Mechanical Measurements of in vitro Tissue by Atomic Force Microscopy
Ryosuke Tanaka, Takaharu Okajima
Graduate School ofInformation Science & Technology, Hokkaido University
Kita 14, Nishi 9, Kita-lm, Sapporo 060-0814, Japan
Abstract:
Atomic force microscopy is a powerful tool for studying
mechanical property of biological samples. However, in
conventional AFM, the scan range is relatively small so that
the AFM is not suitable for the measurement of large-scale
biological samples like tissue. In this study, we developed a
simple AFM system, which allows mapping of the
topography and mechanical property of large-scale
biological samples cultured on dish. Using the AFM, we
succeeded to obtain the mapping images of the relative
height and elastic modulus of hepatocyte co-culture system
in a large area at a subcellular spatial resolution.
1. INTRODUCTION
It is recognized that mechanical interactions between
living cells in tissue are necessary for morphogenesis and
expression of various tissue functions. For example, a stress
or force propagates over a millimeter range during the
collective cell migration in epithelial tissue in vitro [1]. A
detailed knowledge of the alternation of cell stiffness owing
to the external force operating between cells is crucial to
elucidate the dynamics of the tissue morphogenesis.
Atomic force microscopy (AFM) is a powerful technique
for not only imaging the surface topography of living cells,
but also allowing the precise measurement of the mechanical
properties of cells at the single cell level [2]. In most of
previous AFM studies, the relationship between the cell
elastic modulus and the intracellular structures of
cytoskeleton has been extensively studied [3]. Due to a
limitation of the scan range in conventional AFM, however,
the AFM has been less applied to explore the mechanical
property of cell population and tissue. To realize the AFM
measurement of large-scale biological samples, we
developed a simple AFM system, which employs course and
precise scanners to map the topography and the mechanical
property of the whole tissue at the single cell level. A tissue
model of liver was used to investigate the performance of the
AFM.
2. ATOMIC FORCE MICROSCOPY
We developed an AFM system equipped with an upright
optical microscope (Eclipse FNl; Nikon, Japan) (Fig. I (a)).
The force measurements were performed by controlling the
measurement position of sample with a fine scanner where
the cantilever probe was indented to the sample surface with
the Z scanner (P-622.zCL; Physik Instruments, Germany)
and the measurement position was regulated with the XY
scanner (LPS-45 and P-622.2CL; Physik Instruments,
Germany). To map a large-scale biological sample, we
combined a long-range three-dimensional scanner with 400
/--lm in the z-direction and 13 mm in the xy-direction. The
repulsive force applied to the cantilever probe during
indentation was measured from the deflection of cantilever
with an optical lever, in which the laser light was focused on
the cantilever beam through a liquid-immersion objective
lens (Fig. I (b)).
a ge
( ) Cantilever Short-ran
(b)
Fig. I Schematics of AFM system (a) and the optical lever system (b).
Cells dynamically change their mechanical properties.
Therefore, a high-speed measurement is required to clarity
the change in cell mechanical properties. Because a large
scan with the long-range XY scanner may cause an
unexpected mechanical vibration of the AFM, the
short-range scanner was used during the measurement in a
part of the whole mapping area and then the mapping region
was changed with the long-range XY scanner. Thus, we can
perform the mapping experiment of in vitro tissue without
reducing the measurement speed.
Short-range Long-range
XV scanner XV scanner
......... .... .
E ,
::t ......... ....
0 ,
LI) ..... ...... .....
N ,
...............
Fig. 2 Schematic of the AFM mapping with short- and long-scanners.
3. MAPPING IMAGES OF LARGE BIOLOGICAL SAMPLE
To estimate the performance of the developed AFM, we
measured the elastic modules of a co-culture system REFERENCES
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(a) Topography (b) Young's modulus

Endothelial Endothelial Hepatocyte
,......----,., high stiff

low soft

Fig. 3 Topography (a) and Young's modulus (b) images of co-culture

system composed of hepatocyte and endothelial cells. Dashed lines
represent a border between hepatocyte and endothelial regions.

4. CONCLUSION

We developed the AFM system for measuring mechanical

property of large-scale cell samples at the subcellular spatial
resolution without lacking measurement speed. The AFM
presented here can be utilized for measuring spatial
distributions of mechanical property of large-scale tissues.
The AFM system will be extended to investigate the
rheological properties of cells in tissue as the force
modulation mode is introduced [5].

ACKNOWLEDGEMENT

We would like to thank Dr. Yoshikatsu Akiyama, Dr. lun

Kobayashi, and Dr. Masayuki Yamato for providing the
co-culture cell sample.
 Mechanical measurements of in vitro tissue by atomic force microscopy
Ryosuke Tanaka, Takaharu Okajima
Graduate School ofInformation Science & Technology, Hokkaido University
Kita 14, Nishi 9, Kita-ku, Sapporo 060-0814, Japan

Atomic force microscopy is a powerful tool for studying mechanical property of biological
samples. However, in conventional AFM, the scan range is relatively small so that the AFM
is not suitable for the measurement of large-scale biological samples like tissue. In this
study, we developed a simple AFM system, which allows mapping of the topography and
mechanical property of large-scale biological samples cultured on dish. Using the AFM, we
succeeded to obtain the mapping images of the relative height and elastic modulus of
hepatocyte co-culture system in a large area at a subcellular spatial resolution.
XV scanner