IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 7, NO. 3, SEPTEMBER 2008

185

The Effects of Cell Sizes, Environmental Conditions,and Growth Phases on the Strength of Individual
W303 Yeast Cells Inside ESEM
Mohd Ridzuan Ahmad*, Student Member, IEEE, Masahiro Nakajima, Member, IEEE, Seiji Kojima,
Michio Homma, and Toshio Fukuda, Fellow, IEEE

Abstract—We performed in situ measurements of mechanicalproperties of individual W303 wild-type yeast cells by using an
integrated environmental scanning electron microscope (ESEM)nanomanipulator system. Compression experiments to penetrate
the cell walls of single cells of different cell sizes (about 3–6 µm diameter), environmental conditions (600 Pa and 3 mPa), and growthphases (early log, mid log, late log and saturation) were conducted.
The compression experiments were performed inside ESEM, embedded with a 7 DOF nanomanipulator with a sharp pyramidal
end effector and a cooling stage, i.e., a temperature controller.
ESEM itself can control the chamber pressure. Data clearly show
an increment in penetration force, i.e., 96 ± 2, 124 ± 10, 163 ± 1,
and 234 ± 14 nN at 3, 4, 5, and 6 µm cell diameters, respectively.
Whereas, 20-fold increase in penetration forces was recorded at
different environmental conditions for 5 µm cell diameter, i.e.,
163 ± 1 nN and 2.95 ± 0.23 µN at 600 Pa (ESEM mode) and3 mPa (HV mode), respectively. This was further confirmed fromquantitative estimation of average cell rigidity through the Hertz
model, i.e., ESEM mode (3.31 ± 0.11 MPa) and HV mode (26.02 ±3.66 MPa) for 5 µm cell diameter. Finally, the penetration forces atdifferent cell growth phases also show the increment pattern fromlog (early, mid, and late) to saturation phases, i.e., 161 ± 25, 216 ±15, 255 ± 21, and 408 ± 41 nN, respectively.
Index Terms—Cell mechanics, environmental-scanning electron
microscope (SEM), nanomanipulation, single-cell analysis, yeast
cells.

I. INTRODUCTION
HE CONVENTIONAL approach to characterize cellularbiology is called biochemistry. This developed science isused for studying physiological aspects, mainly genetics, bycharacterizing protein and other biomaterials. Since single cells
are difficult to study, a collection of cells is used for characterizing cellular physiology and in turn used to describe the behaviorof a single cell [1]. However, in addition to this advance un-

T

Manuscript received March 25, 2008; revised June 18, 2008. Current version
published August 29, 2008. This work was supported in part by the Malaysian
Government Scholarship under Grant UTM-SLAB and in part by the Ministryof Education, Culture, Sports, Science, and Technology (MEXT.KAKENHI).
Asterisk indicates corresponding author.
*M. R. Ahmad is with the Department of Micro-Nano Systems Engineering, Nagoya University, Nagoya 464-8603, Japan (e-mail: ridzuan@robo.mein.nagoya-u.ac.jp).
M. Nakajima and T. Fukuda are with the Department of Micro-Nano Systems
Engineering, Nagoya University, Nagoya 464-8603, Japan.
S. Kojima and M. Homma are with the Department of Biological Science,
Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
Color versions of one or more of the figures in this paper are available online
at http://ieeexplore.org.
Digital Object Identifier 10.1109/TNB.2008.2002281

