The elasticity of soft biological materials is a critical property to understand their biomechanical behaviors. Atomic force microscopy (AFM) indentation method has been widely employed to measure the Young's modulus (E) of such materials. Although the accuracy of the method has been recently evaluated based on comparisons with macroscale E measurements, the repeatability of the method has yet to be validated for rigorous biomechanical studies of soft elastic materials. We tested the AFM indentation method using colloidal probes and polyacrylamide (PAAM) gels of E < 20 kPa as a model soft elastic material after having identified optimal trigger force and probe speed. AFM indentations repeated with time intervals show that the method is well repeatable when performed carefully. Compared with the rheometric method and the confocal microscopy indentation method, the AFM indentation method is evaluated to have comparable accuracy and better precision, although these elasticity measurements appear to rely on the compositions of PAAM gels and the length scale of measurement. Therefore, we have confirmed that the AFM indentation method can reliably measure the elasticity of soft elastic materials.

References

References
1.
Radmacher
,
M.
,
1997
, “
Measuring the Elastic Properties of Biological Samples With the AFM
,”
IEEE Eng. Med. Biol.
,
16
(
2
), pp.
47
57
.
2.
Costa
,
K. D.
,
Sim
,
A. J.
, and
Yin
,
F. C.-P.
,
2006
, “
Non-Hertzian Approach to Analyzing Mechanical Properties of Endothelin Cells Probed by Atomic Force Microscopy
,”
ASME J. Biomech. Eng.
,
128
(
2
), pp.
176
184
.
3.
Kuznetsova
,
T. G.
,
Starodubtseva
,
M. N.
,
Yegorenkov
,
N. I.
,
Chizhik
,
S. A.
, and
Zhdanov
,
R. I.
,
2007
, “
Atomic Force Microscopy Probing of Cell Elasticity
,”
Micron
,
38
(
8
), pp.
824
833
.
4.
Lin
,
D. C.
,
Dimitriadis
,
E. K.
, and
Horkay
,
F.
,
2007
, “
Robust Strategies for Automated AFM Force Curve Analysis-I. Non-Adhesive Indentation of Soft, Inhomogeneous Materials
,”
ASME J. Biomech. Eng.
,
129
(
3
), pp.
430
440
.
5.
Lin
,
D. C.
,
Dimitriadis
,
E. K.
, and
Horkay
,
F.
,
2007
, “
Robust Strategies for Automated AFM Force Curve Analysis—II: Adhesion-Influenced Indentation of Soft, Elastic Materials
,”
ASME J. Biomech. Eng.
,
129
(
6
), pp.
904
912
.
6.
Simha
,
N. K.
,
Jin
,
H.
,
Hall
,
M. L.
,
Chiravarambath
,
S.
, and
Lewis
,
J. L.
,
2007
, “
Effect of Indenter Size on Elastic Modulus of Cartilage Measured by Indentation
,”
ASME J. Biomech. Eng.
,
129
(5), pp.
767
775
.
7.
Tripathy
,
S.
, and
Berger
,
E. J.
,
2009
, “
Measuring Viscoelasticity of Soft Samples Using Atomic Force Microscopy
,”
ASME J. Biomech. Eng.
,
131
(
9
), p.
094507
.
8.
Kirmizis
,
D.
, and
Logothetidis
,
S.
,
2010
, “
Atomic Force Microscopy Probing in the Measurement of Cell Mechanics
,”
Int. J. Nanomed.
,
5
, pp.
137
145
.
9.
Azeloglu
,
E. U.
, and
Costa
,
K. D.
,
2011
, “
Atomic Force Microscopy in Mechanobiology: Measuring Microelastic Heterogeneity of Living Cells
,”
Atomic Force Microscopy in Biomedical Research: Methods and Protocols
,
C. P.
Braga
and
D.
Ricci
, eds.,
Humana Press
,
Totowa, NJ
, pp.
303
329
.
10.
MacKay
,
J. L.
, and
Kumar
,
S.
,
2013
, “
Measuring the Elastic Properties of Living Cells With Atomic Force Microscopy Indentation
,”
Cell Imaging Techniques: Methods and Protocols
,
J. D.
Taatjes
and
J.
Roth
, eds.,
Humana Press
,
Totowa, NJ
, pp.
313
329
.
11.
Thomas
,
G.
,
Burnham
,
N. A.
,
Camesano
,
T. A.
, and
Wen
,
Q.
,
2013
, “
Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy
,”
J. Visualized Exp.
,
76
, p.
e50497
.
