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RESEARCH ARTICLE
Advanced Science Letters
Vol. 4, 368–376, 2011
Atomic Force Microscopy Study of
Living Baker’s Yeast Cells
L. Mikoliunaite1, A. Makaraviciute1, A. Suchodolskis1, A. Ramanaviciene1, Y. Oztekin12,
A. Stirke1, G. Jurkaite1, M. Ukanis1, G. Carac13, P. Cojocaru14, and A. Ramanavicius1∗
1Center of Nanotechnology and Materials Science – NanoTechnas, Faculty of Chemistry, Vilnius University,
Naugarduko 24, LT-03225, Vilnius, Lithuania
2Department of Chemistry, Faculty of Science, Selcuk University, 42075 Konya, Turkey
3Department of Microbiology, Faculty of Engineering Food, “Dunarea de Jos” University of Galati, 800008 Galati, Romania
4Chemistry, Materials and Chemistry Engineering Department “Giulio Natta”
Politecnico di Milano 20131 Milano, via Mancinelli, 7 Italy
Currently, Atomic Force Microscopy (AFM) has a wide range of applications in a variety of disciplines of science
and industry including biology and medicine. Despite the maturity of the AFM technique, it is still finding new
opportunities in visualization of biological materials and biomolecular processes. The most frequent and ordinary
application of AFM is surface topology and morphological studies. This paper describes educational application
of AFM in observation of a living cell wall structure. For the following study we have selected Sacchoromyces
cerevisiae cells, better known as baker’s yeast cells that are available in any supermarket and widely used for
fermentation control of food and drinks, and as a leavening agent in baking. The sample preparation and mea-
surements are described. Living yeast cells were prepared in solutions containing glucose or sodium chloride.
Proposed AFM based protocol allows the researcher to keep baker’s yeast cells alive, and to observe them
in air conditions. The experiment is suitable for Master and/or Ph.D. students and is designed to show the
main principles of the Bio-AFM operation. In some particular cases, this protocol may be adoptable for bachelor
students, who have already completed an extended course in nanotechnology, biotechnology or microbiology.
To show educational suitability, the protocol has been tested in the environment of educational laboratory and
performed by students studying physics, chemistry, biotechnology and biology.
Keywords: Education, Nanotechnolgy, Nanobiotechnology, AFM, Yeast, Living Cell.
1. INTRODUCTION
Nanotechnology has emerged as a broad and exciting field
of scientific research and technological innovation.1There are
important questions about the technology’s potential economic,
social, and environmental implications.2Many interlinks between
physics and biology that are extremely important for education in
physics and other technological sciences are foreseen, since nature
offers us number of really acting nanodevices. Even Richard
Feynman has not been interested in the creation of miniatur-
ized versions of existing macroscopic machines, but wished to
construct microbiological machines and tools that would enable
scientists to mimic microbiological materials.3The cell is the
structural and functional unit of life and it serves as a “factory”
producing a number of nano-machines, which drive not only
intracellular movements but also the motility of a cell. Among
such nano-machines many proteins and ribosoms are listed.4To
∗Author to whom correspondence should be addressed.
improve the characteristics of living cells foreign components
could be introduced into the cell through membrane/wall holes
opened by transformations, which can be induced by chemical
compounds, strong magnetic field, electrical pulses.5Nanopar-
ticles with special physical properties including fluorescence
(e.g., Q-dots),6–9 catalytic ability10–13 and magnetic properties14–18
could have been applied for modification of living cells. Most
recent topics of yeast cell application in industry is related to gen-
eration of “green-electricity”19–21 therefore we selected yeast cells
for this educational protocol.
Nanotechnological methods based on microelectronic trans-
ducers and actuators are becoming very popular as educational
protocols in physical and life sciences22 including biochemistry
and molecular biology.23 Atomic Force Microscopy (AFM) is a
versatile nanotechnological method, which can be applied in the
study of inorganic,24 polymers25 and even biological samples.26
AFM belongs to the family of scanning probe microscopes and
shares a similar concept with Scanning Tunneling Microscope
(STM). STM has been invented in 198127 and this invention has
368 Adv. Sci. Lett. Vol. 4, No. 2, 2011 1936-6612/2011/4/368/009 doi:10.1166/asl.2011.1236
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RESEARCH ARTICLEAdv. Sci. Lett. 4, 368–376, 2011
been awarded by a Nobel prize in 1986. The problem of a sharp
probe positioning with sub-nanometer scale over an investigated
sample surface has been solved. STM allows one to visualize
and manipulate single atoms on an investigated surface. STM
senses a tunneling current when the probe moves over a surface.
