Visualization of molecular fluorescence point spread functions via remote excitation switching fluorescence microscopy
Identifieur interne : 000735 ( Pmc/Curation ); précédent : 000734; suivant : 000736Visualization of molecular fluorescence point spread functions via remote excitation switching fluorescence microscopy
Auteurs : Liang Su [Belgique] ; Gang Lu [Belgique] ; Bart Kenens [Belgique] ; Susana Rocha [Belgique] ; Eduard Fron [Belgique] ; Haifeng Yuan [Belgique] ; Chang Chen [Belgique] ; Pol Van Dorpe [Belgique] ; Maarten B. J. Roeffaers [Belgique] ; Hideaki Mizuno [Belgique] ; Johan Hofkens [Belgique, Danemark] ; James A. Hutchison [France, Australie] ; Hiroshi Uji-I [Belgique, Japon]Source :
- Nature Communications [ 2041-1723 ] ; 2015.
Abstract
The enhancement of molecular absorption, emission and scattering processes by coupling to surface plasmon polaritons on metallic nanoparticles is a key issue in plasmonics for applications in (bio)chemical sensing, light harvesting and photocatalysis. Nevertheless, the point spread functions for single-molecule emission near metallic nanoparticles remain difficult to characterize due to fluorophore photodegradation, background emission and scattering from the plasmonic structure. Here we overcome this problem by exciting fluorophores remotely using plasmons propagating along metallic nanowires. The experiments reveal a complex array of single-molecule fluorescence point spread functions that depend not only on nanowire dimensions but also on the position and orientation of the molecular transition dipole. This work has consequences for both single-molecule regime-sensing and super-resolution imaging involving metallic nanoparticles and opens the possibilities for fast size sorting of metallic nanoparticles, and for predicting molecular orientation and binding position on metallic nanoparticles via far-field optical imaging.
Url:
DOI: 10.1038/ncomms7287
PubMed: 25687887
PubMed Central: 4339893
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xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Kenens, Bart" sort="Kenens, Bart" uniqKey="Kenens B" first="Bart" last="Kenens">Bart Kenens</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>KU Leuven, Department of Chemistry</institution>, Celestijnenlaan 200G-F, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Rocha, Susana" sort="Rocha, Susana" uniqKey="Rocha S" first="Susana" last="Rocha">Susana Rocha</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>KU Leuven, Department of Chemistry</institution>, Celestijnenlaan 200G-F, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Fron, Eduard" sort="Fron, Eduard" uniqKey="Fron E" first="Eduard" last="Fron">Eduard Fron</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>KU Leuven, Department of Chemistry</institution>, Celestijnenlaan 200G-F, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Yuan, Haifeng" sort="Yuan, Haifeng" uniqKey="Yuan H" first="Haifeng" last="Yuan">Haifeng Yuan</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>KU Leuven, Department of Chemistry</institution>, Celestijnenlaan 200G-F, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Chen, Chang" sort="Chen, Chang" uniqKey="Chen C" first="Chang" last="Chen">Chang Chen</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>IMEC</institution>, Kapeldreef 75, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a3"><institution>KU Leuven, Department of Physics and Astronomy</institution>, Celestijnenlaan 200D, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Van Dorpe, Pol" sort="Van Dorpe, Pol" uniqKey="Van Dorpe P" first="Pol" last="Van Dorpe">Pol Van Dorpe</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>IMEC</institution>, Kapeldreef 75, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a3"><institution>KU Leuven, Department of Physics and Astronomy</institution>, Celestijnenlaan 200D, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Roeffaers, Maarten B J" sort="Roeffaers, Maarten B J" uniqKey="Roeffaers M" first="Maarten B. J." last="Roeffaers">Maarten B. J. Roeffaers</name><affiliation wicri:level="1"><nlm:aff id="a4"><institution>KU Leuven, Department of Microbial and Molecular Systems, Center for Surface Chemistry and Catalysis</institution>, Kasteelpark Arenberg 23, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Mizuno, Hideaki" sort="Mizuno, Hideaki" uniqKey="Mizuno H" first="Hideaki" last="Mizuno">Hideaki Mizuno</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>KU Leuven, Department of Chemistry</institution>, Celestijnenlaan 200G-F, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Hofkens, Johan" sort="Hofkens, Johan" uniqKey="Hofkens J" first="Johan" last="Hofkens">Johan Hofkens</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>KU Leuven, Department of Chemistry</institution>, Celestijnenlaan 200G-F, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a5"><institution>University of Copenhagen, Nano-Science Center/Department of Chemistry</institution>, Universitetsparken 5, 2100 Copenhagen,<country>Denmark</country></nlm:aff><country xml:lang="fr">Danemark</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Hutchison, James A" sort="Hutchison, James A" uniqKey="Hutchison