12.
Gautier
,
H. O. B.
,
Thompson
,
A. J.
,
Achouri
,
S.
,
Koser
,
D. E.
,
Holtzmann
,
K.
,
Moeendarbary
,
E.
, and
Franze
,
K.
,
2015
, “
Atomic Force Microscopy-Based Force Measurements on Animal Cells and Tissues
,”
Methods in Cell Biology
,
K. P. Ewa
,
ed.
, Academic Press, San Diego, CA, pp. 211–235.
13.
Ondeck
,
M. G.
, and
Engler
,
A. J.
,
2016
, “
Mechanical Characterization of a Dynamic and Tunable Methacrylated Hyaluronic Acid Hydrogel
,”
ASME J. Biomech. Eng.
,
138
(
2
), p.
021003
.
14.
Demichelis
,
A.
,
Divieto
,
C.
,
Mortati
,
L.
,
Pavarelli
,
S.
,
Sassi
,
G.
, and
Sassi
,
M. P.
,
2015
, “
Toward the Realization of Reproducible Atomic Force Microscopy Measurements of Elastic Modulus in Biological Samples
,”
J. Biomech.
,
48
(
6
), pp.
1099
1104
.
15.
Butcher
,
D. T.
,
Alliston
,
T.
, and
Weaver
,
V. M.
,
2009
, “
A Tense Situation: Forcing Tumour Progression
,”
Nat. Rev. Cancer
,
9
(
2
), pp.
108
122
.
16.
Levental
,
I.
,
Georges
,
P. C.
, and
Janmey
,
P. A.
,
2007
, “
Soft Biological Materials and Their Impact on Cell Function
,”
Soft Matter
,
3
(
3
), pp.
299
306
.
17.
Rodriguez
,
M. L.
,
McGarry
,
P. J.
, and
Sniadecki
,
N. J.
,
2013
, “
Review on Cell Mechanics: Experimental and Modeling Approaches
,”
ASME Appl. Mech. Rev.
,
65
(
6
), p.
060801
.
18.
Denisin
,
A. K.
, and
Pruitt
,
B. L.
,
2016
, “
Tuning the Range of Polyacrylamide Gel Stiffness for Mechanobiology Applications
,”
ACS Appl. Mater. Interfaces
,
8
(
34
), pp.
21893
21902
.
19.
Tse
,
J. R.
, and
Engler
,
A. J.
,
2010
, “
Preparation of Hydrogel Substrates With Tunable Mechanical Properties
,”
Current Protocols in Cell Biology
,
Wiley
, Hoboken, NJ, pp.
1
16
.
20.
Lakins
,
J.
,
Chin
,
A.
, and
Weaver
,
V.
,
2012
, “
Exploring the Link Between Human Embryonic Stem Cell Organization and Fate Using Tension-Calibrated Extracellular Matrix Functionalized Polyacrylamide Gels
,”
Progenitor Cells: Methods and Protocols
,
K. A.
Mace
and
K. M.
Braun
, eds.,
Humana Press
, Totowa, NJ, pp.
317
350
.
21.
Damljanović
,
V.
,
Lagerholm
,
B. C.
, and
Jacobson
,
K.
,
2005
, “
Bulk and Micropatterned Conjugation of Extracellular Matrix Proteins to Characterized Polyacrylamide Substrates for Cell Mechanotransduction Assays
,”
Biotechniques
,
39
(
6
), pp.
847
851
.
22.
Dimitriadis
,
E. K.
,
Horkay
,
F.
,
Maresca
,
J.
,
Kachar
,
B.
, and
Chadwick
,
R. S.
,
2002
, “
Determination of Elastic Moduli of Thin Layers of Soft Material Using the Atomic Force Microscope
,”
Biophys. J.
,
82
(
5
), pp.
2798
2810
.
23.
Levental
,
I.
,
Georges
,
P. C.
, and
Janmey
,
P. A.
,
2007
, “
Soft Biological Materials and Their Impact on Cell Function
,”
Soft Matter
,
3
(
3
), pp.
299
306
.
24.
Carl
,
P.
, and
Schillers
,
H.
,
2008
, “
Elasticity Measurement of Living Cells With an Atomic Force Microscope: Data Acquisition and Processing
,”
Pflugers Arch. Eur. J. Physiol.
,
457
(
2
), pp.
551
559
.
25.
Lee
,
D.
,
Rahman
,
M. M.
,
Zhou
,
Y.
, and
Ryu
,
S.
