and represents our premier
instrument for force spectroscopy
applications. However, thesefeatures are also applicable
to force measurements madewith other Veeco AFMs like theMultiMode, the BioScope SZ TM,
and the new BioScope IITM. The
analysis examples shown here
were done using NanoScope v.7r.2
software, but many of the samefeatures are also available in v.6
and later versions, with somedifferences.
METHODS

A representative data set of titin
unfolding data was collected asfollows. A recombinant polyprotein
consisting of eight tandem repeatsof the IG27 domain of human titin
was obtained from Athena Enzyme
Systems and diluted to 50µg/ml inPBS. A 50µL drop was incubatedon a fresh template stripped gold
surface for 10 minutes and thenextensively rinsed with PBS andplaced on the PicoForce scanner.
Force-distance curves werecollected with a ramp size of
500nm at a ramp rate of 1000nm/s
using a 3nN relative trigger and a
1s surface delay. A MLCT-AUNM
cantilever was used. Its spring
constant was measured using thethermal tune method and found tobe 35.2pN/nm. The thermal tune

method is fully supported for thePicoForce and other microscopes
running on v.7 NanoScope
software.
Sorting through thousandsof force-distance curves

A total of 1775 force-distancecurves were collected at thisramp rate. Many of these curvescontain no useful data, either
because the tip failed to “catch”
a molecule or because multiple
interactions resulted in a complex
force curve lacking the clean“fingerprint” that indicates asingle protein extension. This isvery typical of these experiments
because the proteins are
randomly distributed on the gold
substrate and attachment to thetip and subsequent extensionof the protein depends on nonspecific physisorption to the tip.
Moreover, the tip can attach to theprotein anywhere along its length,
so there are not always enoughdomains between the substrate
and the tip to give a definitive“fingerprint.” It is not uncommon
then for only a small fraction of
the total force-distance curves
to ultimately be used for the final
data analysis.
Clearly it would be a daunting
task to manually open, examine,and then save or delete eachforce-distance curve file. While
graduate student labor might
be cheap, carpal tunnel surgery
isn’t, so there are two routines
in the NanoScope software tostreamline this chore. The firstis called “Filter Curves” and canbe found under the “File” menu.
This brings up the view shown in
Figure 1. Here, a folder containing
force-distance curves can beselected. Each force curve file
can have up to three independent
plots, so you must also selectwhich plot contains the databy which you wish to sort. A“Minimum Force” and a “MinimumDistance” are then defined thatare used to automatically screenevery force curve file in that
directory. These parameters
are applied to the retract curveof each file, searching whether
the minimum specified adhesion
force can be found at some pointat least the minimum distancefrom the contact point. Note thatthe force is measured relative
to the non-contact deflection
value, not the absolute deflection
value, so it does not matter
whether the baselines of thecurves are at zero. The “MinimumForce” parameter is intended
to allow you to screen for someminimum expected force while theFigure 1:  The Filter Curves view allows force-distance curves that contain no useful data to be
rapidly identified and discarded according to user-defined criteria.
Page         NANOSCOPE SOFTWARE TOOLS FOR FORCE SPECTROSCOPY DATA ANALYSIS

“Minimum distance” parameterallows you to avoid consideringsome non-specific adhesion near
the contact point. As each curveis screened and sorted, they aredisplayed in the view with the“bad” curves on the left and the“good” curves on the right. In thisway, you can quickly get a senseof whether the parameters you
have chosen are appropriate.
Once the routine has finishedsorting through the entire folder, adialog box is displayed requesting
permission to delete the “bad”curves. These files are sent to theWindows recycle bin, but it’s agood idea to back up the unsorted
data in a separate folder anyway
before executing this routine. In
the example shown here, we wereable to quickly reduce our data setof 1775 curves to just 345 curves–
all without harming a graduate
student.

