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Journal of Cell Science 05/2011; 124(Pt 10):1607-11. · 6.11 Impact Factor
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ABSTRACT: The recent advances in optical microscopy enable the simultaneous visualization of thousands of structural and signaling molecules as they dynamically rearrange within living cells. Super-resolution microscopy offers an unprecedented opportunity to define the molecular mechanisms of nanosensing through direct observation of protein movement. This technology provides a real-time readout of how genetically targeted molecular perturbations affect protein interactions. As we strive to meet the challenge offered by the opportunity to ask questions about the mechanism of cell that we never thought we could answer, we need to be aware that the new technologies are still evolving. The current limitations of each technique need to be considered when matching them to specific biological questions. In this review, we briefly describe the principles of super-resolution optical microscopy and focus on comparing the characteristics of each technique that are important for their use in studying nanosensing in the cellular microenvironment.
Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology 03/2011; 3(3):247-55. · 5.19 Impact Factor
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ABSTRACT: A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ∼0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.
Nature Methods 03/2011; 8(5):417-23. · 19.28 Impact Factor
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ABSTRACT: Spinules found in brain consist of small invaginations of plasma membranes which enclose membrane evaginations from adjacent cells. Here, we focus on the dynamic properties of the most common type, synaptic spinules, which reside in synaptic terminals. In order to test whether depolarization triggers synaptic spinule formation, hippocampal slice cultures (7-day-old rats, 10-14 days in culture) were exposed to high K+ for 0.5-5 min, and examined by electron microscopy. Virtually no synaptic spinules were found in control slices representing a basal state, but numerous spinules appeared at both excitatory and inhibitory synapses after treatment with high K+. Spinule formation peaked with approximately 1 min treatment at 37 degrees C, decreased with prolonged treatment, and disappeared after 1-2 min of washout in normal medium. The rate of disappearance of spinules was substantially slower at 4 degrees C. N-methyl-D-aspartic acid (NMDA) treatment also induced synaptic spinule formation, but to a lesser extent than high K+ depolarization. In acute brain slices prepared from adult mice, synaptic spinules were abundant immediately after dissection at 4 degrees C, extremely rare in slices allowed to recover at 28 degrees C, but frequent after high K(+) depolarization. High pressure freezing of acute brain slices followed by freeze-substitution demonstrated that synaptic spinules are not induced by chemical fixation. These results indicate that spinules are absent in synapses at low levels of activity, but form and disappear quickly during sustained synaptic activity. The rapid turnover of synaptic spinules may represent an aspect of membrane retrieval during synaptic activity.
Neuroscience 03/2009; 160(1):42-50. · 3.38 Impact Factor
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Gleb Shtengel, James A Galbraith,
Catherine G Galbraith,
Jennifer Lippincott-Schwartz,
Jennifer M Gillette,
Suliana Manley,
Rachid Sougrat,
Clare M Waterman,
Pakorn Kanchanawong,
Michael W Davidson,
Richard D Fetter,
Harald F Hess
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ABSTRACT: Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.
Proceedings of the National Academy of Sciences 03/2009; 106(9):3125-30. · 9.68 Impact Factor
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ABSTRACT: We demonstrate live-cell super-resolution imaging using photoactivated localization microscopy (PALM). The use of photon-tolerant cell lines in combination with the high resolution and molecular sensitivity of PALM permitted us to investigate the nanoscale dynamics within individual adhesion complexes (ACs) in living cells under physiological conditions for as long as 25 min, with half of the time spent collecting the PALM images at spatial resolutions down to approximately 60 nm and frame rates as short as 25 s. We visualized the formation of ACs and measured the fractional gain and loss of individual paxillin molecules as each AC evolved. By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.
Nature Methods 06/2008; 5(5):417-23. · 19.28 Impact Factor
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ABSTRACT: Much is known about the composition and function of the postsynaptic density (PSD), but less is known about its molecular organization. We use EM tomography to delineate the organization of PSDs at glutamatergic synapses in rat hippocampal cultures. The core of the PSD is dominated by vertically oriented filaments, and ImmunoGold labeling shows that PSD-95 is a component of these filaments. Vertical filaments contact two types of transmembrane structures whose sizes and positions match those of glutamate receptors and intermesh with two types of horizontally oriented filaments lying 10-20 nm from the postsynaptic membrane. The longer horizontal filaments link adjacent NMDAR-type structures, whereas the smaller filaments link both NMDA- and AMPAR-type structures. The orthogonal, interlinked scaffold of filaments at the core of the PSD provides a structural basis for understanding dynamic aspects of postsynaptic function.