When STM works in constant current mode the height of sur-
face features is measured. The drawback is that the technique is
applicable only to conductive samples.
In 1986 AFM emerged when STM was modified to extend
measurements for non-conducting samples.28 In contact mode the
direct contact between a probe and an investigated surface is used
when the probe is drawn and its deflection corresponds to the
height of surface roughness. Several different AFM modes were
developed, which differ in the nature of a probe/sample interac-
tion. In non-contact mode deflections of the probe are propor-
tional to the interaction force between the surface and the probe.
This mode is suitable to measure various physical properties like
magnetic, electrical or probe/sample interaction forces. Another
modification is tapping mode, when oscillating probe only taps
the surface. AFM has a lower resolution than STM, but still has
a higher resolution than the optical microscope. For conventional
probes it is lower than 1 nm in height and can be as low as 5 nm
in the XY direction.
The reader can find a short historical review of AFM regard-
ing the development and application in biology and medicine.29
Despite the short history of AFM, it has become a multifunc-
tional tool in the study of micro- and nano-structures, physical
and mechanical properties of materials and even in investigation
of dynamic processes at surfaces and interfaces of biomaterials.30
AFM is widely spread and routinely used in biological science
for morphological and topographical imaging to reveal structural
parameters at sub-molecular resolution scale.31 One advantage
of AFM application in biology is the ability to perform imag-
ing in liquids, this unique application was firstly demonstrated
in 1987.32 Recent research on the application of the AFM sys-
tems has focused on manufacturing and metrology processes at
a molecular level due to its applicability for intermolecular force
measurements. This includes molecular recognition events when
the AFM offers means to localize specific receptors on cells, such
as cell adhesion proteins or antibiotic binding sites.3334 Chem-
ically modified probes with attached selective functional groups
are used to reveal specific interactions on investigated surfaces
at a molecular resolution.3536 Side by side with the conventional
AFM, the high-speed AFM is under development, which will be
capable of direct and real time visualization of biomolecular pro-
cesses on a millisecond time scales with preserving the resolution
on nano-scale.37
For the current AFM study yeast cells were selected, since
they are very popular in the scientific community for versatile
applications, including bioanalytical applications38 and biological
education.3940 They have complex internal cell structure and are
well suited as a model organism for AFM investigations.41 As an
investigation object the yeast cells distinguish ease of manipula-
tion, they are widely available and rapidly reproduce.
The idea of this paper is to show for students how novel elec-
tronics and micro-engineering based technologies (e.g., Atomic
Force Microscopy (AFM)) can merge with life sciences and how
they are suitable for visualization in life sciences. Particularly,
the aim of this lab protocol is to show the students the appli-
cability of AFM in biology and introduction into major AFM
methods that are suitable for imaging of living cells.
2. EXPERIMENTAL METHODS
2.1. Planning and Preparation for the Experiment
The optimal number of students for this experiment ranges from
two to four. The experiment is divided into two major parts. In
the first part students prepare several different baker’s yeast solu-
tions while in the second part students will use the AFM in order
to observe the surface of the yeast cell wall. The preparations and
arrangement of the experiment take at least 2 hours and depend
on the qualification of a responsible person in the field of the
AFM. All main steps of the experimental procedure are described
below.
2.2. Monitoring Method of Baker’s Yeast Solutions
The BioScope II AFM combined with optical microscope devel-
oped by Veeco Instruments Ltd (Santa Barbara, USA) was
applied for the surface monitoring of baker’s yeast cells. If this
instrument is not available, then any another AFM from any other
manufacturer with the requirement to perform measurements in
a tapping mode (oscillating mode) can be applied. The experi-
ment may be easily modified for measurements in a contact-force
mode. Basically, all novel commercially available AFMs should
be simple in application and as a rule have a contact mode and
the modes that are similar to the tapping mode. In this paper only
basic functions of BioScope II will be presented. We advise one
to refer to the application notes of an appropriate device planned
for application in this experiment to find technical specifications
and specific operation procedures. The experiment is designed to
consume up to 8 hours of students’ time.
