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Jiye Cai Editor
Atomic Force
Microscopy in
Molecular and
Cell Biology
Atomic Force Microscopy in Molecular and Cell
Biology
Jiye Cai
Editor
Atomic Force Microscopy in

Molecular and Cell Biology
Editor
Jiye Cai
State Key Laboratory of Quality Research in Chinese Medicines
Macau University of Science and Technology
Macau, China
Department of Chemistry
Jinan University
Jinan, China
ISBN 978-981-13-1509-1 ISBN 978-981-13-1510-7 (eBook)

https://doi.org/10.1007/978-981-13-1510-7
Library of Congress Control Number: 2018957463
© Springer Nature Singapore Pte Ltd. 2018

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the
editors give a warranty, express or implied, with respect to the material contained herein or for any errors
or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations.
This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore
Preface
The progress in biomedical and life sciences made in twenty-first century is signifi-
cant to human health. On the other hand, the human body is formed by a huge number
of cells and each of them is composed of countless biomolecules. To improve the
efficacy of therapies for human diseases, a better understanding of how different types
of human cells work in both physiological and pathological conditions is crucial. Cell
biomolecules, including proteins, DNA, and RNA, cooperate to form a living system
that can realize cell endocytosis and exocytosis, growth, migration, proliferation, dif-
ferentiation, and apoptosis. Each biomolecule is in the size range of a few nanometers
to dozens of nanometers, and these biomolecules work together like a nanoscale
“robot.” However, nanoscale biomolecules are too small to be imaged with traditional
microscopes, and vacuum-based electron microscopes cannot be used to image living
cells in physiological conditions. Fortunately, the development of scanning probe
microscopy(SPM), such as scanning tunneling microscopy(STM), near-field scan-
ning optical microscopy(NSOM), and atomic force microscopy (AFM), provides a
new type of powerful tool for life science and cell research. Scanning probe micro-
scopes can be used to directly image cell surface at an atomic/molecular level resolu-
tion and detect local cellular properties in different conditions even in solution. With
SPM great achievement has been realized in various research fields.
The aim of this book is to describe AFM and introduce some of recent important
results by using AFM. The main contents include (i) principles of AFM, (ii) in situ
single molecule imaging of cell membranes, (iii) single molecule force spectros-
copy, (iv) high resolution imaging of cells, (v) mechanical property changes of dis-
eased versus healthy cells, (vi) observation of cell-drug interactions, (vii) novel high
speed high resolution AFM techniques, and (viii) established and potential applica-
tions for AFM in molecular and cell biology. The authors are experts in AFM appli-
cation and working in cell research for a long time. I believe this book will provide
a useful reference to the people interested in AFM and its application including but
not limited to cell biomolecules and life science.
Institute of Chemistry, Chinese Academy of Sciences Li-Jun Wan

Beijing, China
v
Acknowledgments
I am grateful to Ms. Becky Zhao and Springer Nature for their many efforts toward
completing this book.
I am grateful to all chapter authors of this book and many journals who permitted
to quote their published papers as references in this book.
I am grateful to State Key Laboratory of Quality Research in Chinese Medicines,
Macau University of Science and Technology, and FDCT(Macau) 028/0416/A1 for
supporting our work and helping us in completing this book.
vii
Contents
1 Principles of Atomic Force Microscopy �� 1

Wanxin Sun
2 Atomic Force Microscopy-Based Single Molecule Force
Spectroscopy for Biological Application�� 29
Chao Tang, Youjie Fan, and Junhong Lü
3 In Situ Single Molecule Detection on Cell Membrane
and Label Molecule Distributions Using AFM/NSOM�� 41
Jiang Pi, Hua Jin, and Jiye Cai
4 AFM Imaging-Force Spectroscopy Combination
for Molecular Recognition at the Single-Cell Level�� 55
Filomena A. Carvalho and Nuno C. Santos
5 Atomic Force Microscopy: A Nanoscopic Application
in Molecular and Cell Biology�� 77
Huai-Hong Cai, Xueyi Zeng, Xiao Tang, and Jiye Cai
6 The Hyphenated Technique of High Speed Atomic Force
Microscopy and Super Resolution Optical Detection System�� 105
Xiao Feng, Yunchang Guo, Hongjie An, and Hongshun Yang
7 AFM and NSOM/QD Based Direct Molecular Visualization
at the Single-Cell Level�� 131
Liyun Zhong, Jiye Cai, and Zhengwei Chen
8 Assessment of Pathological or Drug-Dependent Changes
in Cell Membrane Morphology and Cell Biomechanical
Properties by Atomic Force Microscopy�� 147
Hua Jin, Yue Zhao, Wandang Wang, Jinhuan Jiang, Jiye Cai,
and Colin E. Evans
ix
x Contents
9 In Situ Measuring Mechanical Properties of Normal

and Disease Cells�� 161
Sui-Ping Deng, Yi-Li Yang, Xing-Xing Cheng, Wen-Rong Li,
and Jiye Cai
10 High Resolution AFM and Its Applications�� 179
Hao Sun, Ming Ye, and Wanxin Sun
List of Contributors
Hongjie An Division of Physics and Applied Physics, Nanyang Technological

University Singapore, Singapore, Singapore
Huai-Hong Cai College of Chemistry and Materials Science, Jinan University,
Guangzhou, China
Jiye Cai State Key Laboratory of Quality Research in Chinese Medicines, Macau
University of Science and Technology, Macau, China
Department of Chemistry, Jinan University, Jinan, China
Filomena A. Carvalho Instituto de Medicina Molecular, Faculdade de Medicina,
Universidade de Lisboa, Lisbon, Portugal
Zhengwei Chen Department of Microbiology and Immunology, Center for
Primate Biomedical Research, University of Illinois College of Medicine, Chicago,
IL, USA
Xing-Xing Cheng Department of Chemistry, College of Chemistry and Materials
Science, Jinan University, Guangzhou, People’s Republic of China
Sui-Ping Deng Department of Chemistry, College of Chemistry and Materials
Colin E. Evans Department of Pediatrics, Feinberg School of Medicine,
Northwestern University, Chicago, IL, USA
Youjie Fan JPK Instruments AG, Shanghai, China
Xiao Feng Food Science and Technology Programme, c/o Department of
Chemistry, National University of Singapore, Singapore, Singapore
Yunchang Guo JPK Instruments AG, Shanghai, China
Jinhuan Jiang State Key Laboratory of Quality Research in Chinese Medicines,
Macau University of Science and Technology, Macau, China
xi
xii List of Contributors
Hua Jin State Key Laboratory of Quality Research in Chinese Medicines, Macau
Feinberg School of Medicine, Northwestern University, Evanston, IL, USA
Junhong Lü Shanghai Institute of Applied Physics, Chinese Academy of Sciences,
Shanghai, China
Wen-Rong Li Department of Chemistry, College of Chemistry and Materials
Jiang Pi Department of Immunology and Microbiology, University of Illinois at
Chicago, Chicago, IL, USA
State Key Laboratory of Quality Research in Chinese Medicines, Macau University
of Science and Technology, Macau, China
Nuno C. Santos Instituto de Medicina Molecular, Faculdade de Medicina,
Universidade de Lisboa, Lisbon, Portugal
Hao Sun Bruker Nano Surface, Singapore, Singapore
Wanxin Sun Bruker Nano Surface, Singapore, Singapore
Chao Tang Shanghai Institute of Applied Physics, Chinese Academy of Sciences,
Shanghai, China
Xiao Tang College of Chemistry and Materials Science, Jinan University,
Guangzhou, China
Wandang Wang State Key Laboratory of Quality Research in Chinese Medicines,
Macau University of Science and Technology, Macau, China
Yi-Li Yang Department of Chemistry, College of Chemistry and Materials Science,
Jinan University, Guangzhou, People’s Republic of China
Hongshun Yang Food Science and Technology Programme, c/o Department of
Chemistry, National University of Singapore, Singapore, Singapore
Ming Ye Bruker Nano Surface, Singapore, Singapore
Xueyi Zeng College of Chemistry and Materials Science, Jinan University,
Guangzhou, China
Yue Zhao State Key Laboratory of Quality Research in Chinese Medicines, Macau
Liyun Zhong School of Information and Optoelectric Science and Engineering,
South China Normal University, Guangzhou, Guangdong, China
About the Editor
J iye Cai, Ph.D., Distinguished Professor of State Key

Laboratory of Quality Research in Chinese Medicines,
Macau University of Science and Technology;
Professor of Department of Chemistry, Jinan
University. 1962–1968 studied and graduated from
Department of Physics, Peking University; 1983–1985
visiting scholar at Columbia Radiation laboratory,
Columbia University; 1992–1994 senior visiting
scholar at Department of Chemistry, Stanford
University. He and his collaborator have published
more than 100 SCI papers and 12 patents. He has
trained over 60 graduated students and 25 students
have been professors and senior engineers.
xiii
Chapter 1
Principles of Atomic Force Microscopy
Wanxin Sun
Abstract Clearly understanding the working principles of different modes of

atomic force microscopy (AFM) is important for users to choose suitable measure-
ment modes for their research projects, optimize working parameters, identify arti-
facts, and interpret data. In this chapter, conventional imaging modes and force
modes will be discussed first, followed by the introduction of recent developments
in AFM quantitative nano-mechanical properties measurement.
Since it was invented three decades ago (Binnig G, Quate CF, Gerber C, Phys
Rev Lett 56:930–933, 1986), AFM has been becoming a more and more important
instrument in nano science and technology. The uniqueness of AFM is its capability
of providing nanometer spatial resolution in three dimensions while no vacuum or
contrast reagent is needed. AFM has been extensively used in virtually every branch
of science and engineering and contributes to many discoveries in nanomaterials,
such as the discovery of graphene. In recent years, AFM has been further developed
in three aspects. 1. conveying more material related information, such as mechani-
cal, electrical, magnetic and thermal properties at nanometer scale; 2. integrating
with different advanced optical techniques, including Raman, fluorescence, infrared
spectroscopy; 3. incorporating with environment control for life science and mate-
rial researches, such as temperature, liquid environment with pH and other ion
strength control, light illumination. With these developments, AFM has been
extending it applications beyond topographic imaging, such as polymer phase tran-
sition under different temperature, I-V characteristics in today’s semiconductor
devices, live cell dynamics under different chemical/mechanical stimuli, molecular
dynamics under different temperature and chemical environments.
On the other hand, the expanded capabilities of AFM make it difficult for users
to choose a proper measurement mode, suitable probes and optimize operation
parameters. Many efforts have been made to develop different smart scan modes,
including peak force tapping developed by Bruker, where software can tune opera-
tion parameters to achieve optimized image quality. However, it is still users’ task
W. Sun (*)
Bruker Nano Surface, Singapore, Singapore
e-mail: wanxin.sun@bruker.com
© Springer Nature Singapore Pte Ltd. 2018 1

