Correlative super-resolution fluorescence and metal-replica transmission electron microscopy

Sochacki, Kem A.; Shtengel, Gleb; Engelenburg, Schuyler B. van; Hess, Harald F.; Taraska, Justin W.

doi:10.1038/nmeth.2816

Cited by 125 publications

(129 citation statements)

References 23 publications

(41 reference statements)

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“…In combination with correlative focused ion beam scanning electron microscopy (FIB-SEM), dSTORM can potentially be used also for 3D quantitative super-resolution imaging. Alternatively, the sample can be imaged with two-dimensional tiling and at multiple angles to create 3D tomograms in transmission electron microscopy (TEM) (Sochacki et al, 2014) …”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, super-resolution fluorescence imaging enables higher labeling efficiencies than immunogold EM using fluorophore-tagged antibodies, which facilitates structure determination by localization microscopy. Thus, localization microscopy and EM are complementary methods that can be combined to determine molecular positions in the context of the cellular ultrastructure provided by EM with nanometer resolution (Betzig et al, 2006;Watanabe et al, 2011;Kopek et al, 2012;Nanguneri et al, 2012;Suleiman et al, 2013;Sochacki et al, 2014;Perkovic et al, 2014). In order to perform correlative localization and electron microscopy, several methodological requirements have to be fulfilled.…”

Section: Introductionmentioning

confidence: 99%

“…Second, protein positions determined by localization microscopy have to be located within the EM image across large areas with a precision in the nanometer range. This demand is impeded by a lack of suitable alignment markers that are stationary at the nanoscale range and exhibit good contrast in the two imaging modes (Watanabe et al, 2011;Kopek et al, 2012;Sochacki et al, 2014;Perkovic et al, 2014). Even when such types of markers are available, structural deformations occurring between the two imaging modes can aggravate the overlay of the two images with molecular precision.…”

Section: Introductionmentioning

confidence: 99%

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Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution

Löschberger

Franke

Krohne

et al. 2014

Journal of Cell Science

113

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Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of ,20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution

Löschberger

Franke

Krohne

et al. 2014

Journal of Cell Science

113

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“…The result was disruption of the cell plasma membrane that was not adhered to the glass, that is the dorsal surface, and removal of most of the cells contents, leaving only the ventral plasma membrane as a sheet attached to the coverslip. A significant electron microscopy literature using unroofed cells indicates that they are free from most intracellular membranes and other cytosolic constituents and provide access to the cytoplasmic surface of the plasma membrane (Heuser, 2000;Sochacki et al, 2014). An example using HEK293T/17 cells is shown in Fig.…”

Section: Epifluorescent Patch Imagingmentioning

confidence: 99%

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

Gordon¹,

Senning²,

Aman³

et al. 2016

J Cell Biol

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The plasma membrane is a major site of signal detection, transduction, and propagation in cells. Understanding cell signaling dynamics at the plasma membrane requires approaches that can detect changes in membrane architecture over molecular distances (10-100 Å) and biological time scales (milliseconds to seconds). Methods that provide access to dynamics at the intracellular surface of the plasma membrane would be especially powerful. Fluorescence resonance energy transfer (FRET) was first applied to studies of membrane dynamics by Keller et al. (1977), who recognized that fluorescent probes incorporated into membranes could be used as an indicator of mixing of the two membranes during membrane fusion. FRET occurs when the emission spectrum of a donor fluorophore overlaps with the absorption spectrum of an acceptor, and the donor and acceptor are in close proximity (Lakowicz, 2006;Taraska and Zagotta, 2010). FRET is steeply distance dependent, and each donor-acceptor pair has a characteristic distance at which the FRET efficiency is 50% (R 0 ), where the amount of FRET is optimally sensitive to changes in distance. The R 0 value for fluorescein and rhodamine, for example, is 60 Å (Delgadillo and Parkhurst, 2010), making them an ideal FRET pair for quantifying membrane fusion (Keller et al., 1977).Transition metal ion FRET (tmFRET) is a method to probe the shorter distances typical of the conformational landscape of proteins (Taraska and Zagotta, 2010). tmFRET is a form of FRET that uses a fluorophore as a Correspondence to Sharona E. Gordon: s e g @ u w . e d u ; or William N. Zagotta: z a g o t t a @ u w . e d u Abbreviations used in this paper: FRET, fluorescence resonance energy transfer; PCF, patch-clamp fluorometry; TIRF, total internal reflection fluorescence; tmFRET, transition metal ion FRET.donor and a nonfluorescent transition metal ion as an acceptor (Latt et al., 1970(Latt et al., , 1972Horrocks et al., 1975;Richmond et al., 2000;Sandtner et al., 2007; Taraska et al., 2009a,b;Yu et al., 2013). Transition metal ions such as Ni 2+ , Co 2+ , and Cu 2+ have broad absorption spectra that overlap the emission spectra of visible light fluorophores (see Miessler and Tarr [1999] pages 361-380 for a discussion of electronic spectra of coordination compounds). Transition metals can therefore accept energy transfer from a nearby donor fluorophore and quench the donor's fluorescence. The degree of quenching is a direct measure of the FRET efficiency (Lakowicz, 2006;Taraska et al., 2009a). The FRET efficiency is steeply dependent on distance between the donor and acceptor, allowing tmFRET to serve as a molecular ruler for distances in the membrane. The main advantages of tmFRET over classical FRET methods are (a) R 0 is very short (10-20 Å), allowing short-range interactions to be studied; (b) the transition metals have multiple transition dipoles, reducing the orientation dependence of FRET (Horrocks et al., 1975); (c) the metals can be reversibly bound to native and introduced binding sites; and (d) different...