2.3. Chemicals/Materials
Baker’s yeast (Saccharomyces cerevisiae) was purchased from
food supplier LALLEMAND (Lublin, Poland). For culturing of
the yeast cells solutions containing glucose or sodium chloride,
in distilled water were used. D-(+)-Glucose was purchased from
Carl Roth (Karlsruhe, Germany). All chemicals were of analyti-
cal grade and used as received (if not otherwise stated).
2.4. Preparation Solution Containing
Baker’s Yeast Cells
We recommend to prepare about 10 mL of yeast culture, which
consists of 0.2 g of glucose, and 0.1 g bakers yeast cells dis-
solved in distilled water. This solution should be stirred and kept
in room temperature for 30 min. Additionally, in this study, 0.9%
sodium chloride solution in distilled water was used. The con-
ditions, which are used during incubation period of yeast cells
can be varied, it makes the experiment more diverse and there-
fore very attractive scientifically. For example, the yeast solution
can be kept at 40 C temperature and/or various chemicals, such
as hydrogen peroxide, can be added to induce cell growth or
to modify the wall of yeast cells. Our recommendation for the
bakers yeast culture would be to drie them at low temperatures,
since under higher temperatures yeast cells can lose the rigidity
of the cell wall.
2.5. Preparation of Sample for AFM Imaging
To avoid the influence of surface roughness the silicon wafer from
CrysTec (Germany) has been used as a substrate. If silicon wafer
is not available any other smooth surface could be used as a sub-
strate for the AFM study. However, it is recommended that the
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RESEARCH ARTICLE Adv. Sci. Lett. 4, 368–376,2011
roughness of the surface would not exceed 10 nm. The roughness
can be easily estimated by imaging the substrate surface by AFM
prior to the deposition of the solution. The prepared solution
(10 L) was deposited on the substrate and was let dry for 15 min.
Later the sample (the substrate with the yeast cell solution) was
placed in the measurement compartment of the AFM.
2.6. Preparation of AFM Device for Imaging
The AFM imaging requires some preliminary steps. The most
important steps are (i) sample preparations and (ii) probe selec-
tion, which satisfy imaging conditions (soft or hard sample, tap-
ping or contact mode). The next most important steps are:
(i) data recording: the recorded image refining by supplied soft-
ware (image rotation, flattening, reduction of electro-mechanical
noise etc.),
(ii) data analysis and
(iii) data interpretation.
During the image recording a sharp probe (Fig. 1) moves over
an investigated surface line by line and probe/sample interaction
gathers information about local properties in every probed point of
the sample surfaceThe simplified schematics of AFM is shown in
Figure 2. The AFM consists of three main blocks, which control
the positioning of a probe over a sample and its interaction force
with the sample. These blocks are a scanner, an optical detection
system, and feedback control electronics. The scanner is based
on piezo-transducers, which change their geometrical dimensions
when corresponding voltage is applied. The detection system is
composed of a laser and a special configuration photodiode con-
sisting of four independent sections. The reflected laser beam by
the cantilever falls to the photodiode. The photodiode signal is
proportional to the beam spot position on the light sensitive sur-
face. The beam position depends on deflection (the spot moves
vertically) and bending (the spot moves horizontally) of the can-
tilever. When the height of the sample is measured the photodiode
signal is compared to the reference value and if any difference
is detected the feedback control governs the positioning system
which rises up and down the probe to keep the photodiode signal
equal to the reference signal. In the case of a contact mode, the
reference signal corresponds to the deflection of the cantilever.
In the case of a tapping mode, the reference signal corresponds to
the oscillation amplitude of the cantilever.
The most common structure of the AFM probe is shown in
Figure 1. The probe is composed of a massive base and a long
cantilever with a tip at the very edge. Silicon or silicon nitride is
Fig. 1. Schematic presentation of the standard probe used in the AFM
imaging. The massive base supports the cantilever (top view) with the sharp
needle attached at the very end (side view).
Fig. 2. Schematic presentation of the Atomic Force Microscope. AFM is
composed of three main blocks: (i) The optical detection system (laser, pho-
todiode, cantilever), which tracks the deflection of the cantilever. (ii) The
XYZ positioning system is changing the probe location over the sample and
(iii) the feedback block controls the probe positioning by comparing the pho-
todiode signal with a reference voltage.
used as substrate-materials for AFM probes. The cantilever has
a shape of a triangle or a long rod. The material and the geo-
metrical shape of the cantilever determine mechanical properties
of the probe including its rigidity and resonant frequency. The
radius of the tip apex determines the resolution of an experi-
ment. Probes for common use have the apex radius about 10 nm.