J. Cai (ed.), Atomic Force Microscopy in Molecular and Cell Biology,
https://doi.org/10.1007/978-981-13-1510-7_1
2 W. Sun
to choose measurement modes, identify artifacts, interpret data for their research
projects. All these need users to clearly understand the working principles of
different modes. In this chapter, conventional imaging modes and force modes will
be discussed first, followed by the introduction of recent developments in AFM
quantitative nano-mechanical properties measurement.
1 AFM Working Principles
In AFM, a sharp probe runs a raster scan across the sample surface with a position-
ing accuracy in sub-nanometer level. During the scan, the probe is moved up and
down by a feedback close loop to maintain a constant probe-sample interaction. The
vertical movements are recorded against XY position to form a surface topography
of the sample, as shown in Fig. 1.1. The nano positioning in an AFM is achieved by
Computer
Controller
Laser & Coupling

Optics
Signal
Processing
A B Mirror
C D
XYZ Scanner
Sample
Fig. 1.1 Schematic diagram of an atomic force microscope. (The diagram is not in scale.) The
laser emitted from the laser diode is focused onto the end of the cantilever. The reflected laser beam
is redirected to a quadrant photodiode. The vertical and lateral movements of the cantilever is
detected by the photodiode. The feedback close loop is implemented in the controller. The com-
puter is used to setup parameters for the controller and collect data from it to form images
1 Principles of Atomic Force Microscopy 3
piezoelectric scanners, where the movements in XYZ are driven by high voltages in
the range of hundreds of volts. Intrinsically, piezoelectric material does not response
linearly to the voltage applied. If linear voltages are applied to the scanner, the
recorded images are distorted by the nonlinear motion of piezo scanner. To correct
this issue, there are two prevailing methods in today’s AFMs. One is to add XYZ
position sensors to monitor actual movements of the scanner and correct the nonlin-
earity through a feedback close loop, where the voltages applied to the scanner are
tuned until the scanner reaches the desired positions. This method is usually called
close loop. Different position sensors have been developed for AFM, including
capacitive sensor, inductive sensor, strain gauge, and optical sensors. All of them
work well as long as they are implemented properly. The other approach is to model
the nonlinearity first, and then use nonlinear voltage to drive the scanner to obtain
linear movement. In this approach, a certified calibration grid is scanned by the
scanner with different scan speed, scan size, scan angle etc. After a series of images
are collected, the parameters in the model of scanner movement against applied
voltage and scan conditions are extracted by fitting all the images. This method is
usually called open loop. Traditionally, open loop is used for high resolution scan as
it does suffer from the added noise from the position sensors. With the advances in
sensor developments, close loop in today’s AFM can achieve similar high resolution
performance to open loop. Therefore, more and more users use close loop for rou-
tine sample measurement and open loop for atomic resolution measurement.
The commonly used AFM probes consist of a sharp tip and a micro cantilever, as
shown in Fig. 1.2. The radius of curve of the today’s AFM tip ranges from a few nm
to 30 nm, depending on fabrication process and their applications. The micro canti-
lever is 30–40 μm in width and 125–450 μm in length. The thickness ranges from a
fraction of μm to a few μm.
Fig. 1.2 Scanning electron

microscopic image of a
typical AFM probe,
consisting of a sharp tip
and a micro cantilever
4 W. Sun
The cantilever works as a sensor to detect the probe-sample interaction. The

interaction between the probe and sample surface is complicating and many types
of forces are involved [2]. The detailed discussion about the origins of the interac-
tion forces is beyond the scope of this chapter. Only the aspects directly related to
AFM instrumentation and applications, for example, the magnitude of the overall
interaction forces, cantilever dynamics changes due to force gradient or energy dis-
sipation during probe-sample interaction, will be discussed. The normal force
between the probe and the sample is measured by the cantilever bending, which
simply follows Hook’s law,
F = k Dz (1.1)
where k is the spring constant of the cantilever, Δz is the cantilever bending in nm.
With a variety of cantilevers available commercially, AFM can measure forces
ranging from a few pN to hundreds of μN.
To measure the tiny bending or twisting of cantilever, an optical lever detection
scheme is well adopted in AFM, as shown in Fig. 1.3. The laser beam from a laser
diode is focused onto the end of the cantilever. The reflected beam from the cantile-
ver surface is redirected to a quadrant photodiode, which is usually called position
sensitive photodiode (PSPD) in AFM. When the cantilever bends vertically, the
direction of the reflected laser beam changes accordingly. Then the laser spot on the
PSPD shifts vertically. In the same way, the twist of the cantilever will cause laser
spot on PSPD move laterally. The position of the laser spot is measured by the out-
put of PSPD, i.e. ((A + B)-(C + D))/(A + B + C + D) is proportional to vertical posi-
tion. ((A + C)-(B + D))/(A + B + C + D) is proportional to lateral position. The
Mirror
A B
C D
Fig. 1.3 Schematic diagram of optical lever detection system. The solid line drawing and dashed
line drawing are laser beams for two cantilever deflection states
normalization to the sum signal of (A + B + C + D) is to eliminate the effect of

cantilever reflectivity. It is noted that the optical lever measures the angle of cantile-
ver deflection, not the displacement. Therefore, short cantilever is more sensitive to
detect deflection changes.
2 Contact Mode
In contact mode, the normal force, i.e. the vertical deflection of cantilever, is main-
tained at a constant during scan. When the probe scans across a protruding feature,
the cantilever is pushed up, generating an error in vertical deflection. To eliminate
this error, the controller will lift the probe until the error becomes zero. For a
recessed feature, the probe is lowered to eliminate the deflection error. With an opti-
mized feedback loop, the probe tracks the surface with a constant force to form the
topography of the sample. During the scan, lateral/frictional force always exists
between the probe and sample. Lateral force can form an image mapping the lateral/
friction force distribution across the sample surface, which is called lateral force
microscopy (LFM). This has been used to study self-assembled monolayer [3],
where different function groups show different frictional forces while their heights
are almost the same. While providing rich information about local friction, lateral
force also causes problems, for example, lateral force, if not controlled properly, can
cause damage to delicate samples and wear out the sharp tip. Take live cells as an
example, the measured Young’s modulus is in the magnitude of kPa. Force higher
than a few nN will cause significant deformation, which leads to low resolution,
damage of features and molecules on the cell membrane, and inaccurate morphol-
ogy. In addition, the force exerted by probe can also work as mechanical stimuli,
which may induce a series of responses of cells, including cytoskeleton, focal adhe-
sion complex etc. For biomolecules, large force may induce conformation changes.
For live cells, the force should be control from sub nN to several nN. For biomole-
cules, the force should be ideally controlled to lower than 100pN.
To achieve sub nN force control, cantilever spring constant is a critical parame-
ter. Softer cantilever deflects more under the same load, which is favorable for force
detection sensitivity. However, the thermal noise of cantilever and environmental
interference prevent users from using cantilevers with ultralow spring constant. For
example, cantilevers with a lower spring constant suffer more thermal drift with the
temperature variation. The heat generated by the AFM laser or microscope illumi-
nation often causes significant drift in deflection for cantilevers with spring constant
lower than 0.01 N/m, in the range of a few volts in the PSPD output. Besides tem-
perature, protein adsorption on the cantilever generates stress on one side, resulting
in observable deflection change, which has been used as a sensor for protein detec-
tion [4]. The drift in cantilever deflection causes instability in imaging. For example,
if the cantilever deflection increases with time, the probe will be lifted from the
sample surface gradually. This is because the increase in cantilever deflection means
higher repulsive force to the controller although it is caused by cantilever deflection
6 W. Sun
drift only. The response of the controller is to lift the cantilever. The probe may not
track the surface anymore if the lift is significant, resulting in part of the image not
bearing any sample information. To overcome this issue, higher force must be set to
compensate the cantilever drift. Then the image is not obtained at a constant force.
Higher force in some part of the image will cause the deleterious effects discussed
above. Therefore, too stiff or too soft cantilever is not advisable for live cell imag-
ing. In practice, cantilevers with a spring constant between 0.01 N/m and 0.1 N/m
are usually good for imaging live cells. This kind of cantilever is made of silicon
nitride in V shape, as shown in Fig. 1.4. Silicon nitride is less reflective than silicon.
To increase its reflectivity, a thin layer of Au or Pt is coated on the backside of the
cantilever. The thermal expansion coefficient of metal is different from that of sili-
con nitride, making cantilever bend with temperature change. To relieve the thermal
drift, Bruker released a probe with Au coating only at the end of cantilever for laser
reflection, the rest of cantilever is uncoated. This probe is insensitive to temperature
change and good for cell imaging and force measurement.
AFM can image cells under pseudo physiological conditions with temperature
and chemical environment control. To image with AFM, cells must be attached to a
support surface. 50–70% confluence is a good trial for most cell types. Too low
concentration will make it difficult to locate a cell for AFM imaging. Too high con-
centration sometimes causes cells overlapping with each other or loosely attached.
A large number of cell types can adhere directly onto glass cover slip and plastic
ware, e.g. petridish, by normal cell culturing. Whenever needed, Bunsen burner
flame treatment [5] and adhesives [6, 7] can be used to enhance cell adhesion. The
commonly used adhesives include polylysine, collagen, laminin, Cell-Tak, and PEG
derivatives. To avoid debris or unattached cells sticking to the cantilever during
imaging, it is advisable to rinse the cell preparation with filtered medium or buffer
to remove the cell debris and unattached cells. To maintain the viability of cells,
today’s biological AFM is equipped with temperature control, CO2 atmosphere con-
trol, gas purging, and perfusion apparatus.
Fig. 1.4 Scanning electron