derstanding of cellular genetics, information about mechanicalproperties of cells is also needed. The molecular structure of
the cell wall is only partially understood, and its mechanicalproperties are an area of “near-total darkness” [2]. Moreover,
the approximation of single-cell behavior from a group used inconventional approach also requires further justification whether
it can be applied to all cell types [3], [4]. This knowledge of cellmechanics could be valuable in future for biomedical applications, for example, variations in cell mechanics of healthy andunhealthy cells can be linked to a specific disease. In this paper, we show three kinds of cell mechanic characterization of asingle yeast cell based on cell size, environmental conditions,and growth phases. This information can be used as a standard
procedure for future disease detection.
Numerous methods such as micropipette aspiration, osmotic
swelling/shrinking, cell poking, and cell compression [5] haveenabled the response of the whole Saccharomyces cerevisiae
cells to applied forces to be measured [6]; however, the mechanical properties of different cell sizes remain unknown. Recent advances in atomic force microscopy (AFM) have shown
extensive research progress in the biomechanics of the living
cells [7]–[9], however the effect of environmental conditions
on these properties is still unknown. The mechanical characterization of yeast cells at log and saturation phases have beenreported [6], but detailed mechanics at early, mid, and late log
have not been investigated.
Therefore, we developed an integrated environmental scanning electron microscope (ESEM)-nanomanipulator system.
Unlike the conventional SEM, ESEM enables soft, moist, and/or
electrically insulating materials to be imaged without prior specimen preparation. A low-pressure (up to around 1333 Pa) gas canbe accommodated around the sample. When this gas is water,
hydrated samples can be maintained in their native state [10].
Sample temperature can be controlled by using the coolingstage assembled inside the ESEM chamber. By controlling thechamber pressure and sample temperature, single-cell mechanical characterization and analysis can be conducted by using ananomanipulator and image analysis. By modifying a cantilever
tip using focused ion beam (FIB) etching and/or deposition,
several shapes of indenters for indenter–cell interactions can beapplied. There are two main advantages of the ESEM systemcompared to the AFM system. The first advantage is that the
AFM system is difficult to provide a real-time sample observation and the image is mainly constructed after the scanning of
the AFM tip on the sample surface is finished. Therefore, it is1536-1241/$25.00 © 2008 IEEE
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difficult, if not impossible, to directly observe the response of
the object during the sample manipulation. On the other hand,the ESEM system provides real-time sample observation thatcan be obtained before, during, and after the manipulation. This
real-time observation capability of the ESEM system has many
advantages such as in analyzing the dynamic response of thesample and sample selection/sorting for certain properties likecell size become much easier. The mechanical characterization
and analysis capabilities that can be done on the sample can also
be enhanced.
The second advantage is related to the manipulation aspect.
The AFM system can only provide 2-D manipulation, due tothe difficulty of the real-time observation. Although 2-D manipulation can measure the stiffness property of the sample, it
lacks in the flexibility of manipulation thus limiting the areaof sample that can be analyzed. Unlike the AFM system, the
ESEM-nanomanipulation system can provide both 2-D and 3-D
manipulations on the sample, thus increasing the flexibility ofthe measurement such as the stiffness characterization in different areas of the sample like bottom surface can be realized.
The characterization of mechanical properties of the cell requires no interference from other external pressures or forceson the cell surface. These external forces such as the osmoticpressure may prevent investigation of the true mechanical properties of cell experimentally. The ESEM system can provide
environmental conditions for the cells in their native state without any effect of the osmotic pressure. Cells can be sustained byreleasing low pressure of H2 O molecule gas inside the ESEM
chamber to provide high relative humidity (up to 100%). Therefore, the stiffness data obtained from this experimental setting
show better representation of the mechanical properties of thecell. Our objective is to show the capability and effectiveness of
the ESEM system for reliable mechanical stiffness measurementof biological samples. In the present study, we discuss an in situ
characterization of the mechanical properties of the W303 yeast
cells using ESEM-nanomanipulator under three different cases,
i.e., cell sizes, environmental conditions, and cellular growthphases.

II. ESEM-NANOMANIPULATION SYSTEM
Nanomanipulation is an effective strategy for the characterization of basic properties of individual nanoscale objects
and to construct nanoscale devices quickly and effectively.
We have constructed a hybrid nanorobotic manipulation system integrated with a transmission electron microscope (TEM)nanorobotic manipulator (TEM manipulator) and a scanningelectron microscope (SEM)-nanorobotic manipulator (SEM manipulator) [11]. This system allows effective sample preparation
inside SEM with wide working area and many DOFs of manipulation. It has high-resolution measurement and evaluation ofsamples inside a TEM capability. The sample chambers of theseelectron microscopes are set under the high-vacuum (HV) condition to reduce the disturbance of electron beam for observation.
To observe the water-containing samples, e.g., biocells, drying treatment processes are additionally needed. Hence, direct

Fig. 1.

Overview of the nanorobotic manipulator of the ESEM.

Fig. 2.

Images of W303 yeast cells under. (a) HV mode. (b) ESEM mode.

observations of water-containing samples are normally quitedifficult in these electron microscopes.
In the present study, we used the nanorobotic manipulators inside an ESEM [12]. It has been constructed with three units and7 DOF in total (Fig. 1). The ESEM enables direct observationof water-containing samples with nanometer high resolution bya specially built secondly electron detector. The evaporation ofwater is controlled by both the sample temperature (0 ◦ C–40 ◦ C)
and sample chamber pressure (10–2600 Pa). The temperatureof the sample is controlled by the cooling stage unit (unit 3).
The detail specifications of the manipulator and the ESEM canbe obtained from our previous paper [13]. The following experiments have been conducted through this system.
III. ESEM OBSERVATION OF YEAST CELLSThe unique characteristic of the ESEM is the direct observation of the hydroscopic samples with nondrying treatment. Recently, the evaluation of biosamples has received much attention
for nano-bio applications in nanobiotechnology. Since water is
an important component for chemical reactions, nanomanipulation inside the ESEM could be a very effective tool for awater-containing sample with nanometer resolution.
Wild-type yeast cells (W303) were used for observation andmeasurements by the ESEM nanomanipulation system. The
W303 cells were cultured on YPD plates (1% yeast extract,
2% peptone, 2% glucose, and 2% agar) in a 37 ◦ C incubator for
24 h and dispersed in pure water. Several microliters of dispersed
cells were placed on aluminum block on the cooling stage. The
HV mode is operated at 16.7 ◦ C and 3.03 × 10−3 Pa pressure.
All yeast cells appeared concave and broken under HV mode.
The ESEM mode is operated at 0 ◦ C and ∼600 Pa pressure andacceleration voltage is set at 15 kV. Under the ESEM mode,
decreasing the pressure from ∼700 Pa, water gradually evapo-

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AHMAD et al.: EFFECTS OF CELL SIZES, ENVIRONMENTAL CONDITIONS, AND GROWTH PHASES

Fig. 3. Schematic diagram. (a) Indentation experiment. (b) Sharp. (c) Flat
AFM pyramidal cantilever tips under the ESEM mode. For the HV mode.
(d) Tetrahedral cantilever tip.

rates and the samples can be seen underneath. Their images areshown with HV and ESEM modes, as shown in Fig. 2(a) and (b).
The remaining water can be seen at the intercellular spaces ofyeast cells as black contrast [Fig. 2(b)]. Almost all yeast cells
appear sphere shaped by water-contained condition under the
ESEM operation.