,
2015
, “
Three-Dimensional Confocal Microscopy Indentation Method for Hydrogel Elasticity Measurement
,”
Langmuir
,
31
(
35
), pp.
9684
9693
.
26.
Engler
,
A.
,
Bacakova
,
L.
,
Newman
,
C.
,
Hategan
,
A.
,
Griffin
,
M.
, and
Discher
,
D.
,
2004
, “
Substrate Compliance Versus Ligand Density in Cell on Gel Responses
,”
Biophys. J.
,
86
, pp.
617
628
.
27.
Li
,
C.
,
Allen
,
J.
,
Alliston
,
T.
, and
Pruitt
,
L. A.
,
2011
, “
The Use of Polyacrylamide Gels for Mechanical Calibration of Cartilage–A Combined Nanoindentation and Unconfined Compression Study
,”
J. Mech. Behav. Biomed.
,
4
(
7
), pp.
1540
1547
.
28.
Abidine
,
Y.
,
Laurent
,
V. M.
,
Michel
,
R.
,
Duperray
,
A.
,
Palade
,
L. I.
, and
Verdier
,
C.
,
2015
, “
Physical Properties of Polyacrylamide Gels Probed by AFM and Rheology
,”
Eur. Phys. Lett.
,
109
(
3
), p. 38003.
29.
Buffinton
,
C. M.
,
Tong
,
K. J.
,
Blaho
,
R. A.
,
Buffinton
,
E. M.
, and
Ebenstein
,
D. M.
,
2015
, “
Comparison of Mechanical Testing Methods for Biomaterials: Pipette Aspiration, Nanoindentation, and Macroscale Testing
,”
J. Mech. Behav. Biomed. Mater.
,
51
, pp.
367
379
.
30.
Di Lorenzo
,
F.
,
Hellwig
,
J.
,
von Klitzing
,
R.
, and
Seiffert
,
S.
,
2015
, “
Macroscopic and Microscopic Elasticity of Heterogeneous Polymer Gels
,”
ACS Macro Lett.
,
4
(
7
), pp.
698
703
.
31.
Gavara
,
N.
,
2016
, “
Combined Strategies for Optimal Detection of the Contact Point in AFM Force-Indentation Curves Obtained on Thin Samples and Adherent Cells
,”
Sci. Rep.
,
6
, p.
21267
.
32.
Hutter
,
J. L.
, and
Bechhoefer
,
J.
,
1993
, “
Calibration of Atomic-Force Microscope Tips
,”
Rev. Sci. Instrum.
,
64
(
7
), pp.
1868
1873
.
33.
Pettersson
,
T.
,
Nordgren
,
N.
,
Rutland
,
M. W.
, and
Feiler
,
A.
,
2007
, “
Comparison of Different Methods to Calibrate Torsional Spring Constant and Photodetector for Atomic Force Microscopy Friction Measurements in Air and Liquid
,”
Rev. Sci. Instrum.
,
78
(
9
), p.
093702
.
34.
Tocha
,
E.
,
Song
,
J.
,
Schönherr
,
H.
, and
Vancso
,
G. J.
,
2007
, “
Calibration of Friction Force Signals in Atomic Force Microscopy in Liquid Media
,”
Langmuir
,
23
(
13
), pp.
7078
7082
.
35.
Chung
,
K.-H.
,
Shaw
,
G. A.
, and
Pratt
,
J. R.
,
2009
, “
Accurate Noncontact Calibration of Colloidal Probe Sensitivities in Atomic Force Microscopy
,”
Rev. Sci. Instrum.
,
80
(
6
), p.
065107
.
36.
Pirzer
,
T.
, and
Hugel
,
T.
,
2009
, “
Atomic Force Microscopy Spring Constant Determination in Viscous Liquids
,”
Rev. Sci. Instrum.
,
80
(
3
), p.
035110
.
37.
Kiracofe
,
D.
, and
Raman
,
A.
,
2010
, “
On Eigenmodes, Stiffness, and Sensitivity of Atomic Force Microscope Cantilevers in Air Versus Liquids
,”
J. Appl. Phys.
,
107
(
3
), p.
033506
.
38.
Ryu
,
S.
, and
Franck
,
C.
,
2011
, “
In Situ Hydrodynamic Lateral Force Calibration of AFM Colloidal Probes
,”
Langmuir
,
27
(
21
), pp.
13390
13399
.
39.
Hermanowicz
,
P.
,
Sarna
,
M.
,
Burda
,
K.
, and
Gabryś
,
H.