microscopic image of a
typical silicon nitride probe
suitable for liquid imaging.
This kind of probe usually
has several cantilevers with
different spring constants
As discussed previously, contact force is critically important for live cell imag-
ing. Force adjustment is achieved by changing a parameter called deflection set-
point, at which the cantilever deflection is maintained during scan by the feedback
loop. Larger setpoint means higher force. A force between 200pN and 500pN is a
good starting point to try for contact mode imaging. A scan rate of 0.5–1 Hz is usu-
ally used for cell imaging. In AFM software, the line profiles in both directions
(trace and retrace) are displayed. By adjusting the feedback loop gains and scan
speed, the trace and retrace profiles will overlap with each other. Higher feedback
loop gains make the probe tracking surface faster. On the other hand, oscillation will
happen if the gains are set too high. In real operation, increase the gains until slight
oscillation is observed. Then decrease the gains slightly to eliminate the oscillation.
The highest gains without oscillation are the gains desired. With light force, AFM
can produce the fine details on cell surface and accurate height of the cell. Accurate
cell height is important to measure cell volume change of neurons during apoptosis.
With large force, large deformation on the cell membrane is induced by the probe.
The probe can “feel” the hard cytoskeleton filaments, the deflection error image
provides rich information on cytoskeleton, as shown in Fig. 1.5. Researchers some-
times increase the force intentionally to get better contrast in cytoskeleton images.
In most time, the cantilever deflection baseline (deflection before the probe
touches sample surface) is not zero due to thermal drift or laser adjustment. We need
to consider the baseline when we set the setpoint. For example, if the deflection
baseline is 200pN, and we want to use 300pN force to image, then setpoint will be
500pN. Therefore, deflection baseline must be measured before imaging. Force-
distance curve is well accepted to determine the baseline. It will be discussed in
details in the force measurement section. Here we just briefly introduce how to use
force-distance curve to determine deflection baseline. After the cantilever is engaged
onto the sample surface, the XY scan is stopped and the cantilever is ramped up and
down at a specific point by the Z scanner. The cantilever deflection, which is propor-
tional to the force, is recorded against the Z position of the cantilever. The typical
Fig. 1.5 Cytoskeleton

revealed by deflection error
in contact mode
8 W. Sun
500 Extension
Retraction
400
300
Force (pN)
200
100
-100
-200
-1000 0 1000 2000 3000 4000 5000 6000 7000 8000
Z (nm)
Fig. 1.6 Force-distance curve measured on a live cell. The solid line records the cantilever bend-
ing with the probe approaching the cell. The dashed line records the cantilever bending with the
probe retracting from the cell. The retraction curve shows an adhesion force of about 150pN
force-distance curve on live cell is shown in Fig. 1.6. The flat region on the left is
the cantilever deflection when the probe is not in touch with sample. The average of
the flat region will be used as deflection baseline. After the force-distance curve is
measured and a proper setpoint is set, the AFM is switched back to imaging mode.
3 Tapping Mode
Tapping mode is also known as intermittent contact mode or AC mode, where the
cantilever is oscillated at its resonance frequency or slightly lower frequency. The
oscillation is usually driven by a small piece of piezoelectric material embedded in
the probe holder. The cantilever oscillation is measured by the changes in laser posi-
tion on PSPD. The resonance frequency is found by sweeping drive frequency and
finding the maximum oscillation amplitude. When the probe is brought to the sample
surface by the engaging mechanism, the interaction between tip and sample causes
decrease in oscillation amplitude and time lag between the drive signal and cantilever
oscillation. At the bottom-most point of each oscillation cycle, the tip contacts the
surface instantaneously. That is why tapping mode is also called intermittent contact
mode. The amplitude decreases further when the probe is brought closer to the sur-
face, and vice-versa. Thus, the amplitude is a direct measure of tip-sample interac-
tion and the probe is moved up/down during XY raster scan to maintain constant
amplitude. The up/down movements against XY position are recorded to form
topography. The time lag is measured by the phase difference between drive signal
and cantilever oscillation. Both the amplitude and the phase lag are measured by a
lock-in amplifier in AFM. Phase lag is caused by the energy dissipation during each
oscillation cycle of the cantilever. When the probe contacts the sample surface, the
sample surface undergoes both elastic and plastic deformation, causing energy lost.
When it retracts from the sample surface, it often needs to overcome the attractive
force and adhesive force. The adhesive force is also a cause for energy lost. Phase lag
is related to viscoelastic properties of the sample. Phase lag mapping, also called
phase imaging, has been extensively used to differentiate different materials [8].
In practical AFM operation, the operation amplitude (amplitude setpoint) is cho-
sen at about 80% of the free cantilever amplitude. The origin of the phase shift is the
resonance frequency shift under tip-sample interaction. How much the phase shifts
depends on the material viscoelasticity and the operation parameters. Although
phase image has been used extensively in differentiating different materials, such as
phase separation in copolymer [9], there is ambiguity in material identification due
to its intrinsic nature that phase shift is affected by operation parameters as well as
material viscoelasticity. The contrast in phase image can be reversed under different
operation conditions. Under light tapping, phase contrast is caused by the adhesive
force sensed by the probe. With the increase in tapping force, the material deforma-
tion contributes more in the phase contrast. Therefore, the phase contrast under light
tapping originates from the adhesiveness, which is often affected by relative humid-
ity in ambient environment. In liquid environment, phase contrast under light tap-
ping has been used to recognize molecules [10]. Under hard tapping, the phase
contrast is the combination of adhesive force and material deformation.
Compared with contact mode, the probe contacts sample surface instantaneously
in tapping mode. The lateral force is negligible. Tapping mode is good for loosely
bound samples, which are easily pushed away by the probe in contact mode. In
general, biomolecules and nanoparticles are bound to a substrate loosely. For exam-
ple, proteins and DNAs are usually bound to mica by charges. In contact mode, the
image is not stable as the molecules move with probe during scan. Tapping mode is
well adopted for general imaging because it in general induces less sample damage
and tip wear, and generates sharper images than contact mode. However, tapping
mode is slower than contact mode if the same scanner is used. In principle, it is the
cantilever oscillation amplitude used to control the movement in Z direction in tap-
ping mode. Scanning across a recessed feature, such as a hole, it takes time in the
scale of milliseconds for the cantilever oscillation amplitude to increase. Therefore,
the cantilever dynamics is the bottleneck in tapping mode. No matter how fast the
scanner is, 1–2 Hz is usually used in tapping mode for rough samples. In contrast,
the deflection in contact mode changes about 1000 times faster than amplitude in
tapping mode. The bottleneck is the scanning mechanism rather than the cantilever
itself. For samples with large feature heights, such as cells, contact mode is pre-
ferred as it can scan faster over a large area.
Different cantilevers have different resonance frequency. For each cantilever
mounted into the instrument, its resonance frequency is usually determined by
sweeping frequency and searching the maximum oscillation amplitude, which is
10 W. Sun
done by a function called “auto tune” in software. The viscoelasticity and density of
the medium surrounding the cantilever affect its resonance significantly. In aqueous
solution, the resonance frequency drops significantly compared with that in air. For
example, the resonance frequency in air of a commonly used silicon nitride cantile-
ver is around 50KHz, while it is about 15KHz in water. In air, the response curve in
“auto tune” shows a symmetric Gaussian profile and software can detect its reso-
nance frequency automatically. The same cantilever may show a forest of peaks in
aqueous solution. Traditionally, AFM users follow the guideline in the AFM manual
to choose a suitable frequency. However, with more and more probes are designed
for different applications, it may not be easy to find a recommended frequency in
manuals or references for new probes. In today’s AFM, thermal tune is usually
equipped to determine the cantilever spring constant. The details of thermal tune will
be discussed in the force measurement section. For the sake of easy reading, its prin-
ciple is briefed here. The thermal noise of the cantilever is measured with PSPD after
the tapping piezo is stopped. The power spectra density is calculated for different
frequency. Then the resonance frequency is clearly identified, as shown in Fig. 1.7.
The cantilever oscillation amplitude affects the imaging stability and resolution.
Larger oscillation amplitude is preferred for rough surface, and smaller amplitude is
preferred for high resolution. To make the data consistent and comparable among
different AFM instruments, it is desirable to use the same oscillation amplitude, at
least comparable if not the same. In practical operation, the amplitude measured by
PSPD and lock-in amplifier is a voltage. Different AFM may have different gain in
PSPD amplifier and different optical path in the optical lever detection scheme.
Therefore, the same voltage output from the lock-in amplifier may measure differ-
1.4
Power Spectra Density (pm2/Hz)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0 20 40 60 80 100
Frequency (Hz)
Fig. 1.7 Power spectra density of a silicon nitride cantilever measured in water. The resonance
frequency in water is 15 kHz while it is 57 kHz in air
ent oscillation amplitude. Then the scaling factor between voltage and amplitude in
nm, also called amplitude sensitivity, must be determined for each type of probe.
Here a simple method is described to determine the amplitude sensitivity, the same
method can also tell whether the frequency chosen is suitable for tapping mode
imaging in aqueous solution. Similar to the force-distance curve in contact mode,
the cantilever in tapping mode is ramped by the Z scanner while XY position is
fixed. The oscillation amplitude is recorded against the Z position, as shown in
Fig. 1.8. When the probe is far from the sample surface, the amplitude does not
change significantly with Z position. The slight decrease is caused by the damping
effect from the surrounding medium. The narrower the gap between the cantilever
and sample surface, the stronger the damping. Once the probe touches the surface at
the bottom-most point of each oscillation cycle (shown as mark A in Fig. 1.8), the
amplitude starts to decrease rapidly as it is pushed against the surface further. Mark
B is the position where the probe fully contacts the sample surface. The slope
between A and B is used to measure amplitude sensitivity in nm/V. The steeper the
slope, the better the cantilever for imaging as steeper slope means oscillation ampli-
tude is more sensitive to height changes in sample surface. When there are more
than one peaks in the tuning curve, the one with steepest slope should be chosen for
tapping mode imaging. The amplitude dropping 20% from the corner A is usually a
good setpoint.
0.30
A
0.25
0.20
Amplitude (V)
0.15
0.10
0.05
B
0.00
0 50 100 150 200 250 300