The AFM probes can be modified by various materials, which
improve the distinctive characteristics of a probe and make it
sensitive to magnetic, electrical, and other properties.
The Bioscope II AFM is composed of many units some of
which are directly used to adjust and prepare an imaging proce-
dure and shown in Figure 3. A computer with installed software
supplied by the AFM producer controls all hardware and hence
all measurement steps. As shown in Figure 3(a) the XY AFM
stage (1) with the place for the investigated sample (3) is fas-
tened to the inverse optical microscope (2). A special holder for
the probe is used (Fig. 3(b)). Figure 3(c) shows the optical head
(4) which contains the Zstage with optical detection system and
contacts for the probe holder. As demonstrated in Figures 3(c
and d) the optical head (4) is placed on the alignment stage (5) to
adjustment position (Fig. 3(d)) where a laser beam is positioned
on the very edge of the cantilever by a pair of knobs on the stage.
The process is simplified by direct observation of the laser beam
spot and the cantilever on the LCD screen and on the computer
screen by software (shown on Figs. 3(d and e) by arrow). With
other pairs of knobs the reflected spot is positioned in the center
of the photodiode for tapping mode and slightly below (−2V)
the central line in contact mode. The position of the spot is con-
trolled in the software window, which mimics the geometrical
configuration of the photodiode (Fig. 3(e)). After the adjustment
the optical stage is placed on the XY stage for measurements.
The joystick in all three directions can change the position of the
probe over the sample (Fig. 3(f)). The last step in the procedure
is the adjustment of scanning parameters including:
(i) scan area,
(iii) feedback gains,
(iii) speed of scanning.
As it was mentioned dependently on the type of AFM device
several types of AFM modes could have been applied for this
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RESEARCH ARTICLEAdv. Sci. Lett. 4, 368–376, 2011
Fig. 3. The main parts of the Bioscope II: (a) the XY stage (1) mounted over the optical microscope (2) with the sample compartment (3); (b) the probe
holder; (c, d) the optical head (4), which consists of optical detection system and Zstage and alignment stage (5), which facilitates the laser beam adjustment
on the cantilever; (e) the photodiode adjustment window in the software; (f) the joystick, which controls the movement of the probe over the samples.
type or investigations, in this article we will apply just two of
them: (i) Contact mode and (ii) Tapping mode.
2.7. Principles of the Contact Mode AFM
During a measurement in “contact mode” the tip is always in
contact with a sample surface. When the tip is pressed onto the
sample the cantilever deflects and a feedback loop maintains a
constant deflection moving the probe in Zdirection up/down
compensating the roughness of the surface and hence keeping
the constant probe/sample interaction force. The applied force is
determined by Hooke’s law:
F=kz
where z stands for the deflection of the cantilever and kis a
spring constant of the cantilever, the kdepends on elasticity of
the cantilever. Lower kvalues indicate “softer” cantilever and
then lower force is applied to the measured surface, thus imaging
of soft samples by cantilevers with low kvalues are preferable.
Additionally, to minimize the probe/sample interaction the force
calibration mode can be utilized to adjust the interaction force to
the required level. The lowest kvalues available on the market
now are as low as 10−3N/m.
2.8. Principles of the Tapping Mode AFM
The tip oscillates at the resonance frequency of the cantilever
with an amplitude, which depends on the amount of mechani-
cal energy passed to the probe by the piezo stage. Due to an
intermittent nature of the probe/sample contact the tip induces
mechanical pressure to the investigated surface only for a short
time compared to the contact mode. Tapping mode is often used
for imaging of soft objects.
The tip does not touch an investigated surface when the can-
tilever oscillates with the free vibration amplitude at the resonant
frequency. When the probe gets closer to the surface and/or the
oscillation amplitude is increased, the tip reaches the surface of
the sample and a part of energy is transferred to the surface of the
investigated sample. This results in the reduced free amplitude.
