Development in the STORM

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: .
Developmental Cell (Impact Factor: 9.71). 12/2012; 23(6):1103-10. DOI: 10.1016/j.devcel.2012.10.003
Source: PubMed


The recent invention of superresolution microscopy has brought up much excitement in the biological research community. Here, we focus on stochastic optical reconstruction microscopy/photoactivated localization microscopy (STORM/PALM) to discuss the challenges in applying superresolution microscopy to the study of developmental biology, including tissue imaging, sample preparation artifacts, and image interpretation. We also summarize new opportunities that superresolution microscopy could bring to the field of developmental biology.

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    • "The first involves patterned light illumination, allowing superresolution acquisitions by two fundamentally different methods, stimulated emission depletion (STED; Hell, 2007) and structured illumination microscopy (SIM; Gustafsson, 2000). The second interrogates the precision of fluorophore localization and includes stochastic optical reconstruction microscopy (STORM; Kamiyama and Huang, 2012) and photoactivation localization microscopy (PALM; Sengupta et al., 2012). The above regimes differ in translational and axial resolution, and their temporal efficiency depends on the size of the imaged area. "
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    ABSTRACT: Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for optimal documentation of Arabidopsis hypocotyl epidermal cells, dynamic cortical microtubules labeled with GFP-MBD and GFP-TUA6 markers were comparatively recorded in wild type Arabidopsis plants and in mitogen activated protein kinase mutant mpk4 possessing later microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased abundance and reduced phosphorylation of microtubule-associated protein MAP65-1, thus providing a very useful genetic tool to record intrabundle microtubule dynamics at subdiffraction level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved microtubule branching and release, and finally allowed quantification of individual microtubules within cortical bundles. Time-lapsed SIM imaging allowed visualizing subdiffraction, short-lived excursions of the microtubule plus end and dynamic instability behavior of both ends during free, intrabundle or microtubule-templated microtubule growth and shrinkage. Finally, short, rigid and non-dynamic microtubule bundles in the mpk4 mutant were observed to glide along the parent microtubule in a tip-wise manner. Conclusively, this study demonstrates the potential of SIM for superresolution time-lapsed imaging of plant cells showing unprecedented details accompanying microtubule dynamic organization.
    Plant physiology 03/2014; 165(1). DOI:10.1104/pp.114.238477 · 6.84 Impact Factor
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    • "These clusters appear at a size that is in the order of the diffraction limit of light microscopy ($200 nm in the imaging plane), which restricts the characterization of size and protein numbers. In order to overcome the resolution limit, we use here single-molecule localization-based super-resolution microscopy, which typically achieves a lateral resolution of $20–30 nm (Kamiyama and Huang, 2012). Individual protein clusters which might overlap in a conventional microscopy image can thereby be discerned (van de Linde et al., 2008). "
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    ABSTRACT: Clustering of arrestins upon G protein-coupled receptor stimulation is a phenomenon that is well-known but difficult to describe quantitatively due to the size of the clusters close to the diffraction limit of visible light. We introduce a general method to quantitatively investigate the clustering of arrestin following stimulation of the C-C chemokine receptor 5 (CCR5) using single-molecule super-resolution imaging and coordinate and image-based cluster analysis. We investigated the effect of potent anti-HIV ligands of CCR5 with different pharmacological profiles on arrestin2 cluster formation and found that only the ligands capable of inducing CCR5 internalization induced arrestin2 recruitment and clustering. We further demonstrate that the fraction of arrestin2 molecules found in clusters larger than 100 nm correlates with the magnitude of ligand-induced CCR5 internalization, but not with G protein activation, indicating that recruitment of arrestin2 to CCR5 is independent of G protein activation. Pre-treatment of the cells with the drug cytochalasin D, which blocks actin polymerization, led to the formation of larger clusters, whereas the inhibitor of microtubule polymerization nocodazole had little effect on arrestin2 recruitment, suggesting an active role of actin in the organization and dynamics of these aggregates.
    Journal of Structural Biology 09/2013; 184(2). DOI:10.1016/j.jsb.2013.09.019 · 3.23 Impact Factor
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    ABSTRACT: Over the past decade, developmental neuroscience has been transformed by the widespread application of confocal and two-photon fluorescence microscopy. Even greater progress is imminent, as recent innovations in microscopy now enable imaging with increased depth, speed, and spatial resolution; reduced phototoxicity; and in some cases without external fluorescent probes. We discuss these new techniques and emphasize their dramatic impact on neurobiology, including the ability to image neurons at depths exceeding 1mm, to observe neurodevelopment noninvasively throughout embryogenesis, and to visualize neuronal processes or structures that were previously too small or too difficult to target with conventional microscopy.
    Current opinion in neurobiology 07/2013; 23(6). DOI:10.1016/j.conb.2013.06.008 · 6.63 Impact Factor
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