Z (nm)
Fig. 1.8 Cantilever oscillation amplitude changes with the Z position in ramp mode. Point A
marks the cantilever starts to touch the sample surface at the bottom-most point in each oscillation
cycle. Before that, the oscillation amplitude does not decrease significantly. After that, the ampli-
tude decreases rapidly with further decrease in probe-sample distance, until the probe fully con-
tacts the surface, marked as B in the figure. The slope from A to B is used to determine the
amplitude sensitivity
12 W. Sun
Regarding the optimization of feedback loop gains, the tapping mode is similar
to contact mode. As discussed earlier, cantilever deflection changes much faster
than oscillation amplitude. Contact mode can tolerate higher gains than tapping
mode. For example, the gains high enough to cause oscillation in tapping mode may
not cause any deleterious effect in contact mode. Optimization of gains is more
critical in tapping mode. The oscillation in feedback loop caused by high gains
results in noisy images or artifacts. If the gains are too low, the probe will not track
the surface properly, resulting in artifacts in images, increased tip wear and sample
damage. Therefore, experimental parameters must be tuned more carefully in tap-
ping mode. Among all the parameters, proportional gain, integral gain, setpoint and
scan rate are the top parameters to be taken care in tapping mode.
4 PID Feedback Loop
Due to its simplicity and robustness, proportional–integral–derivative controllers

(PID controllers) are well adopted in AFM feedback loops, including XY lineariza-
tion close loop and feedback loop used for topographic imaging. The principle of
PID controller and tuning procedure can be found in many text books on process
control [11] and articles [12]. Here, we just discuss briefly about its working prin-
ciple and the tuning procedure suitable for AFM. The schematic diagram of a PID
feedback loop is shown in Fig. 1.9.
When the interaction between the tip and sample changes, the output from the
optical lever detection system will deviate from the setpoint. The difference E(t) is
called error signal. It is logical that the adjustment in Z voltage, ΔVz, should be pro-
portional to E(t), i.e. ΔVz = PE(t). If the proportional gain P is set too low, the
response of the system is slow also, and the tip cannot track the sample surface with
Proportional
Setting
E(t) Point
Integral
Differential
Change in Z
Voltage Force
XYZ Scanner
sensor
Fig. 1.9 Schematic diagram of PID feedback loop, E(t) is the error signal
a constant probe-sample interaction. If P is set too high, the system will oscillate. The
system with a proportional gain only is not sensitive to small error signals. Even the
error is small, its integration over time might be large if the error has the same sign.
For example, small steady error caused by smooth but slightly slant surface has the
same sign. In this case, the integration of error is used to eliminate the steady error.
For some small but sharp features, error signal is small but changes rapidly. The
proportional and integral components are insensitive to this kind of error. This kind
of error can be reduced if the derivative of the error signal is included the feedback
loop. Synthesizing the three components, PID feedback loop can be expressed as
0
dE ( t )
DVz = PE ( t ) + I ò E ( t ) dt + D (1.2)
t dt
In order to obtain good AFM images and extend the tip’s life time, the PID
parameters must be optimized. The universal method for generic PID feedback loop
will not be discussed in this chapter. The tuning procedure described in this chapter
is adapted for AFM imaging.
1. Set the scan size to a few μm, say 1–2 μm, even the final scan size is tens of μm.
2. Increase the integral gain until slight oscillation or noise appears in the trace/
retrace profiles. The oscillation or noise can usually be eliminated by increase
the proportional gains gradually. If it cannot be eliminated, integral gain should
be decreased until oscillation/noise disappears.
3. Check the trace/retrace profile, if the probe cannot track the falling slope while
the rising slope is tracked properly, reduce the setpoint in tapping mode (or
increase the setpoint in contact mode) gradually to improve the tracking.
4. Reduce scan rate if the probe tracking cannot be improved further by reducing
setpoint. It should be noted that reducing scan rate must be taken as the last
choice because drift and environmental interference might be pronounced during
the long imaging time, resulting in distorted images.
5. Increase the scan size to the desired size. If the tracking becomes poor, repeat
step 2 to 4 until the image quality is acceptable.
To improve the resolution, sharper probe and less tapping force are always pre-
ferred. Less tapping force is usually achieved by smaller cantilever oscillation
amplitude. Therefore, 1/3 of normal tapping amplitude is usually used in molecular
imaging, e.g. DNA, protein, polysaccharide and other biomolecules. Fig. 1.10
shows a DNA image obtained in tapping mode.
5 Force Mode
In force mode, the AFM probe is ramped up/down by retracting/extending the Z

scanner and the cantilever deflection is recorded against Z position, forming a force-
distance curve. A typical force-distance curve measured on mica is shown in
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Fig. 1.10 DNA image

