JPK instruments nanowizard afm User manual

NanoWizard®AFM
Handbook
Version 2.2a
05 / 2012
© 2004-2012 JPK Instruments AG
all rights reserved

JPK Instruments NanoWizard®Handbook Version 2.2a 1
Contents
1. Introduction...........................................................................................................................................1
1.1 About this handbook........................................................................................................................................ 1
1.2 What is an Atomic Force Microscope? ............................................................................................................ 1
1.3 Scanning Probe Microscopy............................................................................................................................ 2
1.4 Atomic Force Microscopy ................................................................................................................................ 3
1.5 AFM cantilevers............................................................................................................................................... 4
2. Imaging modes.....................................................................................................................................5
2.1 Feedback and imaging control......................................................................................................................... 5
2.2 Amplitude feedback in dynamic modes ........................................................................................................... 5
2.3 Another way of thinking about imaging modes ................................................................................................ 6
2.4 Operation......................................................................................................................................................... 8
2.5 Phase imaging................................................................................................................................................. 9
2.6 Force adjustment in imaging modes.............................................................................................................. 10
2.7 Applications ................................................................................................................................................... 11
3. Force spectroscopy ............................................................................................................................12
3.1 Introduction.................................................................................................................................................... 12
3.2 Data processing for analysis.......................................................................................................................... 13
3.3 Applications ................................................................................................................................................... 16
4. More about cantilevers .......................................................................................................................18
4.1 General points ............................................................................................................................................... 18
4.2 Handling information...................................................................................................................................... 18
4.3 Cantilever types for different imaging modes................................................................................................. 19
4.4 Tip modification ............................................................................................................................................. 20
4.5 Spring constant.............................................................................................................................................. 21
5. Cell imaging........................................................................................................................................24
5.1 AFM in relation to other cell imaging techniques ........................................................................................... 24
5.2Sample preparation ....................................................................................................................................... 25
5.3 Imaging modes.............................................................................................................................................. 27
5.4 Critical imaging parameters........................................................................................................................... 28
5.5 Using the oscilloscope to optimize the imaging parameters.......................................................................... 28
5.6 Artifacts.......................................................................................................................................................... 30
6. Single molecule imaging.....................................................................................................................31
6.1 Preparation is key.......................................................................................................................................... 31
6.2 Imaging hints – intermittent contact mode in liquid........................................................................................ 33
6.3 Imaging hints - contact mode in liquid............................................................................................................ 34
6.4 Imaging hints – imaging in air........................................................................................................................ 35
6.5 Simple DNA protocol for imaging tests.......................................................................................................... 35
7. Artifacts...............................................................................................................................................37
7.1 Tip shape issues............................................................................................................................................ 37
7.2Artifacts from damaged tips........................................................................................................................... 38
7.3 Contamination ............................................................................................................................................... 39

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7.4 Other imaging considerations........................................................................................................................ 39
8. Useful physics for SPM...................................................................................................................... 41
8.1 The cantilever resonance .............................................................................................................................. 41
8.2 Thermal noise spring constant calibration ..................................................................................................... 41
8.3 Young’s Modulus of materials........................................................................................................................ 45
9. Useful chemistry and sample/tip preparations................................................................................... 47
9.1 Cleaning cantilevers and tips......................................................................................................................... 47
9.2 Silanization and APTES treatment ................................................................................................................ 48
9.3 Home made gel packs for cantilever storage................................................................................................. 49
9.4 Suppliers of AFM accessories....................................................................................................................... 49
10. References......................................................................................................................................... 51
10.1 General AFM Papers..................................................................................................................................... 51
10.2 Spring constant calibration references .......................................................................................................... 53
10.3 Books ............................................................................................................................................................ 54

