SAMPLE PREPARATIONHeLa cells were transfected to expressa Green Fluorescence Protein (GFP)
and GPI anchor fusion protein.7 PAis able to recognize all GPI anchoredproteins without any distinction throughthe inositol moiety.5,6 See Figure 1 fora scheme of the interaction process.
DNP cantilevers (Veeco Probes)
were functionalized with PA moieties
according to established protocols.8,9The softest cantilever (0.06 N/m nominal
spring constant) was used to carry out
force volume imaging.
The force measurements wereperformed using a Veeco BioScope II
integrated with a Zeiss Axiovert 200M
and equipped with a Veeco NanoScope Vcontroller. Collected data was analyzed
with NanoScope v7.20 software. A Roper
Scientific Coolsnap camera driven under

Molecular Devices Metamorph software
was used to record epifluorescence
images.

position the AFM probe over a cellof interest to perform force volume
imaging.

RESULTSThe fluorescence images were used
to distinguish between different
expression levels of GFP, with higher
levels of GFP resulting in a higher
fluorescence intensity (Figure 2, topleft). For example, Region A exhibits
no fluorescence while the cell isclearly visible by brightfield imaging(Figure 2, top right and bottom
left images). Region B, however,contains a cell exhibiting a highlevel of fluorescence, thus indicating
expression of the GFP-GPI chimera in
the transfected cell. Once the different
types of cells have been identified in thefluorescence images, the corresponding
brightfield images are then used toTo calculate the forces involved in thebinding of cell surface GPI proteins
and the tip-bound PA, the spring
constant of the cantilever must firstbe determined.10 This is easily andaccurately calculated, in either air orfluid, using the BioScope II thermal tune
function. Before collecting the force
volume data, a reference force curve iscollected on a stiff surface (in this casethe glass bottom of the Petri dish) andused to define the best parameters forforce volume image acquisition.
For force volume imaging, the tip ispositioned at the edge of a cell anda 2x2 μm scan frame (32x32 pixelresolution) was collected over Regions

Figure 2. Force measurements on living Hela. The main part of the figure represents three optical images showing both the cantilever viewed from the top and theliving cells. The top left image is an epifluorescence snapshot exhibiting cells with various fluorescence degrees (the shape of the cantilever has been highlighted inred). Force curves were achieved both on weakly fluorescent or non-fluorescent (zone A) and highly fluorescent cells (zone B). The top right and bottom left images
were taken in brightfield mode, and show the cantilever during a force measurement on the two selected areas. The experience was duplicated and shows the sametendency: for non-fluorescent cells, the number of events dramatically decreases (see histogram) by a factor of 4 to 5, in comparison to measurements achieved onhighly fluorescent cells.

Page 2ATOMIC FORCE MICROSCOPY WITH BIOSCOPE TM II DETECTING SPECIFIC UNBINDING EVENTS ON LIVING CANCER CELLSA and B (Figure 3). Each pixel in theresulting force image represents anapproach-retract event. Considering
only the retract part of each curves,
we can map the unbinding events onthe surface. The darker the pixel, thehigher the force value is. Then the force
curves can be shown individually (singleanalysis), on several spots (multipleanalysis) or on a full range (from 1square to the total surface). Modifying
the threshold for the force image, interms of distance and deflection, theunbinding events on a “slice image”
can be mapped, as shown in Figure
3a (this data was collected on a highly
fluorescent cell). This feature is also
very useful during acquisition ofthe force volume image as a meansof monitoring whether or not theparameters have been set correctly aseach force measurement is collected. A
more complete description of the force
measurement capabilities of NanoScope
software can be found in additional
application notes by Veeco.11
Comparison of the resulting force
volume images acquired for regions
A and B revealed that the number(a)of unbinding events is increased bya factor of 4.7, respectively (94.5 vs.
450 respectively; the experiment wasdone in duplicate). Some of the mostrepresentative curves are shown in
Figure 3b. The shape of the retract
curves clearly shows the interaction
between GPI and PA, with one or moreadhesion or binding events occurring. Inthis case, we selected five representative
retract curves showing a specificunbinding event, and superimposed
them. A zoom-in of the contact area isshown, exhibiting the individual force
curves in cascade. The Peak Detection
Option allows finding the maximum
adhesion peak for each curve (in thiscase, we selected a minimum size of0.1 nN with a percentage of maximumof 70%, and a minimum width of 20 nm).
Considering the spring constant and thedeflection sensitivity, all the unbinding
values were found to be in the range
of a few hundred piconewtons, butwith important variations. This can bedue to the fact that we are conducting
these studies on a live cell system. Assuch, there can be many contributions
to the observed unbinding values suchas the motion of organelles beneaththe cell membrane or changes to thecytoskeleton.
CONCLUSION
In the present study, the force
measurement and navigation
capabilities of Bioscope II were usedin combination with epifluorescencemicroscopy to measure the unbinding
events between GPI-anchored proteinson the surface of living Hela cells
and a proaerolysin-modified AFM
tip. The combination of AFM andepifluorescence enables accurate
identification of cells as a function of
their expression level of the protein ofinterest on which to perform targeted
AFM force measurements. A clear
correlation was demonstrated between
the number of unbinding events and thefluorescence intensities of the targeted
cells. A further study also involving
tip functionalization will demonstrate
how the analysis of the approach andretract portions of a force curve allows
the determination of local elasticity
differences and specific ligand-receptor
interaction.12(b)Figure 3. Force curve analysis on fluorescent living cells. The software offers the possibility to work on force volume images with a high degree of flexibility. As
an example, one can play with the slice image and modify the deflection as a function of the distance threshold. In this case, a threshold at the neighborhood of thecontact point was selected, and each of the dark squares observed on the slice image, considering the retract part of each curve, represented an adhesion event.
Then the best curves (see five examples in the figure) were selected and compiled, and a maximum adhesion peak was extracted for each of them.

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