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“…These proteins are well biochemically characterized, but there appears to be a large amount of redundancy which has made it difficult to parse out their specific roles in endocytosis. The spatial organization of clathrin associated proteins within single clathrin structures is not well studied and may give important structural insight into how this process is regulated.Here, we develop a correlative method that combines 3D interferometric photoactivated localization fluorescence microscopy (iPALM) and platinum replica electron microscopy (PREM) to localize clathrin associated proteins on the topography of mammalian cell membranes [2]. Platinum replicas of mammalian cell cortices viewed by transmission electron microscopy have been used for decades to survey the 3D shape of single clathrin structures across the membrane [3] (Figure 1).…”

mentioning

confidence: 99%

Correlative iPALM and Platinum Replica Electron Tomography to Highlight Single Molecules on Clathrin Endocytic Structures in 3D

et al. 2015

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Clathrin mediated endocytosis is a ubiquitous process used by all eukaryotic cells to internalize material. This is an integral process for neural and endocrine signaling as well as cellular homeostasis. While severe mutations to this process are lethal, minor mutations are associated with several human maladies including cancer and Alzheimer's disease [1]. There are well over 30 known proteins regulating the entire endocytic process including initiation, membrane curvature, pit size, cargo recruitment, and fission. These proteins are well biochemically characterized, but there appears to be a large amount of redundancy which has made it difficult to parse out their specific roles in endocytosis. The spatial organization of clathrin associated proteins within single clathrin structures is not well studied and may give important structural insight into how this process is regulated.Here, we develop a correlative method that combines 3D interferometric photoactivated localization fluorescence microscopy (iPALM) and platinum replica electron microscopy (PREM) to localize clathrin associated proteins on the topography of mammalian cell membranes [2]. Platinum replicas of mammalian cell cortices viewed by transmission electron microscopy have been used for decades to survey the 3D shape of single clathrin structures across the membrane [3] (Figure 1). However, identifying proteins of interest in these images with immunogold requires large metal particles (15 nm) that not only label sparsely but also obstruct the image beneath. iPALM localizes fluorescently labeled proteins to a precision of better than 20 nm in the membrane plane (XY) and 10 nm in the axial (Z) plane [4]. Our iPALM/PREM correlation method allows us to correlate clathrin fluorescence with clathrin viewed with EM, to within 20 nm across a 20 µm cell membrane. We then use this method to find the position of the clathrin adapter protein, epsin 1, with respect to the shape of clathrin structures (Figure 2). We unexpectedly find that epsin 1 is located along the edge of the clathrin structures in XY but along entire height of clathrin pits. While there are several models of how epsin functions in mammalian cells, this localization is most consistent with the recent model that epsin coordinates actin at clathrin sites.As we continue to use this method to study other endocytic proteins, we are finding that different isoforms are localized very differently within cells and that protein localization can be very different in different mammalian cell lines. Additionally, this method is poised to answer many questions about other important processes on the plasma membrane like clathrin-independent endocytosis, exocytosis, cell adhesion, and cell motility.

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Correlative super-resolution fluorescence and metal-replica transmission electron microscopy

Cited by 125 publications

References 23 publications

Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution

Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

Correlative iPALM and Platinum Replica Electron Tomography to Highlight Single Molecules on Clathrin Endocytic Structures in 3D

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