The reflected laser beam by cantilever reveals periodical move-
ments of the tip as shown in Figure 4. Oscillations are converted
to an electronic signal called “Root Mean Square” (RMS) ampli-
tude and this signal is used in feedback electronics to keep the
oscillation amplitude at the same level. The initial amplitude of
oscillation is defined by a value of “Amplitude set point” (Set-
point), which is determined automatically by the system during
an engaging process. The RMS amplitude is constantly compared
to the set-point value. When the probe moves over a height step
the RMS amplitude is decreased as shown in Figure 4. The pro-
portion of RMS/set-point signals is transmitted to the feedback
block, which generates corresponding voltage to the Zstage to
raise the probe to recover the RMS amplitude to the initial level.
The generated voltage is proportional to the step height and is
evaluated as a measurement of the signal indicating the height of
the corresponding sample point.
When a soft sample is investigated it is highly desirable to
minimize the energy, which is dissipated to the sample surface.
The dissipating energy depends on the set-point value (defines the
height of the probe over the investigated surface) and the RMS
amplitude (the oscillation amplitude of the cantilever). Increas-
ing the RMS amplitude when the set-point is fixed enhances the
amount of energy passed to the surface. The same happens when
the set-point is decreased while the RMS amplitude is fixed. As
schematically shown in Figure 4 the RMS amplitude recovery
The step changes
the RMS signal which
is restored moving
probe up
RMS signal detected
on photodiode
Fig. 4. Tapping mode principles. The height step diminishes the amplitude
of the cantilever oscillations (the RMS amplitude detected by the photodiode)
and moving up the cantilever restores the amplitude of oscillations.
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takes some time and the recovery is longer for higher ampli-
tudes. It means that high features on the surface can be distorted
due to the long recovery time. On the other hand, if the RMS
amplitude is set to low and the set-point is quite high the tip can
not track lower features on the sample. The cantilever oscillation
amplitude and the set-point are always determined by the system
during engagement procedure and later they have to be adjusted
dependent on the properties of an investigated surface.
2.9. Set-Up of AFM Imaging in Taping Mode
Attach the holder with mounted probe to the stage of the micro-
scope and put it on the alignment stage. Next, using the LCD
screen adjust the laser beam position on the cantilever and adjust
the position of the reflected beam on the photodiode controlling
adjustment with the photodiode window in the software. Initial-
ize the AFM and find the resonance frequency of the cantilever
by tuning procedure. Place a sample in the measurement com-
partment and later the optical head on the XY stage. Use the
joystick to locate the preferred place for measurements (X,Y
direction) and move down the probe (Zdirection) as low as pos-
sible avoiding probe damage. Set the parameters in software.
Integral gain (slow reaction) and Proportional gain (fast reaction)
0.0 1: Height 4.0 µm
25.0 nm
0.5
nm
0
1
2
3
4
5
6
7
8
9
10
1 1.5 2 2.5 3 3.5 µm
Fig. 5. Roughness of the silicon substrate with the 9 nm height structure.
The bottom plot corresponds to the white line on the image.
determine the strength of the feedback control. Set the Scan Rate
(∼2 Hz). Set the scan size 1 nm to prevent probe damage. Due to
its high features on the surface, during the measurements it can
be adjusted to the required size. Set the image resolution (num-
ber of scanned lines per sample). The software outputs various
types of surface data. For the tapping mode the height and the
amplitude error are the most popular and most informative. The
amplitude error is a derivative of the height signal and empha-
sizes the height variance on the measured surface especially small
features, which can be hidden within the height image. Set the
same trace direction for both windows and set “Plane—Fit to
Fig. 6. Baker’s yeast cells overheated by hot air: (a) the AFM topography
image and (b) the corresponding amplitude error image.
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Fig. 7. Baker’s yeast cells with visible scar structure shown by an outline. (a) Topography and (b) the amplitude error and (c) the amplitude error close up
view of the scar. The smooth surface in the bottom part corresponds to the Si substrate.
Line” and “Offline Plane—Fit to none.” Later the probed image
can be refined by the procedures in the software. All mentioned
parameters could be adjusted during measurements if it is needed.
Set-point, Drive Amplitude, Scan Rate, Proportional and Integral
gains directly determine the quality of the signal and should be
adjusted after the engagement to get a stable signal.