obtained by tapping mode
in buffer solution. The
image size is 2 μm. The
DNA height measured
from the image is more
than 2 nm, showing the
interaction force between
probe and sample is lower
than 300pN, otherwise the
height is in general less
than 2 nm
3
0
Force (nN)
1 2
-5
5
4
-10
0 300 600 900 1200
Distance (nm)
Fig. 1.11 Force-distance curve measured on mica in ambient environment with a relative humid-
ity of 80%. The probe used is a silicon nitride probe (DNP, Bruker)
Fig. 1.11. Before the tip contacts the sample surface, the deflection is a constant,
shown as a flat baseline in the force-distance curve, i.e. the segment 1. When the
probe is close enough to the sample surface, the attractive force gradient may be
greater than the spring constant of the cantilever, snap in will happen, denoted as
2 in the graph. After the probe contacts the surface, the cantilever will bend up with
further push against the surface, shown in segment 3. When the preset force is
reached, the cantilever will retract from the surface, and deflection decreases accord-
ingly, shown as segment 4. The adhesion force between the probe and sample will
pull the cantilever down further till the force generated by the cantilever is equal to
the maximum adhesion force, then the cantilever jumps off from the surface, shown
as segment 5, which is usually used to measure the adhesion force. The adhesion
force is often used to determine the binding force between antigen and antibody,
study surface hydrophobicity by measuring capillary force, and conduct molecular
recognition. In ambient environment, capillary force dominates the overall adhesion
force. The magnitude of capillary force is determined by the relative humidity and
surface hydrophobicity [13]. If the probe and sample are put into controlled envi-
ronment, interaction mechanism, e.g. the types of interaction force and how the
environment changes the interactions, can also be studied. In segment 3, the sample
undergoes deformation as well as the cantilever bends up. By studying the relation-
ship between sample deformation and force, material stiffness and modulus can be
obtained. If sample undergoes significant plastic deformation, segment 3 and 4 will
separate. To obtain modulus under that situation, segment 4 instead 3 will be used
to eliminate the effect of plastic deformation. The area enclosed by the different
segments is the energy dissipated during each ramp cycle. Material mechanical
properties, e.g. Young’s modulus, adhesion force, energy dissipation can be extracted
from the force-distance curve. In force mode, XY scan is stopped and no lateral
force is applied on the sample surface. Scratching on sample surface is rarely
observed, unlike contact mode, where the lateral force exists always. In today’s
AFM, ramps can be programmed at user defined positions or in an array, which are
usually implemented by “point and shoot” or “force volume”. A series of force-
distance curves can be used to construct the topography at a specific force. This
method is usually used to image very soft or sticky samples, which are difficult for
contact mode and tapping mode.
Comparing force distance curves shown in Figs. 1.6 and 1.11, extension curve
and retraction curve are separated in Fig. 1.6. This is because mica is much harder
than live cells and the deformation of mica is negligible while live cell undergoes
significant plastic deformation. Another obvious difference is the adhesion force, as
shown in segment 5 in Fig. 1.11. Mica is hydrophilic and capillary force is strong
when measured in ambient environment. In case of live cells, nonspecific binding
dominates the overall adhesion force with absence of capillary force. The nonspe-
cific binding force is much smaller than the adhesion force caused by capillary in
ambient environment.
Accuracy in force measurement is important for analyzing the interaction mech-
anism. To obtain accurate force, two parameters need to be calibrated, i.e. cantilever
deflection sensitivity and spring constant. Deflection sensitivity measures how
many nm in cantilever deflection correspond to 1 V in the PSPD output. It is mea-
sured by ramping the probe on a hard surface, for example sapphire. The deforma-
tion of such surface is negligible. So the displacement of Z scanner is the same as
the deflection of the cantilever, i.e. the slope of the segment 3 in Fig. 1.11 should be
1. In force-distance curve, deflection sensitivity is determined by fitting the segment
3 to a straight line to obtain the slope. The deflection sensitivity making the slope to
be 1 is the calibrated value. This process has been automated in today’s AFM. To get
accurate and repeatable value, the snap-in point and the approach/retract turning
16 W. Sun
point are usually excluded from the calculation. Deflection sensitivity is determined
by the cantilever length and the sensitivity of PSPD. Short cantilever produces bet-
ter sensitivity and is preferred in measuring displacement in pm range. For a given
AFM, the cantilevers with the same length should have the same deflection sensitiv-
ity. In real operation, the laser spot may not be aligned to the same position on the
cantilever every time. It is normal that the deflection sensitivity of the same cantile-
ver varies slightly after realigning the laser. This is because the change in laser spot
position affects the effective length of the cantilever. It is a good practice to measure
the deflection sensitivity each time after the laser is re-aligned. With the calibrated
deflection sensitivity, how many nm the cantilever deflects can be calculated from
the deflection in voltage.
For a cantilever with rectangular cross section, its spring constant can be
expressed as
Et 3 w
k= (1.3)
4 L3
where E is the Young’s modulus of the cantilever material, e.g. silicon or silicon
nitride,
t, w, and L are the thickness, width, and length of the cantilever respectively.
The width and length of a cantilever can be controlled precisely through micro
fabrication technology, while the thickness bears more deviation due to its manufac-
turing process. 10% thickness error will result in about 30% error in spring constant.
The nominal spring constant on probe boxes can only be used as an indicative value.
Each cantilever must be calibrated to get correct force value. Several methods have
been developed to measure cantilever spring constant. The simplest way is to mea-
sure the resonance frequency f and check the probe factor b from reference book.
The spring constant is calculated by
k = b* f 3 (1.4)
This method is very easy to use as the resonance frequency can be obtained by “auto
tune” in tapping mode. The major drawback is its poor accuracy because the dimen-
sions may be slightly different from those in the reference. For example, the canti-
lever width affects its spring constant, but not its resonance frequency. If the actual
width of cantilever is different from that in the reference, the calculated spring con-
stant will deviate from the real value.
To improve this situation, top view geometry (length and width) is added into the
equation as [14].
r3 3 3
k = 2p 3 L wf (1.5)
E
where ρ and E are the density and Young’s modulus of cantilever material,
L and w are the length and width respectively, usually measured through a well
calibrated micrograph of scanning electron microscopy or optical microscopy.
A more accurate method was developed in reference [14] by adding a known
mass and measuring the resonance frequency shift, i.e.
M
k = ( 2p )
2
2 2
(1.6)
æ 1 ö -æ 1 ö
ç f ÷ ç f ÷
è lø è 0ø
where M is the mass added to the cantilever free end, f0 and fl are resonance frequen-
cies before and after the mass is added respectively.
This method involves gluing a particle with known mass to the end of cantilever.
It is not trivial even with a detailed protocol available [15, 16]. Furthermore, it is
even more troublesome to remove the particle after the spring constant is calibrated.
Therefore, force measurements are usually done before the calibration. After all
experiments are finished, the calibration is carried out and the measurement results
are rescaled with the correct spring constant. This method is complicating and the
cantilever cannot be used after calibration. This method is rarely used in biological
application despite its accuracy.
All the above discussed methods do not measure cantilever in-situ. In practice,
the laser may not be aligned to the same position as in spring constant measurement.
The difference in laser alignment results in difference in cantilever effective length.
With the advances in AFM instrumentation, the thermal noise of cantilever has been
used to calculate its spring constant. After the deflection sensitivity is calibrated, the
cantilever is lifted from the sample surface by at least 100μm and the random motion
of cantilever free end is recorded for a period of time. 10 seconds are usually enough
to get accurate results. The power spectral density (PSD) is then calculated by
Fourier Transformation over the noise recorded, as shown in Fig. 1.7. According to
Equipartition Theorem [17],
1 1
mw02 z 2 = kBT (1.7)
2 2
where m is the effective mass of the cantilever,

ω0 and z are the angular frequency and noise amplitude of cantilever free end
respectively,
kB and T are Boltzman constant = 1.3805 × 10−23joules/Kelvin and absolute tem-
perature in Kelvin,
〈〉means averaging over time, which is obtained by integrating PSD over
frequency.
1 2 1
Considering kz = kBT , the cantilever spring constant k can be obtained
2 2
k = kB T / z 2 (1.8)
18 W. Sun
The mean square of free end amplitude 〈z2〉 is the area under the peak of power
spectra density profile.
This method is well accepted because it neither relies on the cantilever geometry,
as in method 1 and 2, nor demands the complicating procedure, as in the mass add-
ing method. After further improvement by Butt and Jaschke [18] followed by Hutter
[19], the accuracy achieved by the thermal noise method is reliable [20]. In most of
today’s AFM, this method is automated in software. The frequency range covers
from a few KHz to 2 MHz. Virtually all the commercial available cantilevers can be
calibrated with this method. For some ultra soft cantilevers, the resonance frequency
in liquid is below 2KHz. The Z scanner noise and laser pointing noise in some AFM
may limit the accuracy in spring constant measurement. In such case, the spring
constant measured in air is a good estimation.
Determining Young’s modulus through force-distance curve has been exten-
sively studied [21]. Hertz, DMT, JKR and Maugis models are commonly used to
measure Young’s modulus of materials [22]. In all the models, good understanding
of the probe shape and size is as important as accurate measurement of force. For
sample deformation in a few nm, the AFM tip is usually modeled as a sphere. For
polymer samples, a few nm deformation produces enough force for accurate mea-
surement in AFM. DMT model with spherical probe is usually used [2].
4 * 3
F= E Rd 2 + Fad (1.9)
3
where F is the force measured by the probe,

Fad is the adhesion force between tip and sample,
R is the radius of the tip,
d is the sample deformation,
E
E* is the reduced Young’s modulus = , ν is the Poisson Ratio of the mate-
rial. Young’s modulus of the probe material 1 -nis2 usually much higher than that of the
sample. The deformation of probe is negligible.
Tip geometry can be obtained by either high resolution electron microscopy or
tip deconvolution. In the latter method, a reference sample with sharp features is
scanned and the morphological dilation is analyzed to extract the tip geometry [23].
It may cause tip wear to scan over such reference sample as it is rough and very
hard. So the scan parameters must be optimized carefully. As the relationship
between E* and R is linear, R can also be calibrated after sample measurements
and rescale E* with calibrated R .
For soft materials, such as live cells, the deformation is usually in the range of
tens of nm, a conical shape is a good estimation of tip shape. In liquid, adhesion
force is negligible, Hertz mode with conical tip shape is usually used for live cell
measurement,
2 *
F= E ( tan a ) d 2 (1.10)
p
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CHAPTER XVIII.
A SCOUT, AND A DISCOVERY.
Several days passed.

