors imaged numerous NPCs and averaged the images after rotation and alignment to obtain an averaged image for each Nup protein, allowing the determination of the radial distance from the center of the pore. (iii) The distribution of the radii for all of the Nup proteins exhibited a unique pattern which, upon super-position on previously determined electron microscopy (EM) images of the NPC, could only be explained by a head-to-tail orientation of the Nup complex. Adapted from [56]. (B) (i) To map the synapse by SRM, Dani et al. conducted three-color STORM. In this set-up, they used a common reporter dye, Alexa647, and three separate activator dyes, Cy3, Cy2, and Alexa405, which allowed the probes to be distinguished by the wavelength of the activation light. As reference markers, one pre-synaptic and one post-synaptic protein, Basson and Homer1, respectively, were labeled with the Cy3–Alexa647 (colored red in the above image) and Alexa405–Alexa647 (green) probe pairs. The relative position of a third protein labeled with Cy2–Alexa647 (blue) was then determined by conducting three-color stochastic optical reconstruction microscopy (STORM). (ii) By repeating this process for numerous synaptic proteins, a map of their relative positions to Basson and Homer1 was created. Adapted from [73]. (C) (i) Top: conventional fluorescence image of a neuron (actin, green; microtubule associated protein MAP2, magenta). Bottom: STORM image of actin in the area corresponding to the box in yellow from the top image. Actin forms periodic ring structures along the length of the axon. (ii) Two-color STORM of axons (immunostaining indicated in the figure). Actin, adducin, βII-spectrin, βIV-spectrin, and sodium channels (NaV) are distributed along the axon structure in a periodic manner, leading the authors to propose a model (iii) for the cytoskeleton in axons wherein actin filaments, capped by adducin, form ring-like structures with spectrin tetramers connecting adjacent actin/adducin rings. Adapted from [76].
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4. Figure 3
Applications of Super-Resolution Microscopy to Supramolecular Assemblies. (A) Huang et al. conducted two-color stochastic optical reconstruction microscopy (STORM) to study the spatial organization of mitochondria (magenta) and microtubules (green). Previous conventional fluorescence images (left) demonstrated that the two structures co-align, but the distinct interaction sites and degree of interaction has not been clear until a STORM image (right) was obtained. Scale bars, 3μm. Adapted from [110]. (B) The structure of the centrosome pericentriolar material (PCM) was investigated by generating a map of the various protein components by 3D structured illumination microscopy (3DSIM). (i) The PCM proteins were labeled by antibody staining and subsequently imaged. Numerous images were recorded, aligned, and averaged to obtain an initial average structure that was used as a basis for subsequent rounds of alignment and averaging until a final structure was determined, which allowed the measurement of the average radial distance of each protein from SAS6 (spindle assembly abnormal 6), a protein at the center hub of the centrosome. (ii) This methodology was applied to the different PCM proteins to generate a position map of the PCM proteins, which demonstrated that the PCM was not an amorphous structure as previously thought but had defined structural elements. (iii) To obtain higher-resolution images, the centrosome was stained with antibodies against pericentrin-like protein (PLP) and imaged using 3D STORM. The resulting side (left) and top views (right) display the pseudo ninefold symmetry of the centrosome and demonstrate the elongated architecture of PLP. The colored bar indicates distance from the coverslip. Adapted from [116]. (C) Tracking the endosome maturation trajectory by counting single molecules with PALM. (i) The FYVE domain of early endosomal antigen 1 (EEA1) fused to mEos2 serves as a probe to detect accessible phosphatidylinositol 3-phosphate (PI3P) binding sites on endocytic vesicles and endosomes and to resolve their size. (ii) Super-resolution snapshots exhibit broad variability of endocytic vesicles in size and number of accessible PI3P binding sites. (iii) Colocalization with endocytic/endosomal landmark proteins reveals distinct stages of PI3P production and vesicle fusion. (iv) PI3P is produced on newly formed vesicles followed by fusion and maturation to endosomes. Adapted from [34].
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5. Figure I Probes for SRM. Abbreviations: Em, emission; Ex, excitation.
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6. Figure I Multicolor SRM.
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