TOPOGRAPHY AND RECOGNITION IMAGING OF CELLS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 145

on the tip ( Fig. 7.1 , step II). This is always done by amide bond formation,
for which, all PEG linkers possess an activated carboxy (-COOH) group, in
the form of an -hydroxysuccinimide ester (NHS ester). The PEG solution

is normally adjusted to ensure low density of cross-linkers on the Si 3 N 4 tip
surface, and therefore single-molecule detection by the tip is enabled. In the
last step, a ligand molecule is coupled to another free functional end of the
PEG linker as shown in Fig. 7.1 , step III. One of the most suitable PEG linkers
used is an aldehyde linker 10 (abbreviated as NHS-PEG-aldehyde), which can
link underivatized antibodies and other proteins via their lysine residues, of
which 80-90 are found per antibody molecule. Finally, functionalized tips can
be stored in PBS at 4 °C for several weeks until use.

N

7.3 OPERATING PRINCIPLES OF TOPOGRAPHY AND
RECOGNITION IMAGING

In contrast to common recognition imaging based on force spectroscopy a
recently developed AFM imaging technique termed simultaneous topography
and recognition imaging (named TREC) overcomes some of the limitations
regarding lateral resolution and imaging speed by using dynamic force
microscopy with a functionalized sensor tip that is oscillated during scanning
across the surface.
The operating principle of TREC is based on MAC (magnetic alternating
current) mode AFM, 11 where a magnetically coated cantilever is oscillated
through an alternating magnetic ield. The tip functionalized with a ligand
molecule via a short (~8-10 nm) lexible PEG linker (tip functionalization
procedure is described earlier) is oscillated close to its resonance frequency
while scanning over the surface. When such a tip-tethered ligand binds to
its receptor on the sample surface (i.e., when speciic molecular recognition
occurs), the PEG linker will be stretched during upward movement of the
cantilever. The resulting loss in energy will in turn cause the top peaks of
the oscillations to be lowered. The ligand-receptor-binding events thus
become visible because of a reduction in the oscillation amplitude, as a
result of speciic recognition during the lateral scan. In contrast to “normal”
MAC mode imaging, TREC uses the lower part of the oscillation to drive a
feedback loop for obtaining the topography image, whereas the upper part of
the oscillation is used for the generation of the recognition image. Moreover,
using half-amplitude feedback allows accurate determination of the surface
topography. To provide more details, the time-resolved delection signal of

the oscillating cantilever is low-pass iltered to remove the thermal noise and
the DC (direct current) is offset levelled and ampliied before splitting into
the lower (

12

U down ) and upper ( U up ) parts of the oscillations. The signal passes a