J" first="James A." last="Hutchison">James A. Hutchison</name><affiliation wicri:level="1"><nlm:aff id="a6"><institution>ISIS & icFRC, Université de Strasbourg & CNRS UMR 7006</institution>, Strasbourg 67000,<country>France</country></nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a7"><institution>School of Chemistry and Bio21 Institute, University of Melbourne</institution>, Victoria 3010,<country>Australia</country></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Uji I, Hiroshi" sort="Uji I, Hiroshi" uniqKey="Uji I H" first="Hiroshi" last="Uji-I">Hiroshi Uji-I</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>KU Leuven, Department of Chemistry</institution>, Celestijnenlaan 200G-F, B-3001 Heverlee,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a8"><institution>PRESTO, Japan Science and Technology Agency (JST)</institution>, 4-1-8 Honcho Kawaguchi, Saitama 332-0012,<country>Japan</country></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Nature Communications</title><idno type="eISSN">2041-1723</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The enhancement of molecular absorption, emission and scattering processes by coupling to surface plasmon polaritons on metallic nanoparticles is a key issue in plasmonics for applications in (bio)chemical sensing, light harvesting and photocatalysis. Nevertheless, the point spread functions for single-molecule emission near metallic nanoparticles remain difficult to characterize due to fluorophore photodegradation, background emission and scattering from the plasmonic structure. Here we overcome this problem by exciting fluorophores remotely using plasmons propagating along metallic nanowires. The experiments reveal a complex array of single-molecule fluorescence point spread functions that depend not only on nanowire dimensions but also on the position and orientation of the molecular transition dipole. This work has consequences for both single-molecule regime-sensing and super-resolution imaging involving metallic nanoparticles and opens the possibilities for fast size sorting of metallic nanoparticles, and for predicting molecular orientation and binding position on metallic nanoparticles via far-field optical imaging.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Anker, J N" uniqKey="Anker J">J. N. Anker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Atwater, H A" uniqKey="Atwater H">H. A. Atwater</name></author><author><name sortKey="Polman, A" uniqKey="Polman A">A. Polman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Seh, Z W" uniqKey="Seh Z">Z. W. Seh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lin, H" uniqKey="Lin H">H. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Nat Commun</journal-id><journal-id journal-id-type="iso-abbrev">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type="epub">2041-1723</issn><publisher><publisher-name>Nature Pub. Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25687887</article-id><article-id pub-id-type="pmc">4339893</article-id><article-id pub-id-type="pii">ncomms7287</article-id><article-id pub-id-type="doi">10.1038/ncomms7287</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Visualization of molecular fluorescence point spread functions via remote excitation switching fluorescence microscopy</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Su</surname><given-names>Liang</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lu</surname><given-names>Gang</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kenens</surname><given-names>Bart</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Rocha</surname><given-names>Susana</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Fron</surname><given-names>Eduard</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yuan</surname><given-names>Haifeng</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Chang</given-names></name><xref ref-type="aff" rid="a2">2</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Van Dorpe</surname><given-names>Pol</given-names></name><xref ref-type="aff" rid="a2">2</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Roeffaers</surname><given-names>Maarten B. J.</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Mizuno</surname><given-names>Hideaki</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Hofkens</surname><given-names>Johan</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Hutchison</surname><given-names>James A.