JPK Instruments NanoWizard®Handbook Version 2.2a 1
1. Introduction
1.1 About this handbook
Here you can find information about the principles and methods of scanning probe
microscopy. The focus is on applications in biotechnology and life science.
The particular details of the JPK NanoWizard®
AFM system, for both the software
and hardware, can be found separately in the NanoWizard®
User Manual. The aim
of this document is to introduce AFM for those who are not familiar with the
technique, and to provide background information and resources to
aid those who
are familiar with the technique to extend their knowledge of particular applications.
The first sections of this handbook introduce the AFM technique, starting w
ith the
general ideas behind scanning probe microscopy. Later sections of this handbook
provide more detailed information and background for more experienced users.
1.2 What is an Atomic Force Microscope?
AFM is very different from optical microscopy.
Ther
e are no lenses, there is no requirement for a light source to illuminate the
sample, there is no eyepiece to look through; the microscope itself does not even
look like a typical optical microscope. The imaging technique consists of a
mechanical device, w
hich is able to measure very small forces when atoms or
molecules come close together, so it was named atomic force microscopy.
Cantilevers are at the heart of an atomic force microscope.
A critical part of the device called the cantilever is a plate sp
ring, which is fixed at
one end. At the other end it supports a pointed tip.
The tip can be moved across a
sample surface line by line, just like a lawn mower in the garden.
The pointed tip is brought into contact with the sample and moved across the
su
rface. The instrument measures the deflection of the cantilever as it scans, and
from this information about the tip movement a three-
dimensional image of the
sample is built up.

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1.3 Scanning Probe Microscopy
As the name suggests, the heart of an SPM is
a probe that is scanned over the
sample surface to build up some form of image. The type of image you get
depends on the interaction that is measured by the probe. Images can be
produced that reflect many different properties of the sample. The sample h
eight
information (topography), usually forms one aspect of the image, but images can
also be collected that show other properties, including mechanical, electrostatic,
optical, or magnetic information about the sample surface.
Different probes and meas
urement systems are often used for the different
properties, but one requirement is that
the interaction between the probe and the
sample is localized
in some way. This is so that the measured signal is dominated
by some small region of the sample closest
to the tip, so that an image of the
sample can be formed as the tip is scanned over the surface. This implies that the
interaction must have a strong distance dependence, so that only the nearest parts
of the sample contribute to the interaction felt by
the tip. The range of the
interaction will be one factor in the final resolution of the instrument. When the
interaction has a very strong distance dependence, such as the electron tunneling
current used in STM, the resolution can be good enough to “see” individual atoms.
Since the measured signal should be dominated by the small region of probe and
sample that are closest together, the actual probe does not need to be an isolated
point. The probe can be part of some larger structure that is more
convenient to
mount and scan. The size of the probe can be relatively large, perhaps hundreds
of microns or more, but if the interaction has a short enough range then the signal
will be dominated by the very tip region of the probe, so that resolutions c
an still be
achieved in the range from atomic distances to microns.
The idea of a probe measuring a local interaction and building up an image is
relatively straightforward, but the actual implementation of a system with a
resolution in this range is te
chnically challenging. Many factors came together in
the development of scanning probe microscopy, including the development of
piezoelectric materials that made it possible to reproducibly position and scan
components with a sub-nanometer precision.
Th
e following diagram shows some of the different forms of scanning probe
microscopy that have been developed. The techniques are usually named after
the interaction that they measure. The list is not complete, as there are many
different forms of scanning
probe microscopy, and new techniques are still being
developed. The information in this handbook is mainly concerned with Atomic
Force Microscopy.