187

Fig. 4. Typical force—indentation (F–I) curve under ESEM mode using a
sharp tip. The curve shows points before penetration (green arrow) and after
penetration (pink arrow) of the cell. The blue line shows the best fit of the Hertz–
Sneddon model of the experimental data until the penetration point (dotted line).
TABLE IVALUES FOR PARAMETERS USED IN THE ESTIMATION OF THE YOUNG’S
MODULUS OF THE SINGLE CELLS

IV. MECHANICAL PROPERTIES CHARACTERIZATIONOF INDIVIDUAL YEAST CELLS
A. Determination of the Penetration Force and the Cell Stiffness
The indentation experiments [Fig. 3(a)] were conducted byusing the integrated ESEM-nanomanipulation system. The cells
were fixed on the cooling stage in perpendicular direction withan AFM cantilever (0.02 or 0.7 N/m spring constant). The AFM
cantilever is controlled by the nanomanipulator and used toapply the compression force on each cell.
The penetration of a single cell by the AFM cantilever in thisexperiment was based on a direct observation of the cell when it
was bursted by the AFM cantilever. Due to the shape and size ofthe indenters, i.e., pyramidal or tetrahedral tips (sharp or flat),the cell bursted phenomenon cannot be avoided as soon as thecell wall is penetrated [14].
The ESEM can provide real-time observation, i.e., a cell being
approached, touched, indented, and finally, penetrated/bursted
by the AFM cantilever can be seen clearly. Therefore, as far as
this paper is concerned, the penetration of a single cell by the
AFM cantilever was based on the occurrence of the cell bursting
via real-time observation.
Force–cantilever indentation (F –I) curves were determinedfrom the experiment. A typical F–I curve using a sharp cantilever
tip shows a response of a cell before and after the penetration,
as shown in Fig. 4. The value of F was calculated by using (1),where k, ϕ, and L are the spring constant, the displacement angle
in radian, and the length of the cantilever, respectively. Values of
ϕ, L, and I were determined from direct measurements of ESEM
images using an image analysis software (ImageJ, developed at
the National Institute of Health), while k was obtained from the

manufacturer (Olympus Corporation). Equation (1) was derived
from the Hooke’s law, i.e., F = kδ, where δ is the displacementof the cantilever that was obtained by using δ = ϕ(2/3)L: 
2
F = kϕL(1)
3
The Hertz–Sneddon models based on the shape of the tips,
i.e., conical, spherical, and cylindrical, were used to estimate theYoung’s modulus of the cells, as expressed in (2)–(4). Parameters of E, v, α, R, and a are the Young’s modulus, the Poisson’s
ratio (v = 0.5 for soft biological materials [15]) of the elastic
half space (cell’s surface), the half opening angle of a conicaltip, the radius of curvature of a spherical tip and the radiusof a cylindrical tip, respectively. Values for α, R, and a wereobtained from ESEM images. Values for these parameters are
summarized in Table I:
E
2
I2
(2)
Fcone = tan απ(1 − v 2 )Fspherical =E
4
R1/2 I 3/2
3 (1 − v 2 )

(3)

E
aI
(1 − v 2 )(4)

Fcylindrical = 2

The models predict that the load depend on the indentationaccording to a power law related to the tip geometry [15]. In
order to choose the correct tip geometry, an equation of the form

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F = mI b was fitted to force versus indentation curves using
commercial fitting software (DataFit), where the exponent b
depends on the tip shape.
For the sharp pyramidal tip under ESEM mode, we obtained
a value of b close to 3/2, characteristic of the spherical tip.
The curves were then fitted by using the spherical model. Interestingly, under HV mode, the value of b close to 2 was obtained. Therefore, the canonical model was applied under HVmode. Finally, for the flat pyramidal tip, the b value close to
1 was obtained, which is for the cylindrical model. The fitting
model for the sharp tip using the spherical tip was reported by
Lanero et al. [15] while Touhami et al. [16] used the conical tip
as their fitting equation. Obataya et al. [14] used the cylindricalequation to model their flat nanoneedles.
The Hertz–Sneddon models were derived from the classic
Hertz’s mechanics model for linear elastic material [17]. For flat
cylindrical punch relating to an applied force F with an indentation depth I yields the following expression for the initial unloading (load is released after indentation) response, dF/dI =cA1/2 (1 − v 2 )/E, where c is coefficient [c = 2/(π)1/2 ] and A
is the contact area. The equation was based on the flat-ended
punch, but holds true for any punch that can be described by asmooth solid revolution (spherical, conical, elliptical, etc.) [18].
Berkovich and Vickers indenters (three- and four-sided pyramidal cones, respectively) commonly applied in instrumented
indentation techniques cannot be described as bodies of revolution. However, it has been found experimentally and by meansof finite-element simulations that the deviation from the previous equation of pyramidal and other geometrical shapes isnegligible [18]–[20]. The constant c = 1.142 for the pyramidal
indenter and c = 1.167 for tetrahedral indenter differ little fromc = 2/(π)1/2 = 1.1284 from the previous equation. In otherwords, the derived equation for the sharp conical tip can be usedwithout large error, even the sharp indenter is not a true body of
revolution.
The final equations of the Young’s modulus based on the
Hertz–Sneddon models, i.e., (2) and (3), and the parameter’svalues (Table I) for three different tip shapes, i.e., conical (HVmode), spherical (ESEM mode) and cylindrical (ESEM modeand HV mode), are expressed asEcell = (6.681)