With the exception of Sunday, the two wide-awake Bird boys put in
all the time possible in learning the ropes. Whenever the weather
was favorable they might be seen careering around the aviation field
in their gallant little Bleriot monoplane, now rising to greater heights
than they had ever dared venture before, and anon coming down in
some daring “spiral” that evoked loud cheers from those who, from
below, witnessed the new maneuver.
The biplane was also in evidence during these times. Puss Carberry
was wise in his day and generation, however. He had awakened to
the fact that in Frank and Andy Bird he had competitors worthy of his
best steel, and that if he hoped to outgeneral them as rivals on the
day of the race he must get busy.
So he and Sandy, instead of soaring aimlessly about, enjoying
themselves, began to study the humor of their airy steed. They found
plenty to do, for the vagaries of an aeroplane are numerous indeed.
Each pair of aviators had its particular little group of backers in the
shape of boys, who stared and cheered as they watched the
evolutions and stunts which were successfully engineered.
Frank was taking no unnecessary risks. There were numerous things
which of necessity they must learn, since at any moment during a
flight such knowledge might be the means of saving their lives. And
he went about the task of understanding just how volplaning down,
boring up and such maneuvers are successfully accomplished by
expert airmen, just as he did everything else he undertook,
determined to be a master of the science before he finished.
It was now Tuesday. The great day was to be Friday, when on
account of the attractive program laid out, it was believed there
would be a greater crowd present in Bloomsbury than at any other
time during Old Home Week.
By degrees the Bird boys had succeeded in mastering their machine,
so that now even Andy could run it successfully. He was proud of the
fact, and yet it might be noticed that he did not go around boasting.
Truth to tell, Andy had serious thoughts these days. In his mind he
was perhaps picturing the time when he hoped to be able to scour
the country down in that tangled wilderness of the isthmus, where his
father’s balloon had vanished from the sight of mortal man. It was a
solemn duty with Andy, and while he said very little about it, even to
his chum, Frank could tell pretty well what was engrossing his
thoughts.
On this afternoon the breeze had died out altogether, as is often the
case in July. Although it was pretty hot, still the day was an ideal one
for flying.
They had been working on the monoplane most of the morning,
installing a searchlight, arranging a barometer close to the
thermometer, so that the pilot could observe the action of the
mercury without turning his head, and even fixing a new barograph
in place. This last instrument Frank had sent for lately and was much
interested in.
“Pretty soon we’ll want to know how high we are flying, and this will
register the top notch,” he said, as they examined the instrument
with interest.
“Makes me think of the self-registering thermometers I’ve seen,”
declared Andy. “You put one out over night and the mercury pushes
down to a certain degree, which is marked as the limit. Then you can
look at it any old time and know, after which you set the register
again for another shy.”
“Well,” said Frank, “this does its work on the same principle, in that it
registers the highest point reached. When there is a contest on for
height, I believe these registers are placed so there can be no
tampering with them, and afterwards they are taken in charge by the
committee and examined.”
“Say, why not take advantage of this afternoon to make a little trip
over in the direction of Old Thunder Top?” suggested Andy, eagerly.
Frank shook his head as he remarked:
“You forget that one of the conditions of entry to that race is that
each contestant agreed not to go within a quarter of a mile of the
mountaintop before the time set for the trial. The idea is that the one
who succeeds may be the actual first person known to have ever set
foot on the summit of the mountain.”
“Oh, shucks! I haven’t forgotten that!” exclaimed Andy, impatiently.
“But we’ve stuck to our old field here, rising and circling, till I’m sick
of seeing it. To tell the truth, I hunger and thirst for new pastures.
There’s nothing to hinder us from taking a lively spin of twenty miles,
if we want, so long as we keep away from that plateau up yonder.
Say yes, Frank!”
Of course the idea appealed to his cousin with equal force and he
could not resist Andy’s plea.
“All right, then. I suppose it would be a good idea to take a little spin
somewhere,” he said. “We’ve mastered the machine pretty well for
novices.”
“Yes,” continued Andy, with a sly grin, “and it ain’t any harder off
there in the forest, in case we fall from five hundred feet, than here in
the open. Fact is, we might have a better chance by lighting in the
branches of a big oak or a chestnut.”
“Well, let’s get ready to make the venture,” said Frank, rising to his
feet; “but I hope all the same that we may not have to try that drop
you joke about. One thing we can do is to climb up so as to get a line
on how high Old Thunder Top really is. There’s always been a hot
dispute about that, you know.”
“That’s so,” observed the other, scurrying around in search of things.
“I’ve heard a high school teacher say it was not over seven hundred
feet, as distance was so deceptive. And on the other hand, a
surveyor who was used to judging distance told me he felt sure it
would go much beyond a thousand feet. Now we’ll get a line on it,
and the day of the race the facts will be known to a dead certainty.”
In a short time both air boys were ready for the flight.
“Put on your coat, Andy,” advised Frank, on seeing that his chum
was about to take his place in his shirt sleeves. “While it’s
oppressively hot down here, don’t you know that five hundred feet up
the air will be chilly. And if we are moving at the rate of a mile a
minute, you’ll be wishing you had your sweater on, mark my words.”
Andy smiled, but all the same he donned his coat. Frank knew, for
there was precious little that escaped him. And Andy, as a rule, was
quite satisfied to take pattern by his wide-awake cousin.
There were a few fellows hanging around. It was holiday time and on
such a hot day they cared precious little for baseball or any other
sport requiring exertion. In their minds it was much finer watching
what the Bird boys would do next, for by now all of juvenile
Bloomsbury had been seized with a severe attack of what Andy
called “aviatus,” and numerous projects for building aircraft were
being discussed in homes and on the commons.
As usual there was a whoop of pure delight from the gathered boys
when the little monoplane started upward. None of them had been
taken into the secret of what new stunts were to be undertaken, but
they anticipated that Frank and Andy must have something up their
sleeves—they looked so mysterious after that conference.
The aeroplane rose with not the slightest hitch. They were getting so
accustomed to handling the machine by now that it seemed second
nature to accomplish a successful rise. Indeed, as yet there had not
been the slightest serious accident to alarm them, since the engine
was cut off so suddenly on that first day.
After circling the field a few times at furious speed, “just to get his
hand in,” as Frank said, the pilot turned the nose of the little flier
straightaway in an easterly direction.
To their ears was borne the faint whoops from the cluster of envious
boys left on the aviation field, as they realized what sort of new
program the young aviators had on tap.
“How high are we now?” asked Andy, as he craned his neck and
looked down upon the country that lay far below, and which was
spread out for miles in all directions around them.
Frank laughed.
“Why,” said he, “you seem to forget that it isn’t necessary any longer
to depend on guesswork, when you’ve got that hunky little barograph
almost under your nose. Suppose you just take a squint and inform
me.”
“Sure, pop!” exclaimed Andy, radiantly. “I had just forgotten all about
that new contrivance. Hey! what d’ye think Frank, we’re sailing along
right now about seven hundred feet up. Phew! that’s going some for
new beginners, eh?”
“It’s fine. I don’t think we’d better go any further just now, but drop
down to within a couple of hundred, and see how the lake and the
woods look up this way.”
“But before you begin to go down, Frank, I want you to take a squint
over toward Old Thunder Top. Just notice, will you, that while we’re
now up seven hundred, that summit is still higher—several hundred
feet, I reckon.”
“Which shows how little that teacher knew,” Frank observed. “Why,
we’ve been up to the foot of that big cliff many a time and if we’d only
had this little business along we’d soon found out that it was close on
a thousand there. Now sit close, for I’m going to spiral down a bit.”
They began to move around in circles in dropping, as the engine was
throttled down. By now Frank had this little trick down to a science,
though he refused to try and show off at any time and thus take
unnecessary risks.
When the aeroplane had reached a level of about two hundred feet
from the earth, the engine was once more accelerated and they
resumed their steady onward progress.
“It’s sure a treat to look down on the lake like this. Beats the view
from up on the side of the mountain all hollow,” presently remarked
Andy.
“Why, yes,” said his companion, as he swerved just a trifle to follow
the contour of the water lying below; “because in this way we can
see the shore line all around.”
“And now, as we’re at the head of the lake, which way shall we
return, Frank?”
“Let’s veer off and cover a few miles of country beyond the
mountain,” the pilot ventured. “It’s wild up here and as a rule none of
us know much about it. Look at the woods, how thick they are, with
only a little opening here and there. I remember once how we came
up here to camp and the snakes were so bad we soon tired of it.”
“That’s so!” Andy exploded. “I hate snakes worse than I do skunks.
Looky here! if that ain’t our old friend Baldy, skating around to take a
squint at the new-fangled bird that’s invaded his exclusive territory.
Hey! won’t he be surprised, though, when we land up there on Old
Thunder Top Friday? Perhaps the old chap may tackle us; we’d
better take a gun along to be prepared.”
But Frank laughed at the idea.
“That would hardly be according to the rules of the race. If we want
to make fast time we don’t want to load ourselves down with all sorts
of traps, Andy.”
“I guess you’re on,” the other remarked. “But say, this country is
pretty rough, all right. I was just thinking what a splendid thing an
aeroplane must be in wartimes. A fellow could soar over the enemy
and learn everything that was going on. I can see a big change in
our ways of fighting when the next war comes along.”
“Oh!”
“What ails you, Frank?” demanded Andy, as he heard his companion
give vent to this exclamation, for naturally his nerves were all on
edge with excitement.
“I saw something just then, that’s all. When you look down don’t
bend over and give the fact away that you’re looking. We’re just
passing over a little opening in the dense woods. I remember that it
is close to that old dilapidated cabin we found when camping up
here. Used to be occupied by charcoal burners once on a time. They
had a scrap and one man was badly hurt, while the other
disappeared.”
“Sure, I remember,” said Andy; “but what of that, Frank?”
“Look in that glade and you’ll get a glimpse of two fellows hiding.
They dropped down in the grass at sight of the airship and are
watching us,” said Frank, quickly.
“I declare that’s so; but who are they, and why do you act that way?”
asked the puzzled Andy.
“Now we’ve passed over. Don’t turn your head back for anything,
because, Andy, that was Jules and his pal, the robbers Chief Waller
has been hunting high and low for so long!”
CHAPTER XIX.
HELPING OUT THE CHIEF.
“Wow! that’s some news, Frank!” cried Andy, doubtless thrilled by

