In recent years single-molecule localization super-resolution microscopy (SMLM) has become an indispensable tool for many fields of research. Here, for any image of a single molecule one determines its center position with much higher accuracy than the size of that image itself. A challenge of SMLM is to achieve super-resolution also along the third dimension. Recently, Metal-Induced Energy Transfer or MIET [1,2] was introduced. It exploits the energy transfer from an excited fluorophore to plasmons in a thin metal film. Similar to Förster Resonance Energy Transfer (FRET), this coupling shows a strong distance dependence, but over a range up to 150 nm and enables axial localization of fluorophores with 5-6 nm resolution at a photon budget of 1000 photons. [3,4] Here, we show that using a graphene layer the localization accuracy of MIET reaches Ångström accuracy. At such accuracy, minute details such as nanometer-scale roughness of the sample surfaces becomes important.
For proof of principle, we determined absolute distances of single molecules from a surface for samples with an a priori well-known sample geometry. We spin-coated fluorescent dye molecules (Atto655) on top of three different substrates with spacer thickness values of 10, 15, and 20 nm, defining the distance of the molecules from the graphene layer. Next, we determined the thickness of supported lipid bilayers (SLBs) by localizing fluorescent dyes attached to lipid head groups in the bottom and top leaflet of the SLB.
We have demonstrated that by using graphene as the energy acceptor in MIET, the axial localization accuracy and resolution reaches sub-nanometer levels at photon budgets which are typical in conventional SMLM experiments. An interesting feature of graphene-MIET is that it provides an axial localization accuracy which now surpasses significantly that of lateral localization provided by most SMLM approaches.
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Proc. SPIE. 10071, Single Molecule Spectroscopy and Superresolution Imaging X
KEYWORDS: Surface plasmons, Metals, Luminescence, Molecules, Energy transfer, Dielectrics, Distance measurement, Resonance energy transfer, Fluorescence resonance energy transfer, Molecular energy transfer
We present a new concept for measuring distance values of single molecules from a surface with nanometer accuracy using the energy transfer from the excited molecule to surface plasmons of a metal film . We measure the fluorescence lifetime of individual dye molecules deposited on a dielectric spacer as a function of a spacer thickness. By using our theoretical model , we convert the lifetime values into the axial distance of individual molecules. Similar to Förster resonance energy transfer (FRET), this allows emitters to be localized with nanometer accuracy, but in contrast to FRET the distance range at which efficient energy transfer takes place is an order of magnitude larger. Together with orientation measurements , one can potentially use smMIET to localize single emitters with a nanometer precision isotropically, which will facilitate intra- and intermolecular distance measurements in biomolecules and complexes, circumventing the requirement of the knowledge of mutual orientations between two dipole emitters which severely limits the quantification of such distances from a conventional single-pair FRET (spFRET) experiment.
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