Fcone
I2

Fspherical
I 3/2

F

 Ecell = (1.187 × 106 ) cylindricalIFcylindrical6 Ecell = (1.825 × 10 )I

(5)Ecell = (3.977 × 103 )(6)(ESEM mode)(7)(HV mode)

Fig. 5 shows typical F –I curves using the spherical and cylindrical models under ESEM mode while the inset shows a curveusing the conical model under HV mode as obtained in our experiments. The curves are shown until the bursting point of thecells, which is the maximum force which the cell can sustain
before the cell is bursted. After the bursting point, the force will
drop for a while due to the reversed force and will increase again

Fig. 5. Typical force—indentation (F–I) curves under ESEM mode using
spherical and cylindrical models for sharp and flat tips, respectively. Inset shows
the F–I curve under HV mode using conical model.

Fig. 6. Penetrations of yeast cells by different cantilever tips, i.e., (a) Sharp
cantilever tip. (b) Flat cantilever tip.

when the tip traverses inside the cell or touches the bottom of
the cell wall (Fig. 4). Starting from Fig. 5, data after the bursting
points are not shown in the following figures. The reason forthis exclusion is that the data were no longer representing thecell stiffness property since the cells were seriously damaged.
The curve leading up to the failure is expected to give a combined elastic property of indenting the whole yeast cell and the
localized deformation of the surface ruled by the properties of
the cell-wall surface. Therefore, the reported E values in thispaper indicate the maximum strength of the cell before burstingfrom a local indentation point. The E values must be higher
than the local elastic cell property as the strength of the cell isdominated by the whole cell stiffness [6]. Nevertheless, examining the cell strength based on the Young’s modulus parameter
per se is not adequate since its value may remain constant at
different situations, e.g., cell growth phases [6]. Therefore, thepenetration force was used to determine the cell strength under
several factors, i.e., cell sizes, environmental conditions, andcellular growth phases.
B. Effect of Cell Sizes on the Strength of the Cells
The penetrations of single yeast cells using sharp and flat
cantilever tips [Fig. 3(b) and (c)] are shown in Fig. 6(a)and (b). The required force to penetrate a single yeast cell under
the ESEM mode using a sharp tip for different cell sizes are

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TABLE IIYOUNG’S MODULUS OF THE SINGLE CELLS AT DIFFERENT CELL SIZES

Fig. 7. Penetration force using a sharp tip at different cell sizes. Experimental
data are fitted using the spherical model. The curves are shown until the penetration points. Inner bracket () indicates the cell number for each cell size. Cellsize is in µm.

Fig. 8. Comparison of penetration force using sharp (using the sphericalmodel) and flat tips (using the cylindrical model) for 4 and 5 µm cell sizes
range. Young’s modulus of the cells remains constant at about 3 MPa. The
curves are shown until the penetration points.

shown in Fig. 7. The comparison between sharp and flat tips
to penetrate each cell is shown in Fig. 8. The environmental
settings for ESEM mode are set to 600 Pa and 0 ◦ C. These parameters are chosen to vaporize water so that the majority of thecell surface can be seen for mechanical manipulation.
Results clearly show a strong relationship between the cellsize and strength, the latter increases with an increase in thecell size (Fig. 7). Yeast cell walls consists predominantly of
glucan with (1,3)-β and (1,6)-β linkages, and mannan covalently linked to protein (mannoprotein) [21]–[23]. Mechanicalproperties are a function of β-glucans, whereas mannoproteins
control porosity [22], [24]. Thus, it can be inferred that the
β-glucans composition increases when the yeast cell size increases. This prediction is consistent with other studies statingthat small and large yeast cells exhibit similar glucan and mannan contents but further fractionation of the glucan showed fivetimes less alkali-insoluble glucan in the smaller sized cells [25].
This may be another reason why the large yeast cells appear

