Figure 5

Combinatory fluorescence-AFM on bioconjugated protein-quantum dot (QD) system. Reprinted with permission from [14], 2011 Elsevier. (A) Registration of raw QD fluorescence signals (yellow-red) with AFM topography (grey scale) of the same sample area (8 × 8 μm²). The fluorescence signals were fit by 2D Gaussians to determine their centers with nanometer accuracy, a technique also known as fluorescence imaging with one nanometer accuracy (FIONA). In (B), FIONA signals are shown in red color, indicating localization probability of the fluorescence centers. The red box in (B) indicates the QD-protein-DNA complex shown magnified in (C and D) as top view and 3D representation, respectively. The scale bar in (D) corresponds to 30 nm. These zoom in figures demonstrate good FIONA-AFM overlay accuracy, allowing the identification of a fluorescently tagged molecule in the AFM topography from its fluorescence signal. (E) Schematic of Fluorescence-AFM set-up. The sample is deposited on a mica substrate (inset zoom, not to scale), excited from below by total internal reflection (TIR) fluorescence (black arrows) and mechanically scanned from above by the AFM. Excited fluorescence (grey arrows) is filtered through a narrow bandwidth emission filter and recorded by a CCD camera attached to the microscope tri-occular port behind a 1× to 4× pre-magnifier.