,
2014
, “
AtomicJ: An Open Source Software for Analysis of Force Curves
,”
Rev. Sci. Instrum.
,
85
(
6
), p.
063703
.
40.
Sneddon
,
I. N.
,
1965
, “
The Relation Between Load and Penetration in the Axisymmetric Boussinesq Problem for a Punch of Arbitrary Profile
,”
Int. J. Eng. Sci.
,
3
(
1
), pp.
47
57
.
41.
Heuberger
,
M.
,
Dietler
,
G.
, and
Schlapbach
,
L.
,
1995
, “
Mapping the Local Young's Modulus by Analysis of the Elastic Deformations Occurring in Atomic Force Microscopy
,”
Nanotechnology
,
6
(
1
), p.
12
.
42.
Puricelli
,
L.
,
Galluzzi
,
M.
,
Schulte
,
C.
,
Podestà
,
A.
, and
Milani
,
P.
,
2015
, “
Nanomechanical and Topographical Imaging of Living Cells by Atomic Force Microscopy With Colloidal Probes
,”
Rev. Sci. Instrum.
,
86
(
3
), p.
033705
.
43.
Mahaffy
,
R. E.
,
Shih
,
C. K.
,
MacKintosh
,
F. C.
, and
Käs
,
J.
,
2000
, “
Scanning Probe-Based Frequency-Dependent Microrheology of Polymer Gels and Biological Cells
,”
Phys. Rev. Lett.
,
85
(
4
), pp.
880
883
.
44.
Mahaffy
,
R. E.
,
Park
,
S.
,
Gerde
,
E.
,
Käs
,
J.
, and
Shih
,
C. K.
,
2004
, “
Quantitative Analysis of the Viscoelastic Properties of Thin Regions of Fibroblasts Using Atomic Force Microscopy
,”
Biophys. J.
,
86
(
3
), pp.
1777
1793
.
45.
Boudou
,
T.
,
Ohayon
,
J.
,
Picart
,
C.
, and
Tracqui
,
P.
,
2006
, “
An Extended Relationship for the Characterization of Young's Modulus and Poisson's Ratio of Tunable Polyacrylamide Gels
,”
Biorheology
,
43
(
6
), pp.
721
728
.
46.
Yeung
,
T.
,
Georges
,
P. C.
,
Flanagan
,
L. A.
,
Marg
,
B.
,
Ortiz
,
M.
,
Funaki
,
M.
,
Zahir
,
N.
,
Ming
,
W.
,
Weaver
,
V.
, and
Janmey
,
P. A.
,
2005
, “
Effects of Substrate Stiffness on Cell Morphology, Cytoskeletal Structure, and Adhesion
,”
Cell Motil. Cytoskeleton
,
60
(
1
), pp.
24
34
.
47.
Janmey
,
P. A.
,
Georges
,
P. C.
, and
Hvidt
,
S.
,
2007
, “
Basic Rheology for Biologists
,”
Methods in Cell Biology
,
Academic Press
, San Diego, CA, pp.
1
27
.
48.
Saha
,
K.
,
Keung
,
A. J.
,
Irwin
,
E. F.
,
Li
,
Y.
,
Little
,
L.
,
Schaffer
,
D. V.
, and
Healy
,
K. E.
,
2008
, “
Substrate Modulus Directs Neural Stem Cell Behavior
,”
Biophys. J.
,
95
(
9
), pp.
4426
4438
.
49.
Legant
,
W. R.
,
Miller
,
J. S.
,
Blakely
,
B. L.
,
Cohen
,
D. M.
,
Genin
,
G. M.
, and
Chen
,
C. S.
,
2010
, “
Measurement of Mechanical Tractions Exerted by Cells in Three-Dimensional Matrices
,”
Nat. Methods
,
7
(
12
), pp.
969
971
.
50.
Guo
,
S.
, and
Akhremitchev
,
B. B.
,
2006
, “
Packing Density and Structural Heterogeneity of Insulin Amyloid Fibrils Measured by AFM Nanoindentation
,”
Biomacromolecules
,
7
(
5
), pp.
1630
1636
.
51.
Proksch
,
R.
,
Cleveland
,
J.
,
Callahan
,
R.
, and
Viani
,
M.
,
2004
, “
Finite Optical Spot Size and Position Corrections in Thermal Spring Constant Calibration
,”
Nanotechnology
,
15
(
9
), p.
1344
.
52.
Rico
,
F.
,
Roca-Cusachs
,
P.
,
Gavara
,
N.
,
Farré
,
R.