to be stronger than the smaller cells [25]. The alkali-insoluble
glucan plays a role in maintaining cell rigidity [26]. Nevertheless, the elastic properties of the cells at different sizes remainunchanged (Table II). The small and large yeast cells containdifferent composition levels of the alkali-insoluble glucan [25].
The increase in this glucan in the large cells does not change
the properties of the molecule itself but rather increases thenumber of molecules, without changing its elasticity properties.
Consequently, the elasticity of the whole cell also remains unchanged and independent of the cell size. On the other hand,since there are more glucan molecules in the larger cells, thethickness of the cell wall also increases [6]. Hence, the amountof force needed to penetrate the cell also increases due to more
molecule barriers during the indentation that leads to penetration. The average penetration forces for 3, 4, 5, and 6 µm cell
diameter ranges are 96 ± 2, 124 ± 10, 163 ± 1, and 234 ±14 nN, respectively (Fig. 7). This was expected since the yeast’s
cell wall is remarkably thick (100–200 nm) [27], whereas its
plasma membrane is only about 7 nm thick [27]. These results are comparable to reported penetration force of 1 nN fora human epidermal melanocytes that is the only cell membranewithout cell wall structure [8]. Since the mechanical strength ofthe cell is provided by the cell wall and not the cell membrane,
therefore, the melanocytes are easily penetrated compared toyeast cells. Additionally, the effect of local (small contact area
between the tip and cell surface and global (large contact area
between tip and cell surface) penetrations is also an importantfactor in the amount of applied force. This can be seen fromour results for the local penetration (96–234 nN) compared toreported average of 149 ± 56 µN for global bursting of the yeast
cell [6]. The average Young’s modulus obtained from (6), i.e.,
3.24 ± 0.09 MPa is reasonable compared to reported local cell
stiffness values of 0.73 MPa [7] and 1.12 MPa [9], since the Evalues obtained in this paper represent not only the local elastic
property of the cell but also the whole cell stiffness.
In addition to the cell size factor, we showed that the shapeof the indenter also plays an important role in predicting thepenetration force of a single cell. This can be confirmed from
Fig. 8 where the penetration force can be reduced to 40%–50%
by using a sharp cantilever tip compared to a flat tip. This provides a good indicator for future cell surgery where a minimal
penetration force is required for preventing serious cell damage
or undesirable mechanotransductions due to a large cell deformation [28]. The Young’s modulus (E) of the cells estimatedusing the spherical model (sharp tip) and the cylindrical model

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Fig. 9. Force-indentation curves until the penetration points using a sharp tip(conical model) for about 5 µm cell size under HV mode.
Fig. 10. Force-indentation curves until the penetration points using a flat tip(cylindrical model) for 4 to 6 µm cell sizes under HV mode.

(flat tip) obtained from (6) and (7), respectively, showed that the
E values of the cell remained constant at about 3 MPa.
The link between the cell strength with the increase cellsize as reported in this paper is somehow against the Laplacelaw. Laplace law governs the tension of an ideal thin-wallsphere, i.e., Wall tension = Transmural pressure × Radius of
the curvature × 1/2 [29]. From this equation, the more curved amembrane (small cell) is the less tension is experienced by thecell wall for a given transmural pressure (the pressure difference
between inside and outside of the cell). The less is the tension
the cell wall, the less is the possibility for the cell wall to fail.
This means that smaller cell is less likely to be ruptured than alarger cell.
This notion could be true if the thickness of the cell remains
constant independent of cell sizes. Since the cell wall plays amain role in cell stiffness than the membrane cell, any differencein the cell wall thickness would give different stiffness propertyof the cell. Smith et al. [6] showed that the thickness of thecell wall increases with the cell growth. Kliss et al. [21] also
revealed that cells increase their size during their lifetime before
undergoing a cell division. During this growing process, thecomposition of the β-glucan is increasing that, in turn, increases
the thickness of the cell wall [6]. The heterogeneity of the cellwall composition and cell wall thickness will give different
stiffness properties to the cell, and therefore, the Laplace lawthat governs the surface tension of the cell may play a minor
role in predicting the strength of the cell.
C. Effect of the Environmental Conditions on the Strength of
the Cells
The required force to penetrate a single yeast cell under HVmode using sharp and flat tips for different cell sizes are shownin Figs. 9 and 10, respectively. Insets are images of penetration of a single yeast cell using sharp (Fig. 9) and flat (Fig. 10)
cantilever tips. Higher spring constant of the cantilever, i.e.,
0.7 N/m was used for the compression experiment as lower
spring constant values failed to penetrate the cell. Fig. 3(d)shows the image of a tetrahedral cantilever tip used in this ex-

TABLE IIIYOUNG’S MODULUS OF THE SINGLE CELLS AT DIFFERENTENVIRONMENTAL CONDITIONS

periment. The penetration force to burst a cell wall increases
about 20 times from ESEM mode to HV condition (cells with
5 µm diameter were used for comparison and analysis) (Figs. 7
and 9). Since these data under HV condition are reported forthe first time, it is difficult to compare with other reports. Nevertheless, the increase in penetration force from ESEM to HVmodes that was obtained in our experiment can be logicallyunderstood. Cell wall construction is tightly controlled since
polysaccharide composition, structure, and thickness of the cellwall vary considerably, depending on the environmental conditions [21]. Therefore, we can infer that under extreme conditionof HV, the yeast cell tries to survive by increasing its wall thickness as suggested by the increased compression force needed topenetrate its cell wall (Figs. 9 and 10). Other possibility is that
under HV condition, cells become too dry, which eventually
increases their cell hardness. These findings reveal that besides
the shape of a cantilever tip, other external factors such as environmental conditions also influence the penetration force ofa single cell. Like the ESEM-mode, the cell size remains theinternal factor that influences the penetration force under HV
condition. By using a sharp tip, an average of 3 µN penetration
force was required for a 5 µm diameter cell. As expected, thisforce increased (up to 14 µN) when a flat cantilever tip was
applied, for cells with an average diameter of 4–6 µm.
For a quantitative evaluation of the elasticity of the cell, theHertz–Sneddon mechanics model for conical and cylindricaltips, as expressed in (2) and (4), are used for sharp and flat
tips, respectively. Average elastic properties of yeast cells under
the influence of two environmental conditions are shown in

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191

Fig. 11. Growth phase cycle for W303 yeast cells based on the OD6 6 0 n m
absorbance measurement.

