KEYWORDS: Energy transfer, Biophysics, Molecules, Fluorescence lifetime imaging, Acquisition tracking and pointing, Photonics, Near field optics, Near field, Molecular energy transfer, Microscopy
Graphene-induced energy transfer (GIET) was recently introduced for sub-nanometric axial localization of fluorescent molecules. GIET exploits the near-field energy transfer from an excited fluorophore to a single sheet of graphene. This alters the fluorescence decay-time of the emitter and can be easily determined by fluorescence lifetime imaging microscopy (FLIM). The axial resolution of GIET implies to study of biological membranes.
We present the measurement of the thickness of synthetic model membranes and demonstrate changes upon the addition of cholesterol. Furthermore, we are able to show the flipping of lipids from one leaflet to the other and determine the rates of this dynamics.
In addition, we used GIET for mapping quasi-stationary states of the mitochondrial membranes before and during ATP synthesis. Upon activation, the inner membrane clearly approaches the outer membrane and the inter-membrane space is reduced by ∼2 nm.
KEYWORDS: Energy transfer, Molecules, Graphene, Super resolution microscopy, Super resolution, Plasmons, Metals, Thin films, Resonance energy transfer, Fluorescence resonance energy transfer
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.
[1] A. I. Chizhik et al. Nat. Photonics 8, 124 (2014).
[2] S. Isbaner et al. Nano Lett. 18, 2616 (2018).
[3] N. Karedla et al. ChemPhysChem 15, 705 7 (2014).
[4] N. Karedla et al. J. Chem. Phys. 148, 204201 (2018).
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.