Sorting through hundreds offorce-distance curves

Of course it would still be very
unpleasant to manually open
and sort through even 345 force
curve files. But at some point
it does become more efficientand effective to screen theforce curves visually rather
than depending on a softwarealgorithm to appreciate thenuances of the curves. At thispoint the screened data from theFilter Curves routine will stillcontain curves that have multiple
interactions instead of a clean
pulling “fingerprint.” In order toeliminate these, we include the“Review Curves” routine, whichis also found under the “File”
menu. This view, shown in Figure2, allows you to rapidly page
though the force curve files usingthe keyboard arrow keys andmark “bad” curves for deletion byhitting the keyboard “Delete” key.
Like the “Filter Curves” view, you
select the plot number by which
you wish to sort. There are also
other view options, as shown, tohelp make the curves easier tosort. After paging through eachfile, pressing the “Delete files”
button will prompt to request
permission to delete the files
that you have tagged for deletion.
Before that time, you are free topage back to previous files in case
you mistakenly mark a file fordeletion. Compared to individually
opening every file, this greatly
reduces the time required for thisfinal sorting step. Instead of 20–30
seconds to open, view, close, andthen save or delete each file, thesorting time is typically reduced toFigure 2:  The Review Curves view allows the user to rapidly sort through force-distancecurves without individually opening the files.
NANOSCOPE SOFTWARE TOOLS FOR FORCE SPECTROSCOPY DATA ANALYSIS        Page only 1–2 seconds per curve. While
not fully automated, it certainly
helps to quickly reduce the data
set to one containing only usefuldata. In the case shown here, westarted with 345 files after theinitial screening and end up with
only 25 files that contained good,clean titin pulling curves. Clearly
this “good”-to-“bad” ratio will
depend on the initial quality of thedata as well as how stringently
you sort out imperfect curves.
Some groups choose to reduce
the surface concentration of theirproteins to maximize the chancethat only single proteins will bestretched when an interaction
does occur. Other groups
prefer to increase the chance ofinteractions by increasing thesurface concentration, though thisalso tends to increase the chanceof multiple interactions. A suitablebalance between the two cases
must be made. We suggest thatthe first case– fewer but cleaner
interactions– may be preferable
since the Filter Curves routine
is very efficient at screening out
curves containing no interactions(or only a non-specific interaction),while curves containing multiple
interactions must be removed
using the more user-intensive
Review Curves routine.

Visualizing many force
curves at one time

Once you are finished sorting yourdata set, it is nice to be able tolook at the remaining curves in amanner that gives a good overview
of the data. The “Browse” view,found under the “View” menu, isconvenient for this purpose. Its
thumbnail view, shown below inFigure 3, does not display the data
quantitatively or in high resolution,
but it is useful for spotting any
obviously bad curves that you
might have missed or finding that
one really nice curve at which
you would like to take a closer
look. Of course you can also load

individual files or delete, move, or
copy them. We will discuss next
how you can analyze multiplecurves from this view and thenlater how you can batch export
force-distance data from this view.
Extracting quantitative datafrom many force-distancecurvesOnce the force curves havebeen sorted so that only thosecontaining clean, distinct“fingerprints” remain, theresearcher is still left with thetask of measuring and tabulating
the values for each unfolding
peak. In principle, this can be done

Figure 3:  The Browse view lets you visualize many force-distance curves at once and carry out
many normal file management tasks.

Page         NANOSCOPE SOFTWARE TOOLS FOR FORCE SPECTROSCOPY DATA ANALYSIS

by individually loading each fileand measuring the forces in thenormal offline force curve view.
But as with the sorting tasks, thisis a tedious job. The “MultipleCurve Analysis” routine makes
this analysis much simpler.
This view is hidden away in theBrowse view that was describedearlier. To access it, select ablock of force curve files fromthe Browse view, then right-click
and select “Curve Analysis.” Thecurves will display, overlaying
on one another. Because thenon-contact deflection value isarbitrary, there will probably beconsiderable offsets between
them. Therefore, one of the firstthings you will want to do is toturn on “Auto-align curves” forboth the X- and Y- axes. Click“Refresh Plot” to update the view.
The individual force curves shouldalign with one another according
to their contact points, as shown in
Figure 4. Up to fifty curves can bedisplayed at one time.