</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a6">6</xref><xref ref-type="aff" rid="a7">7</xref></contrib><contrib contrib-type="author"><name><surname>Uji-i</surname><given-names>Hiroshi</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a8">8</xref></contrib><aff id="a1"><label>1</label><institution>KU Leuven, Department of Chemistry</institution>, Celestijnenlaan 200G-F, B-3001 Heverlee,<country>Belgium</country></aff><aff id="a2"><label>2</label><institution>IMEC</institution>, Kapeldreef 75, B-3001 Heverlee,<country>Belgium</country></aff><aff id="a3"><label>3</label><institution>KU Leuven, Department of Physics and Astronomy</institution>, Celestijnenlaan 200D, B-3001 Heverlee,<country>Belgium</country></aff><aff id="a4"><label>4</label><institution>KU Leuven, Department of Microbial and Molecular Systems, Center for Surface Chemistry and Catalysis</institution>, Kasteelpark Arenberg 23, B-3001 Heverlee,<country>Belgium</country></aff><aff id="a5"><label>5</label><institution>University of Copenhagen, Nano-Science Center/Department of Chemistry</institution>, Universitetsparken 5, 2100 Copenhagen,<country>Denmark</country></aff><aff id="a6"><label>6</label><institution>ISIS & icFRC, Université de Strasbourg & CNRS UMR 7006</institution>, Strasbourg 67000,<country>France</country></aff><aff id="a7"><label>7</label><institution>School of Chemistry and Bio21 Institute, University of Melbourne</institution>, Victoria 3010,<country>Australia</country></aff><aff id="a8"><label>8</label><institution>PRESTO, Japan Science and Technology Agency (JST)</institution>, 4-1-8 Honcho Kawaguchi, Saitama 332-0012,<country>Japan</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>hutchison@unistra.fr</email></corresp><corresp id="c2"><label>b</label><email>hiroshi.ujii@chem.kuleuven.be</email></corresp></author-notes><pub-date pub-type="epub"><day>17</day><month>02</month><year>2015</year></pub-date><volume>6</volume><elocation-id>6287</elocation-id><history><date date-type="received"><day>05</day><month>05</month><year>2014</year></date><date date-type="accepted"><day>14</day><month>01</month><year>2015</year></date></history><permissions><copyright-statement>Copyright © 2015, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>The enhancement of molecular absorption, emission and scattering processes by coupling to surface plasmon polaritons on metallic nanoparticles is a key issue in plasmonics for applications in (bio)chemical sensing, light harvesting and photocatalysis. Nevertheless, the point spread functions for single-molecule emission near metallic nanoparticles remain difficult to characterize due to fluorophore photodegradation, background emission and scattering from the plasmonic structure. Here we overcome this problem by exciting fluorophores remotely using plasmons propagating along metallic nanowires. The experiments reveal a complex array of single-molecule fluorescence point spread functions that depend not only on nanowire dimensions but also on the position and orientation of the molecular transition dipole. This work has consequences for both single-molecule regime-sensing and super-resolution imaging involving metallic nanoparticles and opens the possibilities for fast size sorting of metallic nanoparticles, and for predicting molecular orientation and binding position on metallic nanoparticles via far-field optical imaging.</p></abstract><abstract abstract-type="web-summary"><p><inline-graphic id="i1" xlink:href="ncomms7287-i1.jpg"></inline-graphic>Plasmonic nanoparticles can dramatically enhance the optical properties of molecules but background scattering is a limiting factor. Su <italic>et al.</italic> use remote excitation by plasmons on nanowires to better access single fluorophore point spread functions for improved sensing and super-resolution imaging.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>Wide-field excitation of switching localization microscopy.</title><p>(<bold>a</bold>) Schematic illustration of wide-field excitation of the Alexa 647-labelled silver nanowire. Legends on the right denote the components for labelling the Ag nanowire (details in Methods). (<bold>b</bold>) The fluorescence image of an Alexa 647-labelled Ag nanowire excited by wide-field excitation in switching buffer. Strong background emission/scattering from the nanowire is clearly observable, together with bright satellite emission spots due to Alexa 647 emission. (<bold>c</bold>) Excitation (blue) and fluorescence (red) spectra of Alexa 647 in PBS solution, a fluorescence spectrum of an Alexa 647-adsorbed Ag nanowire in switching buffer (black dots), and an emission/Raman scattering spectrum of an Alexa-free nanowire (green dots) in switching buffer. (<bold>d</bold>–<bold>g</bold>) High-resolution reconstructed images taken with wide-field excitation (bin size 32 nm), each observed PSF was fit to a single 2D Gaussian function, with the molecule localized at the centroid of the function. Localized single-molecule density maps of a 110-nm diameter nanowire (<bold>d</bold>) and a 250-nm diameter nanowire (<bold>e</bold>) and localized single-molecule intensity histograms of the 110-nm nanowire (<bold>f</bold>) and 250-nm diameter nanowire (<bold>g</bold>). The insets in <bold>d</bold> and <bold>e</bold> display the SEM images of the corresponding nanowires. (<bold>h</bold>) and (<bold>i</bold>) are histograms of the localized single-molecule density maps along cross-sections perpendicular to the long axis of the nanowires displayed in <bold>d</bold> and <bold>e</bold>, respectively. The grey shaded areas represent the actual nanowire cross-sections measured by SEM. Scale bars, 1 μm.</p></caption><graphic xlink:href="ncomms7287-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>Remote excitation of switching localization microscopy.