what the pilot of the monoplane had just said.
“I’m positive about it,” the other went on. “Of course, it would be hard
to tell a fellow standing up when you were sailing over his head and
two hundred feet high, but in this case these fellows were lying
down. And I saw their faces, too.”
“Well, why not?” remarked his cousin, thoughtfully. “We’ve believed
all along they must be hiding out somewhere in the woods. And
Frank, what better place could they find than that old cabin? It’s
lonely enough, goodness knows. And there are farms not more than
two miles away, where they might forage for chickens, eggs and
such things.”
“Just what they’re doing, I guess,” remarked Frank.
“And say, didn’t I just hit it right when I remarked about the value of
aeroplanes in wartime?” observed the passenger, with a self-
satisfied chuckle.
“You certainly did; but then, that has been recognized as a fact for
some time now. They’re even using flying machines down on the
Mexican border to locate the doings of the hostile forces. Our
government has a regular aeroplane corps, you know, Andy. And
after this no nation dare go without, for that would be putting them at
a terrible disadvantage.”
“And so those rascals have been bunking in that old cabin all this
time?” mused Andy. “Like as not they went there right after missing
our airship that night. But if they’re hanging around here, Frank, don’t
you think they mean to make another try for this craft? Some fine
night they’ll break in again and give us a scare for our money.”
“Well,” said Frank, quietly, “you know since that time I’ve never failed
to fix it every night so that the engine could not be run. It’s easy as
falling off a log to hide some important little part and render the
whole thing useless. But now that we’ve got a hunch about their
hiding place, we must let Chief Waller know. He can come up here
this very night and grab the precious pair.”
“I agree with you,” Andy hastened to say. “To tell the honest truth,
Pard Frank, I’ll never be easy till Jules and Jean are safely in the
cooler. I’m afraid they’ve got it in for a couple of fellows I know. And if
they crept in on us some night they’d just make you tell where you
had put that missing part of the engine, even if they had to torture us.
I know the breed. They’re a cruel, cold-blooded lot, and I want to see
’em caged!”
“Oh, well,” Frank continued, “it’ll be up to the chief. Unless he makes
a foozle of the whole business he ought to gather them in easy. But
let’s turn now.”
“Are you going back the same way?” asked the other, burning with
eagerness.
“I think not, Andy. That might make them suspect we had glimpsed
something and were coming to make sure. We’d better fight shy of
that glade and take a wide sweep around. Besides, it’s a farming
country over yonder and worth looking down at.”
“Yes,” said his cousin, quickly, “and it’s sure a sight to see the rustics
breaking their blessed necks looking up. Everybody runs out of the
house like it was afire. I only hope we don’t come across such a fool
as one I read about the other day.”
“Why, what did he do?” queried Frank.
“Hanged if he didn’t blaze away with a shotgun at a poor aviator.
Lucky the man happened to be up too high, or he’d have been filled
full of bird shot. There’s no telling what some of these jay fools might
do. They think it’s a big hawk, perhaps one of those giant roc birds
old Sindbad the Sailor used to ride on. But look down, Frank; there’s
the first farm. See the men in the field shaking their fists at us! Now,
what in the dickens are they doing that for, d’ye suppose?”
Frank laughed as he replied:
“I’ve an idea that perhaps they’ve been missing poultry of late and
take it for granted we’re the up-to-date thieves. They believe
anything bad of people who ride in automobiles or fly in new-fangled
airships, you know.”
“Oh, you mean that Jules and his pal have been doing their little
stunt around here?” said Andy, jumping at conclusions. “And now
we’ve got to shoulder the blame of that bad business? Well, it’s one
consolation, then, that after the pair have been jugged we can drop
down and tell this family all about it. I’d like to clear my skirts of any
such nasty reputation.”
Frank gradually veered the course until they were heading for the
home field.
“All told,” he said, “I think our little excursion was a success.”
“A howling success,” grinned Andy; “especially the last part, where
the farmer and his family gave us a tongue lashing which was all a
jumble to us.”
“By the way, I don’t think we’d better say anything about those two
men while the boys are around. A little later, Andy, we can get on our
wheels and take a spin to town, where we will tell the chief all about
it.”
“Just as you say,” returned Andy. “I was thinking myself that one of
the boys might happen to leak and some friend of the fellows get
wind of it. Then he’d warn them and the police would have all their
trouble for their pains. But you really do believe they’re camping out
in the old Badger shack, don’t you?”
“Looks like it,” was all Frank would say.
Presently they came in sight of the field and the hangar. The boys
were still on deck to receive them and ask a myriad of questions.
“There goes the biplane off,” remarked Andy, as they started to
wheel their little airship back into its snug quarters, after Frank had
closely examined every part, machinery and framework, to make
sure it was in “apple-pie” trim.
“Yes,” returned his cousin, pausing to look, “and they’re going to take
something of the same trip we made. Puss evidently don’t want the
Bird boys to get ahead of him, if he can help it. And I must say he’s
doing far better handling that biplane of his than I ever dreamed he
could.”
Ten minutes later they fastened the doors of the shed.
“Elephant says he’ll hang around while we go to town,” remarked
Andy, who did not want to miss the treat of seeing the pompous
chief, when he learned that after all his labor in trying to locate the
hiding rascals it was the two Bird boys who had found out where
they were in concealment.
“That’s all right,” Frank went on. “I was a trifle uneasy about leaving
things alone here, though, of course, it’s ridiculous to think any harm
could come to the shed in broad daylight.”
“Well,” remarked the doubting Andy, “there’s a crowd that has little
use for us, you know, and some of them are thick with Sandy and
Puss. I feel dead sure there are one or two among the lot so low
down, that if they had the chance they’d just like to throw a lighted
match in our shed and watch things burn from the woods over
yonder. I hate to say it, but that’s a fact.”
“Well, I’m not disputing you, because I’ve known them to do some
mighty mean things myself. But get your wheel, Andy. I’m glad now
you brought mine over yesterday when you dropped in to carry my
message to father. He thinks it just the boss thing for me to be
sleeping outdoors every night. You know his fad, though; and it’s pie
for me.”
It took them but a short time to arrive in the heart of the town.
Dropping off at police headquarters they entered the building. The
chief was there luckily, and at sight of the Bird boys he showed
sudden signs of interest. Somehow, people had of late awakened to
the fact that things were generally moving when Frank and Andy
were around. There was “something doing,” so to speak. And so
people began to expect more or less of them.
“Glad to see you, boys,” said Chief Waller, unbending his dignity
enough to extend a hand to each and even smile. “Dropped in to
have a talk about those clever rascals who tried to do you out of your
new aeroplane? Sorry I can’t say they’re in the cooler. My men have
several clues they’re running down, and we hope to be able to report
something soon.”
It was always “something doing soon” with the chief. He had a way
of forever being on the point of making a grand coup, but singular to
relate, no one could remember of past successes that had rewarded
his vigilance. Still, he was useful in his way and had a pull with the
town fathers that kept him his job year after year.
“Oh!” burst out the impulsive Andy, “we just dropped in to say that we
believe we know where those bad men are hiding!”
“Have chairs,” exclaimed the chief. “Sit down and tell me all about it,
please.”
“Why,” Andy went on, Frank having motioned to him to do the
talking, “this afternoon, as we were taking a spin in our aeroplane,
we happened to pass over the woods up near that old Badger shack,
and we saw two men lying down in the grass, trying to hide so we
wouldn’t notice ’em. It was Jules and Jean, as sure as shooting, and
we believe they’re camping in that cabin!”
The police head showed signs of sudden animation.
“Why, boys,” he said, slapping his hand down on his knee, “would
you believe it, now. I was just thinking of that old shack when you
came in. See, here’s a chart of the whole county and that’s one of
the places I had my eye on. But all the same, I’m real glad you came
to tell me, boys. Now I’ll feel positive and we can surround the place.
Would you like to go along with us tonight, Frank?”
But Frank shook his head in the negative as he replied:
“Thanks, but I guess we’ve got all the work cut out for us we can
handle, chief. We believed you’d want to keep it a secret, for fear
that they had some friends in town who might carry a warning. But
we’ll move on now. Hope you have success and make a haul. Come
on, Andy, let’s get back home!”
CHAPTER XX.
THE AEROPLANE RACE.
“Frank, we’re in great luck!”

