Simultaneous topography and recognition imaging using force microscopy

Abstract

We present a method for simultaneously recording topography images and localizing specific binding sites with nm positional accuracy by combining dynamic force microscopy with single molecule recognition force spectroscopy. For this we used lysozyme adsorbed to mica, the functionality of which was characterized by enzyme immunoassays. The topography and recognition images were acquired using tips that were magnetically oscillated during scanning and contained antibodies directed against lysozyme. For cantilevers with low Q-factor (approximately 1 in liquid) driven at frequencies below resonance, the surface contact only affected the downward deflections (minima) of the oscillations, whereas binding of the antibody on the tip to lysozyme on the surface only affected the upwards deflections (maxima) of the oscillations. The recognition signals were therefore well separated from the topographic signals, both in space (Delta z approximately 5 nm) and time (approximately 0.1 ms). Topography and recognition images were simultaneously recorded using a specially designed electronic circuit with which the maxima (U(up)) and the minima (U(down)) of each sinusoidal cantilever deflection period were depicted. U(down) was used for driving the feedback loop to record the height (topography) image, and U(up) provided the data for the recognition image.

FIGURE 1

Enzyme immuno assay of HyHEL5-lysozyme binding. (A) Mica sheet 1 and 2 were successively coated with lysozyme, anti-lysozyme (HyHEL5), and peroxidase conjugated anti-mouse IgG. Sheets 3 and 4 were only coated with lysozyme and peroxidase conjugated anti-mouse IgG. The higher absorption values of samples 1 and 2 compared to samples 3 and 4 reveals the specific binding of HyHEL5 to its antigen. (B) Absorption time-scan of peroxidase conjugated anti-mouse IgG in solution used as calibration for the determination of the number density of active surface-bound lysozyme.

FIGURE 2

Topography images of lysozyme molecules imaged using the MAC mode. (A) Single molecule preparation. The cross section shows single lysozyme molecules with 1–2 nm in height and 15–20 nm in width. (B) A dense monolayer of lysozyme molecules adsorbed onto a mica surface as it was used for force spectroscopy experiments.

FIGURE 3

Force-distance cycle of a single molecular lysozyme-HyHEL5 unbinding event at 50 pN unbinding force and 20 nm unbinding length. Lysozyme is adsorbed onto a mica surface and the antibody HyHEL5 is attached to an AFM tip via a cross-linker molecule (PEG derivative). The inset shows a force-distance cycle measured when the binding is blocked by adding free antibody in solution.

FIGURE 4

Force volume mode data using lysozyme adsorbed onto a mica surface and HyHEL5 antibody attached to the tip. Binding sites on the lysozyme layer were detected in A and significantly blocked with free HyHEL5 in solution. (B) The unbinding forces in the pixels are scaled in gray scale values (0–100 pN). (C) Pdf of the unbinding forces observed in absence (solid line) and in the presence of free HyHEL5 antibody (dotted line). Areas are scaled to binding probabilities. The most probable unbinding force for the specific HyHEL5-lysozyme interaction was ∼60 pN (solid curve), whereas unspecific adhesion forces were slightly lower (dotted curve).

FIGURE 5

(A) Deflection signal of a magnetically oscillated cantilever during scanning along the surface when the feedback was switched off. The signal was recorded on a sound card over a time range of 4 ms. Peak to peak amplitude was 5 nm. (B) and (C) Signal as shown in A over a full scan line (500 nm scanned in 1 s). Due to the resulting compression of the time axis, only the extrema of the oscillation periods remain visible. (B) Bare tip on lysozyme molecules adsorbed onto a mica surface. (C) HyHEL5-antibody coated tip on lysozyme molecules.

FIGURE 6

(A) Principle of recording repetitive traces while scanning lysozyme molecules adsorbed onto mica surfaces. (B) The slow scan axis was disabled and the deflection signal was recorded on a sound card. Minima (left panels) and maxima (right panels) of the oscillation amplitudes were depicted using MATLAB. The traces are shown in false color gray code (see height bar, 0–1 nm) over a full scan line (500 nm).

FIGURE 7

Signal processing for simultaneously obtaining topography and recognition images. The raw cantilever deflection signal obtained in the MACmode is fed into the TREC box, where the maxima (U_up) and minima (U_down) of each oscillation period are depicted and used for the recognition and topography image, respectively.

FIGURE 8

Simultaneously acquired topography (A) and recognition (B) image using a HyHEL5 antibody-coated tip on lysozyme molecules adsorbed onto mica surface. (A) Topography image showing single lysozyme molecules. (B) Recognition image. The black dots indicate positions of antigenic sites. The correlation between topography and recognition image is indicated with black arrows, showing that at least two-thirds of the lysozyme molecules are recognized in the recognition image at the same position.