Table III, where the cell stiffness increases about 10 times underthe HV mode. The cell surface becomes rigid when it is placedin the HV mode. It is believed that in the absence of water, thecell surface becomes harder than in the presence of water. This
leads to a significant increase in the Young’s modulus (26.02 ±3.66 MPa) obtained from (5) under the HV mode as comparedto the ESEM mode (3.31 ± 011 MPa) as expected.
D. Effect of Growth Phases on the Strength of the Cells
All cells have a unique growth phase curve during their life cycle that is normally divided into four phases: lag, log, saturation,and death phases. In the present study, we did not investigate thedeath phase. These phases can be easily identified from the optical density (OD) absorbance–time curve (Fig. 11). Lag phase islocated at the initial lower horizontal line where the cells adapt
themselves to growth conditions and the cells mature and divide
slowly. Log phase can be seen from an exponential line where
the cells have started to divide and grow exponentially. The actual growth rate depends upon the growth conditions. Saturation
phase resembles a final upper horizontal line where the growthrate slows and ceases mainly due to lack of nutrients in themedium. During the death phase, all of nutrients are exhausted
and the cells die [30]. It is believed that the mechanics of thenormal cells have different properties at each of the phases. In
this section, the mechanical properties of W303 yeast cells atfour different growth phases (early, mid, late log and saturation)
are discussed.
Since each cell has a unique growth phase, we determined thegrowth phase of W303 yeast cells by measuring the absorbanceat 660 nm using a spectrophotometer (Hitachi: U-2000) at 1 h
intervals (Fig. 11). The growth rate (absorbance per hour) was
about 0.23, as can be seen from the slope of the curve. Samples
were stored at −80 ◦ C (Sanyo: MDF-291AT) to hold its growth
before the compression experiment.
The compression experiments were done at 6 h intervals starting at early log phase and ending at saturation phase. Penetration
force was analyzed for each phase as shown in Fig. 12(a)–(d).
As expected, the force needed to penetrate a single cell increased
from early log to saturation phase (161 ± 25, 216 ± 15, 255 ±21, and 408 ± 41 nN), whereas the elastic properties of the cells

Fig. 12. Force-indentation curves (fitted using spherical model) for single cells
at each growth phases. (a) Early log. (b) Mid log. (c) Late log. (d) Saturation.
The curves are shown until the penetration points.

seem constant for all the phases obtained from (6), i.e., 3.28 ±0.17, 3.34 ± 0.14, 3.24 ± 0.11, and 3.38 ± 0.11 MPa (Table IV).
These mechanical properties of the W303 yeast cells at different
growth phases are in an agreement with reported increment of
average surface modulus of Scerevisiae cell wall to be 11.1 ±0.6 N/m (log phase) and 12.9 ± 0.7 N/m (saturation phase) with

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192

IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 7, NO. 3, SEPTEMBER 2008

TABLE IVYOUNG’S MODULUS OF THE SINGLE CELLS AT DIFFERENT CELLGROWTH PHASES

no significant increase in the elastic modulus of the cell, i.e.,
112 ± 6 MPa (log phase) and 107 ± 6 MPa (saturation phase)[6]. Their values for elastic modulus are quite high is also reasonable since they measured the whole elastic properties of thecell by compressing a single cell between two big flat indenters
as compared to a local cell indentation in our case.
From this analysis, it can be inferred that the thickness of theW303 cell wall is increased during the growth phase since thepenetration force increases at each phase, and at the same time,
maintaining their elastic properties during the growth phaseas verified by the constant elastic modulus values shown in
Table IV. The increase in the cell thickness does not affectthe elastic properties of the cell but the penetration force will
be increased since more molecules need to be penetrated. We
believe that the increase in the thickness of cell wall is mainly
due to an increment of β-glucans molecules [22].
V. CONCLUSION
To the best of our knowledge, we, for the first time, havedemonstrated the effect of the internal influences (cell size andgrowth phases) and the external influence (environmental conditions) on the cell strength. The penetration force is proportional
to the increase in the cell size, during the cell growth and under
HV condition. The elastic modulus of the cell increases dramatically under HV condition but remains unaffected during
the cell growth. In the future, we are going to characterize themechanics of diseased cells and these data can be compared tothe mechanical properties of the normal cells, and eventually,some relation can be made between the cell mechanics and particular human disease. The advantage of the integrated ESEMnanomanipulation system relies on its capability to performin situ local direct observation and manipulation of biologicalsamples and its ability to control environmental conditions.
ACKNOWLEDGMENT
The authors would like to thank Prof. Toshifumi Inada at
Nagoya University for providing the yeast wild-type strain
W303 cells.
REFERENCES[1] B. F. Brehm-Stecher and E. A. Johson, “Single-cell microbiology: Tools,
technologies, and applications,” Microbiol. Mol. Biol. Rev., vol. 68,
pp. 538–559, 2004.
[2] F. M. Harold, “Molecules into cells: Specifying spatial architecture,” Microbiol. Mol. Biol., vol. 69, pp. 544–564, 2005.