“I think so myself, Andy,” replied the taller of the cousins, as he
coolly bent down to adjust some little thing about the engine of the
monoplane.
“Here it’s past noon on the great day! Six thousand good people
have gathered on the field here and on the neighboring fences just to
see us fly, and there isn’t a mite of wind. I say it’s the greatest luck
ever,” and Andy, who was bubbling all over with enthusiasm, turned
to look around at the vast throngs.
The two aeroplanes had been taken to the field where the sports of
the wonderful Old Home Week at Bloomsbury were being carried
out.
All through the morning there had been foot racing, athletic games,
and a host of similar amusements that gave the people many thrills
and made them glad that they were in the home town at such a time.
But the greatest event of the whole week was scheduled for that
afternoon at two, when, wind and weather permitting, two home-
manufactured aeroplanes, piloted by boys of Bloomsbury, expected
to compete for a silver cup, supposed to have been offered by the
committee, but which we happen to know had been purchased by
genial old Colonel Josiah Whympers, guardian of our hero, Andy
Bird.
The conditions of the race were well understood. They had even
been printed, so that no one might rest in ignorance concerning their
nature.
Both aeroplanes were to be started at the same time, so there would
be no advantage on either side. Then the first to successfully land on
the summit of Old Thunder Top mountain and plant an American flag
where the foot of man had, up to now, never touched, was to be
declared the winner.
There was to be no jockeying or trickery. The committee, again
inspired by the colonel, who understood the ways of boys and
especially of the Puss Carberry stripe, had made it plain that any
attempt to interfere, outside of an accident, would disqualify the
guilty party from receiving the fine prize.
Of course, there were few among the vast throng who had ever set
eyes on an airship, save as they may have seen either of the
contestants flying about Bloomsbury while trying out their machines.
The enthusiasm was intense. Everywhere people gathered in
clusters to compare notes. Of course, there were adherents of both
contestants. Sometimes this came through personal feeling, each
pilot having his circle of champions among the boys of the town,
although four out of five favored Frank, on account of his fine, manly
disposition.
Nevertheless, there were many who believed that a biplane could
easily outdistance a monoplane in a race, and it was laughable to
hear these people, who were in reality so densely ignorant on the
subject, standing up staunchly for what they believed to be the better
type of air flier.
A rope had been stretched around the space where the two
aeroplanes rested and no one was allowed within this enclosure,
unless at the wish of the contestants. Chief Waller had all his force
on the ground and a number of temporary policemen had been taken
on for the week of hilarity, so that the crowds were handled fairly
well.
At a quarter to two the people were to be pressed back all along the
line, for when the word was given the contesting air craft would
require a certain space in which to flash over the field before taking
wings and soaring aloft.
No wonder Andy trembled with excitement as he gazed upon the sea
of upturned and expectant faces that centered on the enclosure as
the time drew near for the start.
“It’s only wanting ten minutes now, Frank!” he remarked, trying to
keep his voice steady.
“Brace up, Andy,” said the other, with a frown. “You mustn’t allow
yourself to get in that shape. Remember all you’ve learned and how
you managed to keep your head cool yesterday in a trying time.
Make up your mind we’re just going to get up on that old mountain
before the other fellows have a chance to beat us. There’s Puss, just
as excited as you, but he never shows it. Don’t let them see that you
are trembling all over. It weakens our chances!”
Somehow it seemed to give Andy’s heart a new impetus to know that
he was not the only one who was in a quake.
“All right, Frank, I’ll be in trim once we get a decent start. But I’m in a
funk now for fear something will go wrong with the glide-off. If they
get a start of us we’re going to be in the soup!” he said, anxiously.
“Oh, rats! Don’t you know they’re just as possible to have trouble in
making the start as we are? Just make up your mind both of us are
going to get away as neatly as we usually do, and that the race is
going to be to the swift.”
“But I’ve heard lots of people sing the praises of that Gnome engine
they’ve got in the biplane. It does its work with a rush!” observed
Andy.
“All right. We simply believe we’ve got a much better one in the new
and light little Kinkaid. And we’re just going to prove it!” continued
Frank, positively.
“Well, it hasn’t failed us even once so far, for a fact,” admitted Andy;
“and I guess I’m a fool for doubting it now. I’m hoping that later on
that same motor will be buzzing away down in a country where there
won’t be any crowd to watch how we get along. Five minutes now,
Frank!”
“Get in your place then and be ready. Seconds may count with us
and we don’t ever want to be caught unprepared!” said the pilot of
the monoplane, as he cast a last look over everything that would
have to do with the success or failure of the flight.
That was always Frank Bird’s slogan—be prepared! He hit the nail
on the head when he declared that readiness was almost half the
battle. That was what won the war for Germany, when France
started operations so suddenly away back in 1870. The army north
of the Rhine was a vast machine and every single member of it knew
just what was expected of him when the time came!
So wise Frank had carefully sized up the present situation. He had
mapped out in his mind just what sort of emergencies were liable to
occur in a race of this kind, and settled in advance how he could best
meet, and grapple with them in case of need.
He knew just how the wind was apt to strike them after they had
risen to a certain height, and also what sort of clever maneuvers
would best counteract the effect of a head breeze, that must
increase as they neared the mountain crest.
On the other hand, Puss Carberry never bothered his head about
such trifles. He expected the superior speeding capacity of his big
biplane to carry him to victory, regardless of anything his rival might
attempt in the way of aerial strategy.
Possibly, ere the competition came to a close, Puss might have
learned a lesson he would not be apt to soon forget—that often
these same trifles may win or lose a battle, whether on the field of
war, or in an aeroplane competition for a prize.
The five minutes was gradually growing less, and of course the
excitement increased in consequence, until it was at fever heat.
Heads were craned, and people pushed hither and thither, seeking
new points of advantage. For while they readily understood that both
airships could be easily watched, once they rose from the ground,
everybody wanted to witness with their own eyes the curious and
interesting dash along the field that would be a necessary forerunner
of the flight.
All sorts of instruments had been fetched along, which might prove
useful when the contestants were nearing the summit of Old
Thunder Top. Here a lady had a pair of pearl-mounted opera
glasses, that perhaps had once been leveled at a Patti, perhaps
dating back even to a Jenny Lind. Beside her would be a farmer,
who had a telescope of brass, one of the ancient kind used on board
ships many decades back, and which was undoubtedly an heirloom
in his family.
Then there were numerous up-to-date field and marine glasses;
while others depended on their clear eyesight to tell them which
aeroplane touched first the up-to-now never explored crown of the
grim old mountain, with its range of cliffs rebuffing would-be climbers.
Two minutes more!
A great hush had fallen on the throng. Every eye doubtless was
glued on the little enclosure, and the two distinct groups collected
about the rival air craft.
The master of ceremonies had assumed charge. He warned both
contestants once more to play the game fairly, telling them that the
danger was great enough without either taking unnecessary risks in
making a foul.
Signals had been arranged whereby the discharge of cannon were
to serve as a warning against interference. One shot would direct
Frank to steer clear, while a double report meant that the biplane
was cutting dangerously across the path of its rival, and must sheer
off under penalty of being disqualified.
They knew Puss in Bloomsbury. His reputation as a lover of clean
sport was none too good. There were many who anticipated that if
he saw that his chances in the race were practically hopeless, Puss
Carberry would not hesitate to do some mean trick, and stoutly claim
that it was an accident—that his steering apparatus had fouled,
causing him to collide with the slighter monoplane.
Of course this was a most foolhardy thing to dream of doing, since a
foul in midair might not only send his rivals down to death, but imperil
his own life. And yet there were those among his own chums who
winked knowingly as they talked over things in low voices, and
assured each other that Puss was bound to win, or know the reason
why.
Frank had his own private opinion on this subject. He had long made
Puss Carberry a study, and believed he had the other pretty well
sized up. He was a peculiar combination of a boy, bold at times
almost to rashness; and again cautious beyond imagination. And
Frank knew that there was constant war within him between the
good and the bad, though on nearly all occasions the latter
predominated.
He had made up his mind that Puss was too shrewd to risk such a
thing as a collision in midair. If he attempted any of his customary
dirty work it would come in an altogether different way.
All who were not to have a part in the starting of the aeroplanes had
been pressed beyond the ropes. To have no advantage on either
side, and make the start exactly fair, a certain number had been
limited as assistants.
“Are you ready for the start?”
It was the master of ceremonies who called this out in a loud voice.
“Silence! silence!” went from lip to lip; and several groups of giggling
girls felt the rebuke enough to subside.
“Ready!” called out Frank, promptly, while Andy took a big breath,
and gripped the upright beside him nervously.
Both of them had their eyes on the course in front. Frank saw that it
was practically clear of people, so that the chances of any trouble
were reduced to a minimum. He was glad of this, because if the
contestants were recalled to try for another start the strain would be
almost unbearable.
He heard Puss answer “all ready, Judge,” after a trifling delay, as
though he might have thought of some slight change at the very last
second.
Then came a brief period of suspense. Perhaps it was only a few
seconds, while the starter glanced to right and left to make sure that
everything was as it should be, and the track clear. But to poor Andy
it seemed as though the delays were interminable, and that an hour
must have elapsed.
He was impelled to turn his head, and see what the Judge could be
doing; but remembering the positive instructions given by his cousin,
he resolutely shut his jaws, and refrained.
Would the signal never come? Those who were to assist in
launching the aerial craft had assumed an attitude of expectancy.
Their muscles were set, and they stood ready to give the initial push
as the propellers started whirling.
Suddenly the loud voice of the starter rang out, sharp and clear:
“Then go!”
CHAPTER XXI.
HEADED FOR THE SUMMIT OF OLD THUNDER
TOP.
Instantly, as it seemed, there were loud exclamations, accompanied

by all signs of bustle and excitement. The engines began to snap,
and both aeroplanes started down the slight descent, which had
been selected as best suited for their initial forward movement.
Whether the boys who handled the biplane were more alert, or it was
really true that they started a fraction of a second before the signal
was given, as some afterward affirmed—no matter, the one result
was that Puss and Sandy left the ground just a trifle in advance of
their competitors.
Perhaps this trifling advantage might prove of great benefit to them
ere the end of the race came—no one dared prophesy just then.
But now both airships were rapidly rising, and heading in almost a
direct line for the distant mountain. A roar of shouts arose from all
over the wide territory, where thousands of intensely interested
people clustered. Hats and handkerchiefs were wildly waved in the
air, and some of the more enthusiastic boys threw their head gear as
high as they could.
There was no answer from either of the whizzing air craft. Those
who navigated the upper regions of space had learned long before
now that under no circumstances must they allow their attention to
be diverted for even a second from the business in hand.
Even Andy had his duties to perform. Frank had exercised his
inventive genius, and arranged some sort of pendulum contrivance
underneath the body of the monoplane, that in a measure did away
with the dizzy rolling motion of which his companion had formerly
complained. It had been tested on the preceding day, and appeared

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Atomic Force Microscopy in Molecular

and Cell Biology Jiye Cai

Atomic Force Microscopy: Methods and Protocols Nuno C.

Atomic Force Microscopy: Fundamental Concepts and

Stem Cell Renewal and Cell Cell Communication Methods

International Review of Cell and Molecular Biology 1st

Euglena Biochemistry Cell and Molecular Biology 1st

International Review of Cell and Molecular Biology 1st

International Review of Cell and Molecular Biology

International Review of Cell and Molecular Biology 1st

Atomic Force Microscopy in

ISBN 978-981-13-1509-1 ISBN 978-981-13-1510-7 (eBook)

Library of Congress Control Number: 2018957463

© Springer Nature Singapore Pte Ltd. 2018

Institute of Chemistry, Chinese Academy of Sciences Li-Jun Wan

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9 In Situ Measuring Mechanical Properties of Normal

Hongjie An Division of Physics and Applied Physics, Nanyang Technological

J iye Cai, Ph.D., Distinguished Professor of State Key

Abstract Clearly understanding the working principles of different modes of

© Springer Nature Singapore Pte Ltd. 2018 1

1 AFM Working Principles

Laser & Coupling

Fig. 1.2 Scanning electron

The cantilever works as a sensor to detect the probe-sample interaction. The

normalization to the sum signal of (A + B + C + D) is to eliminate the effect of

Fig. 1.4 Scanning electron

Fig. 1.5 Cytoskeleton

0 50 100 150 200 250 300

4 PID Feedback Loop

Due to its simplicity and robustness, proportional–integral–derivative controllers

In force mode, the AFM probe is ramped up/down by retracting/extending the Z

Fig. 1.10 DNA image

0 300 600 900 1200

where m is the effective mass of the cantilever,

where F is the force measured by the probe,

Several days passed.

“Wow! that’s some news, Frank!” cried Andy, doubtless thrilled by

“Frank, we’re in great luck!”

Instantly, as it seemed, there were loud exclamations, accompanied

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Institute of Chemistry, Chinese Academy of Sciences Li-Jun Wan

1 Principles of Atomic Force Microscopy �� 1

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J iye Cai, Ph.D., Distinguished Professor of State Key

1 AFM Working Principles

4 PID Feedback Loop