</title><p>(<bold>a</bold>) Schematic illustration of RE-SFM, the Ag nanowire is irradiated at the left end with focused laser, launching SPPs that propagate along the surface of the nanowire (purple curly arrow), and excite the surface-bound Alexa molecules. (<bold>b</bold>) RE-SFM images showing two fluorescence spots along the nanowire (enclosed by the white square) bleaching separately. (<bold>c</bold>) RE-SFM images showing two fluorescent spots along the nanowire (enclosed white square) appearing, dimming and disappearing (blinking off) at the same time in subsequent images. Scale bars, 2 μm.</p></caption><graphic xlink:href="ncomms7287-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>Multi-spot fluorescence PSFs of single molecules near a Ag nanowire.</title><p>A series of PSFs of single-molecule fluorescence in the vicinity of a silver nanowire (~250 nm diameter) observed during RE-SFM measurement (image sizes: 1.94 × 1.94 μm<sup>2</sup>, the parallel dashed lines indicate the edges of the nanowire). (<bold>a</bold>) A 2D Gaussian-shaped PSF at the middle of the nanowire. (<bold>b</bold>) A 2D Gaussian-shaped PSF at the side of the nanowire. (<bold>c</bold>,<bold>d</bold>) Elongated PSFs at the side of the nanowire. (<bold>e</bold>) A symmetric PSF with two 2D Gaussian-shaped spots on opposite sides of the nanowire. (<bold>f</bold>) An asymmetric PSF with two 2D Gaussian-shaped spots on opposite sides of the nanowire. (<bold>g</bold>) A symmetric PSF with two 2D Gaussian-shaped spots along one side of nanowire. (<bold>h</bold>) A PSF with four 2D Gaussian-shaped spots, two on either side of the nanowire. (<bold>i</bold>) High-resolution (bin size 32 nm) reconstructed single-molecule localization density map of an ~250-nm diameter nanowire from RE-SFM (SEM image inset) for which a molecule is assumed to be localized at the centroid of each fluorescence spot observed. PSFs were fit by single- or multi-component 2D Gaussian functions and a ‘molecule’ localization recorded at the centroid of each peak (that is, a two-spot PSF leads to the localization of two ‘molecules’). The ‘bare’ spot in the image at the left end of the wire is where the excitation laser was focused, background emission and scattering overwhelm Alexa 647 emission in this region. The spots on the far left of the image, and far from the nanowire position, are due to residual Alexa 647 that was not washed completely off the substrate.</p></caption><graphic xlink:href="ncomms7287-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>Simulated fluorescence PSFs for single molecules on Ag nanowires.</title><p>(<bold>a</bold>) Sketch of the simulation geometry, the double-headed arrow indicates the position and orientation of the oscillating electric point dipole that simulates a single fluorophore in the vicinity of the nanowire (~280 nm diameter). (<bold>b</bold>–<bold>e</bold>) Far left: the position and orientation of the point dipole (indicated by the double-headed arrow) relative to the pentagonal cross section of the silver nanowire. Left centre: the simulated far-field emission PSF colour map for the point dipole imaged in the sample plane (nanowire edges indicated by dashed lines in (<bold>b</bold>), top colour map). The image size is 3 × 3 μm<sup>2</sup>. Right centre: polar plot of the angular dispersion of point dipole emission in the <italic>xz</italic> plane, Far right: polar plot of the angular dispersion of the point dipole emission in the <italic>yz</italic> plane.</p></caption><graphic xlink:href="ncomms7287-f4"></graphic></fig><fig id="f5"><label>Figure 5</label><caption><title>Localization of simulated fluorescence PSFs.</title><p>(<bold>a</bold>,<bold>b</bold>) Simulated PSFs of a point dipole oriented perpendicular to the long axis of the nanowire and placed at the apex or on the adjacent facet of a 110-nm diameter (<bold>a</bold>) and 250 nm diameter (<bold>b</bold>) nanowire (colour maps, centre, the white lines indicate the edges of the nanowire). The PSFs are fit by single or multi-component 2D Gaussian functions (colour maps, right) and ‘molecule’ localizations recorded at the centroid of each Gaussian peak (that is, assuming that each fluorescent spot observed is from a separate molecule at its centroid). For instance, fitting the two-spot PSF at the top of <bold>b</bold> yields two localized ‘molecules’ (localization positions indicated by the diamonds in the schematic cross-section of the nanowires on the left) (<bold>c</bold>,<bold>d</bold>) Cross-sectional histograms of ‘molecule’ localizations perpendicular to the nanowire long axis for the simulated PSFs of the full range of molecular adsorption positions and orientations shown in <xref ref-type="fig" rid="f4">Fig. 4</xref> for nanowires of 110 nm (<bold>c</bold>) and 250 nm (<bold>d</bold>), diameter respectively. The histograms are fit by Gaussian distributions (red line) indicating a full width at half maximum, and thus predicted nanowire diameter, of ~160 nm for the hypothetical 110 nm diameter nanowire (<bold>c</bold>) and two lines separated by ~500 nm surrounding a line centred along the nanowire long axis for the hypothetical 250 nm diameter nanowire (<bold>d</bold>). Scale bars, 500 nm.</p></caption><graphic xlink:href="ncomms7287-f5"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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