,
Rotger
,
M.
, and
Navajas
,
D.
,
2005
, “
Probing Mechanical Properties of Living Cells by Atomic Force Microscopy With Blunted Pyramidal Cantilever Tips
,”
Phys. Rev. E
,
72
(
2
), p.
021914
.
53.
Derjaguin
,
B. V.
,
Muller
,
V. M.
, and
Toporov
,
Y. P.
,
1975
, “
Effect of Contact Deformations on the Adhesion of Particles
,”
J. Colloid Interface Sci.
,
53
(
2
), pp.
314
326
.
54.
Johnson
,
K. L.
,
Kendall
,
K.
, and
Roberts
,
A. D.
,
1971
, “
Surface Energy and the Contact of Elastic Solids
,”
Proc. R. Soc. A
,
324
(
1558
), pp.
301
313
.
55.
Frey
,
M. T.
,
Engler
,
A.
,
Discher
,
D. E.
,
Lee
,
J.
, and
Wang
,
Y. L.
,
2007
, “
Microscopic Methods for Measuring the Elasticity of Gel Substrates for Cell Culture: Microspheres, Microindenters, and Atomic Force Microscopy
,”
Methods in Cell Biology
,
W.
Yu-Li
and
E. D.
Dennis
, eds.,
Academic Press
, San Diego, CA, pp.
47
65
.
56.
Heuberger
,
M.
,
Dietler
,
G.
, and
Schlapbach
,
L.
,
1996
, “
Elastic Deformation of Tip and Sample During Atomic Force Microscope Measurements
,”
J. Vac. Sci. Technol., B
,
14
(
2
), pp.
1250
1254
.
57.
Darling
,
E. M.
,
Topel
,
M.
,
Zauscher
,
S.
,
Vail
,
T. P.
, and
Guilak
,
F.
,
2008
, “
Viscoelastic Properties of Human Mesenchymally-Derived Stem Cells and Primary Osteoblasts, Chondrocytes, and Adipocytes
,”
J. Biomech.
,
41
(
2
), pp.
454
464
.
58.
Janmey
,
P. A.
, and
Miller
,
R. T.
,
2011
, “
Mechanisms of Mechanical Signaling in Development and Disease
,”
J. Cell Sci.
,
124
(
1
), pp.
9
18
.
59.
Richards
,
E. G.
, and
Temple
,
C. J.
,
1971
, “
Some Properties of Polyacrylamide Gels
,”
Nature
,
230
(
12
), pp.
92
96
.
60.
Baselga
,
J.
,
Hernandez-Fuentes
,
I.
,
Pierola
,
I. F.
, and
Llorente
,
M. A.
,
1987
, “
Elastic Properties of Highly Crosslinked Polyacrylamide Gels
,”
Macromolecules
,
20
(
12
), pp.
3060
3065
.
61.
Baselga
,
J.
,
Hernandez-Fuentes
,
I.
,
Masegosa
,
R. M.
, and
Llorente
,
M. A.
,
1989
, “
Effect of Crosslinker on Swelling and Thermodynamic Properties of Polyacrylamide Gels
,”
Polym. J.
,
21
(
6
), pp.
467
474
.
62.
Charest
,
J. M.
,
Califano
,
J. P.
,
Carey
,
S. P.
, and
Reinhart-King
,
C. A.
,
2012
, “
Fabrication of Substrates With Defined Mechanical Properties and Topographical Features for the Study of Cell Migration
,”
Macromol. Biosci.
,
12
(
1
), pp.
12
20
.
63.
Oyen
,
M. L.
,
2014
, “
Mechanical Characterisation of Hydrogel Materials
,”
Int. Mater. Rev.
,
59
(
1
), pp.
44
59
.
64.
Wen
,
J. H.
,
Vincent
,
L. G.
,
Fuhrmann
,
A.
,
Choi
,
Y. S.
,
Hribar
,
K. C.
,
Taylor-Weiner
,
H.
,
Chen
,
S.
, and
Engler
,
A. J.
,
2014
, “
Interplay of Matrix Stiffness and Protein Tethering in Stem Cell Differentiation
,”
Nat. Mater.
,
13
(
10
), pp.
979
987
.
65.
Stellwagen
,
N. C.
,
1998
, “
Apparent Pore Size of Polyacrylamide Gels: Comparison of Gels Cast and Run in Tris-Acetate-EDTA and Tris-Borate-EDTA Buffers
,”
Electrophoresis
,
19
(
10
), pp.
1542
1547
.
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