[3] M. B. Elowitz, A. J. Levine, E. D. Siggia, and P. S. Swain, “Stochastic
gene expression in a single cell,” Science, vol. 297, pp. 1183–1186, 2002.
[4] H. M. Shapiro, “Microbial analysis at the single-cell level: Tasks andtechniques,” J. Microbiol. Methods, vol. 42, pp. 3–16, 2000.
[5] Y. C. Fung, Biomechanics: Mechanical Properties of Living Tissue, 2nded. New York: Springer-Verlag, 2005.
[6] A. E. Smith, Z. Zhang, C. R. Thomas, K. E. Moxham, and A. P. J.
Middelberg, “The mechanical properties of Saccharomyces cerevisiae,”
Proc. Nat. Acad. Sci., vol. 97, pp. 9871–9874, 2000.
[7] A. E. Pelling, S. Sehati, E. B. Gralla, J. S. Valentine, and J. K. Gimzewski,“Local nanomechanical motion of the cell wall of Saccharomyces cerevisiae,” Science, vol. 305, pp. 1147–1150, 2004.
[8] I. Obataya, “Nanoscale operation of a living cell using an atomic force
microscope with a nanoneedle,” Nano Lett., vol. 5, pp. 27–30, 2005.
[9] T. S. Lanero, “Mechanical properties of single living cells encapsulated in polyelectrolyte matrixes,” J. Biotechnol., vol. 124, pp. 723–731,
2006.
[10] A. M. Donald, “The use of environmental scanning electron microscopy
for imaging wet and insulating materials,” Nat. Mater., vol. 2, pp. 511–
516, 2003.
[11] M. Nakajima, F. Arai, and T. Fukuda, “In situ measurement of Young’s
modulus of carbon nanotube inside TEM through hybrid nanorobotic
manipulation system,” IEEE Trans. Nanotechnol., vol. 5, no. 3, pp. 243–
248, May 2006.
[12] M. Nakajima, F. Arai, and T. Fukuda, “Nanofixation with low melting
metal based on nanorobotic manipulation,” in Proc. 6th IEEE Conf. Nanotechnol. (IEEE Nano 2006), pp. 925–928.
[13] M. R. Ahmad, M. Nakajima, S. Kojima, M. Homma, and T. Fukuda,“In situ single cell mechanical characterization of W303 yeast cells inside environmental-SEM,” in Proc. 7th IEEE Int. Conf. Nanotechnol.,Hong Kong, 2007, pp. 1022–1027.
[14] I. Obataya, C. Nakamura, S. W. Han, N. Nakamura, and J. Miyake,“Nanoscale operation of a living cell using an atomic force microscopewith a nanoneedle,” Nano Lett., vol. 5, pp. 27–30, 2005.
[15] T. S. Lanero, O. Cavalleri, S. Krol, R. Rolandi, and A. Gliozzi, “Mechanical properties of single living cells encapsulated in polyelectrolyte
matrixes,” J. Biotechnol., vol. 124, pp. 723–731, 2006.
[16] A. Touhami, B. Nysten, and Y. F. Dufrene, “Nanoscale mapping of theelasticity of microbial cells by atomic force microscopy,” Langmuir,
vol. 19, pp. 4539–4543, 2003.
[17] I. N. Sneddon, “The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile,” Int. J. Eng.
Sci., vol. 3, pp. 47–57, 1965.
[18] G. M. Pharr, W. C. Oliver, and F. R. Brotzen, “On the generality of therelationship among contact stiffness, contact area, and elastic modulus
during indentation,” J. Mater. Res., vol. 7, pp. 619–617, 2002.
[19] R. B. King, “Elastic analysis of some punch problems for a layered
medium,” Int. J. Solids Struct., vol. 23, pp. 1657–1664, 1987.
[20] M. Dao, N. Chollacoop, K. J. Van Vliet, T. A. Venkatesh, and S. Suresh,“Computational modelling of the forward and reverse problems in instrumented sharp indentation,” Acta Mater., vol. 49, pp. 3899–3918, 2001.
[21] F. M. Klis, A. Boorsma, and P. W. J. D. Groot, “Cell wall construction in
Saccharomyces cerevisiae,” Yeast, vol. 23, pp. 185–202, 2006.
[22] G. Lesage and H. Bussey, “Cell wall assembly in Saccharomyces cerevisiae,” Microbiol. Mol. Biol. Rev., vol. 70, pp. 317–343, 2006.
[23] P. N. Lipke and R. Ovalle, “Cell wall architecture in yeast: New sturctureand new challenges,” J. Bacteriol., vol. 180, pp. 3735–3740, 1998.
[24] H. Zlotnik, M. P. Fernandes, B. Bowers, and E. Cabib, “Saccharomyces
cerevisiae mannoproteins form an external cell wall layer that determines
wall porosity,” J. Bacteriol., vol. 159, pp. 1018–1026, 1984.
[25] T. Srinorakutara, “Study on the effect of diluent osmotic pressure on yeast
cell strength and cell size using a novel micromanipulation technique,”Int. J. Microbiol. Biotechnol., vol. 14, pp. 719–725, 1998.
[26] G. H. Fleet and D. J. Manners, “Isolation and composition of an alkalisoluble glucan from the cell walls of Saccharomyces cerevisiae,” J. Gen.
Microbiol., vol. 94, pp. 180–192, 1976.
[27] G. M. Walker, Yeast Physiology and Biotechnology. Chichester, U.K.:
Wiley, 1998.
[28] H. Huang, R. D. Kamm, and R. T. Lee, “Cell mechanics and mechanotransduction: Pathway, probes, and physiology,” Amer. J. Physiol. Cell
Physiol., vol. 287, pp. C1–C11, 2004.
[29] C. E. Morris and U. Homann, “Cell surface area regulation and membranetension,” J. Membr. Biol., vol. 179, pp. 79–102, 2000.
[30] G. J. Tortora, B. R. Funke, and C. L. Case, Microbiology: An Introduction,
8th ed. Redwood City, CA: Benjamin Cummings, 2003.