3. RESULTS AND DISCUSSION
Before deposition of prepared cell solution on silicon surface its
roughness was estimated as it is shown in Figure 5. The AFM
topography image shows very smooth silicon surface with the
roughness below 1 nm as shown below the image in the profile
section, which corresponds to the white line on the image. Due
to such small roughness values are close to the limit of our AFM
resolution and due to certain environmental conditions (poor tem-
perature isolation, vibrations, and noise) some features appear on
the image to prove that measurement is correct. If such features
do not appear, it indicates problems in imaging (e.g., the AFM
probe is not properly touching the surface, etc.).
We measured baker’s yeast cells in air environment. Prepared
solution after the deposition on the substrate was dried by hot
air. If cells were overheated their walls lose the rigidity and
bend/arch/incurvate as shown in Figure 6. Two AFM images are
presented, which correspond to topography (Fig. 6(a)) and ampli-
tude error (Fig. 6(b)). The former allows precise measuring of
geometrical size of the investigated features in all three dimen-
sions. The latter is a derivative of the topography image, which
emphasizes any features on the surface including an estimated
geometrical shape of features on the plane.
Figure 7 presents baker’s yeast cell topography and amplitude
error images. A bud scar is detected, which is emphasized by
outline. The close up image in Figure 7(c) reveals a structure of
the scar.
Figure 8 shows the topography image with the extracted cross
section on the plot below corresponding to the white line. The
profile allows determination of geometrical sizes of measured
features on the sample surface. As it is seen with the width of
the three cells it is confined in 1–2 m range. The bud on the
top of the cell is 0.25 m height and 1 m width. To show such
structures for the students it is especially useful since the buds
are growing over time and demonstrating “nature in action;” it
increases the fascination of this experiment and makes it very
attractive for the students.
Figure 9 presents two amplitude error images, which show
the same place measured in “soft tapping” and “hard tapping”
modes. A yeast cell wall is relatively rigid but it can be damaged
by the probe. In the first case (Fig. 9(a)), the dissipation of the
Fig. 8. Calculation of geometrical sizes of measured features from the AFM
topography image.
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Fig. 9. The AFM amplitude error images measured in soft tapping mode
(a) and in hard taping mode. (b) The outline shows induced mechanical
damages to the cell membrane by probe.
mechanical energy to the surface is low and the probability of an
induced damage to the surface is quite low. In the second case,
the contact force of the tip/sample is so high that the membrane
of the cell is broken as shown by an outline in Figure 9(b). Two
bud scars can be distinguished on both images (Fig. 9(b)).
We expected this cell would start reproduction. Baker’s yeast
cells bud when a small bud emerges from the surface of the
parent cell and enlarges until it is almost the size of the par-
ent. However, after drying them in the air, only the bud scars
or very small buds—the marks of reproduction were obvious.
Despite the fact that the yeasts during the measurement are in
ambient air conditions, sometimes the reproduction processes
could continue for some time. This could be obviously seen in
Figure 10(a). The insert shows the same bud imaged 5 min later
and the Figure 10(b) presents the enlarged view of the bud. As
one reproduction cycle lasts for about 20 min. in physiological
liquids, the growth of the bud in air stops or is very slow.
As stated in the methods portion baker’s yeast cells have been
kept in different solutions for later studies by AFM. Figure 11
shows cells, which have been kept in glucose (Fig. 11(a)) or
sodium chloride solutions (Fig. 11(b)). After drying a visible
Fig. 10. (a) The reproduction of baker’s yeast cell; (b) The insert shows the
growing bud, shown in “a” image.
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Fig. 11. The traces of (a) glucose; (b) sodium chloride crystals on yeast cell
membranes formed after drying the yeast cells, which were kept in glucose
and sodium chloride solutions correspondingly.
amount of glucose and sodium chloride from the solutions is left
on the surface of yeast cell walls that is apparently seen in both
images. The glucose crystals are well revealed by the phase view
while sodium chloride crystals on cells are clearly observed in
the amplitude error image.
4. CONCLUSIONS
The results presented clearly show that AFM can be applied in
versatile educational protocols. The AFM method gives a much
higher resolution if compared to the usual optical techniques. If
applied for study of biological objects, the living cells and even
smaller structures like bud scars, the described experiment here
can be presented in detailed images. We believe that in the near
future AFM will be widely applied for educational purposes as a
visualization tool, which offers unique resolution and/or specific
surface characterization capabilities.
Acknowledgments: The study was supported by Lithua-
nian State Science and Studies Foundation according to contract
number S-19/2008 and by Research Council of Lithuania accord-
ing to contract number MOS-9/2010.
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