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Scanning Tunneling Microscope –STM
H. Rohrer, G. Binnig (1981)
The family of Scanning Probe Microscopes -SPMs
Scanning Near-field
Optical Microscope
–SNOM
Photon Scanning
Tunnelling Microscope
-PSTM
Atomic Force Microscope
–AFM
G. Binning, C. Quate, C
Gerber (1986)
Magnetic Force Microscope
-MFM
Electrostatic Force Microscope
-EFM
Shear Force Microscope
-ShFM
Scanning Ion Conductance
Microscope
-SICM
Scanning Capacitance
Microscope
-SCM
Scanning Chemical Potential
Microscope
-SCPM
Scanning Thermal Microscope
-SThM
1.4 Atomic Force Microscopy
The atomic force microscope (AFM) is one of the fam
ily of scanning probe
microscopes, and is widely used in biological applications. The AFM uses a
flexible cantilever as a type of spring to measure the force between the tip and the
sample. The basic idea of an AFM is that the local attractive or repulsiv
e force
between the tip and the sample is converted into a bending, or deflection, of the
cantilever. The cantilever is attached to some form of rigid substrate that can be
held fixed, and depending whether the interaction at the tip is attractive or
repulsive, the cantilever will deflect towards or away from the surface.
This cantilever deflection must be detected in some way and converted into an
electrical signal to produce the images. The detection system that has become
the standard method for A
FM uses a laser beam that is reflected from the back of
the cantilever onto a detector. The optical lever
principle is used, which means
that a small change in the bending angle of the cantilever is converted to a
measurably large deflection in the position of the reflected spot.
The attractive or repulsive force between the tip and the sample causes a
deflection of the cantilever towards or away from the sample. As the cantilever
deflects, the angle of the reflected laser beam changes, and the spot f
alls on a
different part of the photodetector. The signals from the four quadrants of the
detector are compared to calculate the deflection signal.
Most AFMs use a photodiode that is made of four quadrants, so that the laser spot
position can be calcula
ted in two directions. The vertical deflection (measuring the
interaction force) can be calculated by comparing the amount of signal from the
“top” and “bottom” halves of the detector. The lateral twisting of the cantilever can
also be calculated by comparing the “left” and “right” halves of the detector.
AFM is particularly suited for biological applications, because the samples can be

4 JPK Instruments NanoWizard®Handbook Version 2.2
imaged in physiological conditions. There is no need for staining or coating, and
no requirement that the sample sh
ould conduct electrons. Therefore high
resolution imaging is possible in physiological buffer or medium, and over a range
of temperatures. Living cells can be imaged, as well as single molecules such as
proteins or DNA. The force contrast gives 3-dimens
ional topography information,
as well as the possibility to access other information such as the mechanical
properties or adhesion.
1.5 AFM cantilevers
Cantilevers are fabricated on chips
What you get when you order cantilevers is a small micro-precision-
machined
rectangular or triangular piece of silicon or silicon nitride with a shiny surface. The
minute cuboid you can see is not the cantilever itself, but the chip that holds the
cantilever. Generally you need a magnifying glass to see the cantilever at
the
narrow side of the chip. Sometimes there are two or more cantilevers attached to
the narrow edges of the chip.
What you are unable to see without a good optical microscope is the tip at the end
of the cantilever. Typically the tip is a few microns
long, and shaped like a pyramid
or a cone. The radius and angle of the end of the tip determines the imaging
quality.
Cantilevers can be thought of as springs.
From physics lessons in school, you may recall that the extension of springs can be
described by Hooke's Law F = - k * s.
This means: The force F
you need to extend the spring depends in a linear way on
the distance sthat you extend it. This linear behavior
just means that if you double
the deflection of the spring, the force is also doubled.
The four damping springs of a car's wheels have a higher spring constant than the
spring in your ball pen. The spring constants of the commercially available
cantilevers vary over four orders of magnitude; cantilevers with spring constants
between 0.
005 N/m and 40 N/m are commercially available. You can deduce the
properties of a cantilever from its outer shape. Thicker and shorter ones tend to be
stiffer and have higher resonant frequencies.
chip
cantilever