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193

Mohd Ridzuan Ahmad (S’07) received the B.S.
and M.S. degrees in electrical engineering fromthe Universiti Teknologi Malaysia (UTM), Skudai,
Malaysia, in 2000 and 2004, respectively. He is currently working toward the Ph.D. degree in the Department of Micro-Nano Systems Engineering, Nagoya
University, Nagoya, Japan.
From May 2000 to December 2001, he received a
National Science Fellowship from the Ministry of
Science, Technology, and Environment, Malaysia.
From June to November 2002, he was a Research
Officer in the Department of Robotics and Mechatronics, UTM. Since 2003,
he has been a Lecturer in the Faculty of Electrical Engineering, UTM. He is
also involved in robotics research field especially in multiagent robotics system.
His current research interests include micro/nanomanipulation, nanobiology,
biomechanics, and nanodevices.

Michio Homma received the B.S. degree in biology from International Christian University, Tokyo,
Japan, in 1980, and the M.S. and Dr. Science degrees
in biology from Tokyo University, Tokyo, in 1982
and 1985, respectively.
From 1985 to 1992, he was a Postdoctoral Associate in the Department of Molecular
Biophysics Biochemistry, Yale University. From1988 to 1992, he was a Lecturer in the Research Institute for Disease Mechanism and Control, School of Medicine, Nagoya University, Nagoya,Japan. From 1992 to 1998, he was with the Department of Molecular
Biology, Faculty of Science, Nagoya University. He has been a Professor in
the Department of Biological Science, Graduate School of Science, Nagoya
University. His current research interests include energy transduction and sensory transduction in bacteria.

Masahiro Nakajima (M’03) received the B.S. degree in mechanical engineering from Shizuoka University, Shizuoka, Japan, in 2001, and the M.S. and
Dr. Eng. degrees in mechanical engineering from
Nagoya University, Nagoya, Japan, in 2003 and 2006,
respectively.
From April to May 2006, he was a Research
Fellow in the Ministry of Education, Culture, Sports,
Science, and Technology (MEXT), Nagoya University, where he was also a Research Associate from
June 2006 to March 2007. Since April 2007, hehas been an Assistant Professor in the Department of Micro-Nano Systems
Engineering, Nagoya University. His current research interests include micro/nanomanipulation, nanoassembly, nanofabrication, nanodevices, nanomechanics, and nanobiology.

Toshio Fukuda (M’83–SM’93–F’95) received theB. S. degree from Waseda University, Tokyo, Japan,
in 1971, and the M.S. and Dr. Eng. degrees from the
University of Tokyo, Tokyo, Japan, in 1973 and 1977,
respectively.
From 1977 to 1982, he was with the National Mechanical Engineering Laboratory, Tsukuba, Japan.
From 1982 to 1989, he was with the Science University of Tokyo, Tokyo. Since 1989, he has been with
Nagoya University, Nagoya, Japan, where he is currently a Professor in the Department of Micro-Nano
Systems Engineering. His current research interests include intelligent robotic
systems, cellular robotic systems, mechatronics, and micronanorobotics.
Prof. Fukuda was the President of the IEEE Robotics and Automation Society during 1998–1999, the Editor-in-Chief of IEEE/ASME TRANSACTIONSON MECHATRONICS during 2000–2002, the Director of the IEEE Division X,
Systems and Control during 2001–2002, and the President of the IEEE Nanotechnology Council during 2002–2005.

Seiji Kojima received the B.S., M.S. and Ph.D. degrees in biological sciences from Nagoya University,
Nagoya, Japan, in 1994, 1996, and 1999, respectively.
From April 1999 to February 2004, he was a
Postdoctoral Fellow in the Department of Biology,
University of Utah, USA. From March 2004 to
September 2005, he was a Postdoctoral Fellow at the
Dynamic Nanomachine Project, ICORP JST, Japan.
Since October 2005, he has been a Research AssistantProfessor in the Department of Biological Science,
Nagoya University. His current research interests include bacterial physiology, protein chemistry and structure, and visualization ofprotein behavior by fluorescent probes especially membrane proteins involvedin energy conversion.

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