Figure 4:  The Multiple Curve Analysis view offers functions that automatically align force
curves by their contact points, finds adhesion peaks according to user-defined criteria, andcan create a report of the tabulated peak forces and peak-to-peak distances.

If you set “Select Mode” to “On,”the force curves will all become
the same color. But if you selectan individual file in the list
below then that curve will turna different color. If you wish todisplay just a few curves, you canmake a graph like that shown in
Figure 5 by setting “Y axis AutoZero” to “Cascade” and setting thedesired “Cascade Offset.” This isconvenient for comparing a small
subset of the curves. The mostpowerful feature in the Multiple
Curve Analysis view, however,is the ability to automaticallyfind, measure, and tabulate thesize and position of the adhesion
peaks. This is done by setting a“Minimum size” and “MinimumFigure 5: The Multiple Curve Analysis view can also display force curves in a “cascade” styleview, which makes comparing several curves more convenient.

NANOSCOPE SOFTWARE TOOLS FOR FORCE SPECTROSCOPY DATA ANALYSIS        Page Figure 6:  A Histogram of the peak force
data is easily created from data file exported
from the Multiple Curve Analysis view.

Width” for a peak to be counted.
The minimum width is measured
at a user-defined point definedby a percentage of the peakmaximum. One these are set,click “Find Peaks” and the peak
information fills the bottom file
list, as shown in Figure 4. This
list can then be exported to adelimited text file by clicking the“Export…” button. This file is theneasily loaded into an analysis
package like Igor Pro or even
Excel to create a histogram of theunfolding forces likes that shown
here in Figure 6.
The parameter values needed tomost reliably find the peaks willvary. It is helpful to try different
values with just a few curves
before analyzing a large groupso that you can more easily see
which peaks are found. Note thatthe final large pull-off peak will
also be tabulated. These values
will appear in the histogram at
higher forces and can simply bediscarded.
Advanced analysis ofindividual force curves

There are a number of analysis
options available when you loada single force curve from theBrowse view. For example, by
dragging markers in onto thegraphs you can read values
from the curves for the X andY positions, delta X and Y, andslopes. Be sure that the “ActiveCurve” parameter is set to thecorrect curve, Extend or Retract.
Besides these simple features,
it is also possible to fit a function
to the curve between the twomarkers. You can choose between
either a line fit or a worm-likechain fit. The fit parameters are
output to the parameter list on
the right side of the display. Thefit function itself is overlaid on theactual data in the plot, as shownin Figure 7. The worm-like chain
equation is:where b is the persistence length(treated as a user-defined value,not a fit variable) and L is thecontour length.

Figure 7:  Worm-like chain fit of a titin extension curve

Page         NANOSCOPE SOFTWARE TOOLS FOR FORCE SPECTROSCOPY DATA ANALYSISBATCH EXPORTING FORCEDISTANCE CURVE DATA

Finally, for analyses not directly
supported in the NanoScope
software, it is easy to batch
export your force curve data.
This can be done by selecting agroup of files from the Browse
view and then right-clicking andchoosing Export…ASCII. Thisdisplays the dialog box shown inFigure 8, where one can selectthe exact channels and units tobe exported. The full header canalso be exported, although this is
not normally required and is notrecommended if the files are to
be loaded as simple delimited text
files.

Figure 8: Batch export dialog box

SUMMARY

This Application Note has
described a number of features
available within the NanoScope
software that make offline
analysis of force spectroscopy
data much easier and faster.
These include features to help
sort through large data sets offorce-distance curves and remove
those that contain no usefuldata. Analysis functions were
described, including the Multiple
Curve Analysis view, which can
automatically find and tabulate
peak values and positions inmany force curves. We hope thatyou find these features helpful
in making your research moreproductive.

NANOSCOPE SOFTWARE TOOLS FOR FORCE SPECTROSCOPY DATA ANALYSIS        Page 