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2. Imaging modes
2.1 Feedback and imaging control
The detection system measures the cantilever deflection as the tip is moved over
the surface by the scanning system. It is possible to scan laterally over the surface
without c
hanging the height of the cantilever and just measure this deflection
signal. This is known as “constant height” imaging, but is not the most common
solution. The force applied by the cantilever depends on the deflection, so higher
parts of the sample will experience higher forces in this mode.
It is much more common to use some form of feedback loop to monitor the
cantilever response, and adjust the height of the cantilever accordingly to take
account of the changes in surface height. In this case, th
e base of the cantilever is
moved up and down over higher and lower parts of the sample. All parts of the
sample should now experience the same force, if the system is well set up, and the
maximum force can be controlled.
A “PI” controller is often used
to control the imaging, which means that
proportional-
integral feedback is used. The difference between the setpoint and
actual values is used to change the height position of the cantilever. There are two
values to set how the height position is update
d; a time constant for the integrator
and a value for the proportional gain. These two values control how quickly the
feedback responds to a change in sample height. The actual values need to be
optimized for different imaging conditions, depending on th
e sample topography
and scan speed for example.
If a value of the cantilever deflection is selected then the feedback system adjusts
the height of the cantilever to keep this deflection constant as the tip moves over
the surface. Thus the microscope ima
ges using “constant force” rather than
constant height. When the deflection of the cantilever is used as the feedback
signal, this is usually known as contact mode imaging.
2.2 Amplitude feedback in dynamic modes
There are other ways of operating the syst
em, using dynamic modes where the
cantilever vibrates, and this oscillation of the cantilever is measured rather than the
static deflection of the tip. There are different ways to excite the oscillations -
the
cantilever substrate can be shaken directly,
or a magnetic field can be used to
drive the cantilever itself if it is coated with a ferromagnetic layer. In aqueous
conditions, the most common technique is to drive the cantilever acoustically
through the liquid. In all these cases, however, the measu
rement of the cantilever
oscillation and control systems are similar, and the cantilever is usually driven
close to resonance.
In these dynamic modes, a setpoint amplitude is chosen, and the height adjusted
to match this amplitude through the feedback system. In addition to the height and
error signal information from this constant amplitude mode, the phase between the

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drive signal and the cantilever can also be measured. There are several different
dynamic modes, depending on how much of the oscillation
cycle the tip actually
makes contact with the surface.
Intermittent contact mode is widely used, and can give a combination of the
benefits of the other modes. The cantilever oscillates and the tip makes repulsive
contact with the surface of the sample
at the lowest point of the oscillation. The
lateral forces can be much lower than contact mode, since the proportion of the
time where the tip and sample are in contact is quite low. There may be a higher
normal force between the tip and sample when they are in contact, however.
In non-
contact mode the cantilever oscillates close to the sample surface, but
without making contact with the surface. This mode is not so widely used, since
the attractive force means that there is a possibility of the tip ju
mping into contact
with the surface. The capillary force makes this particularly difficult to control in
ambient conditions. Very stiff cantilevers are needed so that the attraction does not
overcome the spring constant of the cantilever, but the lack of
contact with the
sample means that this mode should cause the least disruption.
Another mode is possible, where the tip does not leave the surface at all during the
oscillation cycle. This is something like a dynamic form of contact mode, and is
usually called force modulation mode.
2.3 Another way of thinking about imaging modes
The imaging modes can also be thought of in terms of the forces between the tip
and surface. Generally, when two objects are brought together, the long range
forces are attra
ctive, and the force becomes repulsive when the objects are close
together. The longer-
range attractive forces are usually van der Waals forces and
capillary forces, and then the repulsive interaction takes over at short ranges, when
the objects are in “c
ontact” and the electron orbitals begin to overlap. The situation
may be a lot more complex, however, when electrostatics and other interactions
from soft samples in liquid are taken into account.
Broadly, though, a general curve can be drawn of the tip-
sample force against
distance, and the different operating modes can be matched with different parts of
the curve. An example is shown, which demonstrates the main features. The
curve is a general approximation, however, and different samples will have
very
different curves in practice. Negative force (below the axis) is attractive in this
diagram, and positive force (above the axis) is repulsive. As the tip and sample
approach from a long distance, the attractive force increases to some minimum in
th
e curve. Approaching beyond this minimum reaches a relatively sharp upwards
part of the curve into the repulsive regime.