Proceedings of the National Academy of Sciences 04/2008; 105(11):4453-8. · 9.68 Impact Factor
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ABSTRACT: Accurate determination of the relative positions of proteins within localized regions of the cell is essential for understanding their biological function. Although fluorescent fusion proteins are targeted with molecular precision, the position of these genetically expressed reporters is usually known only to the resolution of conventional optics ( approximately 200 nm). Here, we report the use of two-color photoactivated localization microscopy (PALM) to determine the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resolution in whole, fixed cells of approximately 20-30 nm at acquisition times of 5-30 min. We apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, we find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resolution, and speed of the technique all suggest its potential to directly visualize molecular interactions within cellular structures at the nanometer scale.
Proceedings of the National Academy of Sciences 01/2008; 104(51):20308-13. · 9.68 Impact Factor
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ABSTRACT: Migrating cells extend protrusions, probing the surrounding matrix in search of permissive sites to form adhesions. We found that actin fibers polymerizing along the leading edge directed local protrusions and drove synchronous sideways movement of beta1 integrin adhesion receptors. These movements lead to the clustering and positioning of conformationally activated, but unligated, beta1 integrins along the leading edge of fibroblast lamellae and growth cone filopodia. Thus, rapid actin-based movement of primed integrins along the leading edge suggests a "sticky fingers" mechanism to probe for new adhesion sites and to direct migration.
Science 03/2007; 315(5814):992-5. · 31.20 Impact Factor
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ABSTRACT: We compared the distribution of three scaffolding proteins, all belonging to a family of membrane-associated guanylate kinases, thought to have key roles in the organization of the postsynaptic density (PSD). Isolated PSDs readily adhered to treated glass coverslips where they were labeled with immunogold and rotary shadowed for analysis by EM. The distribution of proteins within individual PSDs were measured by counting and mapping individual immunogold particles. PSD-95, as previously described, is distributed evenly throughout the PSD. We find here that PSD-93 has a nearly identical distribution suggesting that PSD-95 and PSD-93 could perform similar roles. SAP97, in contrast, is concentrated near edges of cleft sides of the PSDs, and in small clumps on their cytoplasmic sides. The homogenous distribution of PSD-95 and PSD-93 throughout the PSD is consistent with their being part of a backbone that stabilizes their various binding partners within the PSD. The distribution of SAP97 confirms that this protein is actually an integral component of the PSD, and suggests that it may have a role in inserting or stabilizing its main binding partner, Glu-R1, at the edge of the PSD.
Brain Cell Biology 01/2007; 35(4-6):239-50. · 3.25 Impact Factor
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ABSTRACT: Controlled damage by light energy has been a valuable tool in studies of cell function. Here, we show that the Ti:Sapphire laser in a multiphoton microscope can be used to cause localized damage within unlabeled cells or tissues at greater depths than previously possible. We show that the damage is due to a multiphoton process and made wounds as small as 1 microm in diameter 20 microm from the surface. A characteristic fluorescent scar allows monitoring of the damage and identifies the wound site in later observations. We were able to lesion a single axon within a bundle of nerves, locally interrupt organelle transport within one axon, cut dendrites in a zebrafish embryo, ablate a mitotic pole in a sea urchin egg, and wound the plasma membrane and nuclear envelope in starfish oocytes. The starfish nucleus collapsed approximately 1 h after wounding, indicating that loss of compartmentation barrier makes the structure unstable; surprisingly, the oocyte still completed meiotic divisions when exposed to maturation hormone, indicating that the compartmentalization and translocation of cdk1 and its regulators is not required for this process. Multiphoton excitation provides a new means for producing controlled damage deep within tissues or living organisms.
Molecular Biology of the Cell 06/2003; 14(5):1808-17. · 4.94 Impact Factor
