1. Draft 100330
Atomic Force Microscopy Imaging of Biological Samples under
Dry and Liquid Conditions
Larissa Clark
Chemical and Materials Engineering
Atomic Force Microscopy
Michel Goedert, Ph.D., Manager
San José State University
September 14, 2009
Corrections March 30, 2010

2. ii – Draft 100324
Table of Contents
1.0 Introduction..........................................................................................................................................1
1.1 AFM Basics.......................................................................................................................................1
1.2 Biological Sample Background.........................................................................................................3
1.2.1 Chromosomes.............................................................................................................................3
1.2.2 HeLa Cells..................................................................................................................................4
2.0 Materials and Methods.........................................................................................................................5
2.1 Samples and Sample Preparation ......................................................................................................5
2.2 AFM Equipment................................................................................................................................6
3.0 AFM Imaging Procedures....................................................................................................................6
3.1 Contact Mode in Dry Conditions with the PNP-DB Probe...............................................................6
3.2 Contact Mode in Liquid Conditions with the PNP-DB Probe ..........................................................7
3.3 Intermittent Contact Mode in “Just Dry” Conditions with PPP-NCHR Probe .................................7
4.0 Data and Analysis ................................................................................................................................8
4.1 Dry AFM Imaging in Contact Mode with the PNP-DB Probe ........................................................9
4.2 Comparison of Dry and Liquid Imaging with the PNP-DB Probe in Contact Mode......................11
4.3 “Just Dry” Imaging with the PPP-NCHR Probe in Intermittent Contact Mode..............................12
4.4 Effects of Gain with the PNP-DB Probe in Liquid using Contact Mode........................................14
4.5 Comparison with Previous Studies..................................................................................................15
5.0 Future Work .......................................................................................................................................17
6.0 Conclusion .........................................................................................................................................17
7.0 Acknowledgments..............................................................................................................................18

3. iii – Draft 100324
List of Figures
Figure 1. AFM probe operation..................................................................................................................2
Figure 2. Artist’s rendition of an animal cell and formation of a chromosome. ........................................3
Figure 3. Stages of coiling from DNA to a metaphase chromosome.........................................................4
Figure 4. AFM images of Abbott chromosomes and 30-100 nm chromatin fiber in dry conditions.........9
Figure 5. AFM images of HeLa chromosomes and 30-70 nm fiber in dry conditions ............................10
Figure 6. AFM images of Abbott normal male metaphase chromosomes in dry and liquid conditions..11
Figure 7. AFM image of three HeLa cells................................................................................................12
Figure 8. Effects of gain adjustments on image quality and feature height on the Abbott chromosomes14
Figure 9. AFM images of the Ushiki and Hoshi dry sample prepared by critical-point drying...............16
Figure 10. AFM images of Ushiki and Hoshi chromosomes in hexylene glycol buffer............................16
List of Tables
Table I. Sample Preparation and Final Feature Sizes..................................................................................8
Table II. Feature Sizes for Contact Mode Imaging in Dry and Liquid Conditions with PNP-DB Probe...11
Table III. Effects of Gain on Image Quality and Feature Height in Liquid using Contact Mode. ..............14
Table IV. Sample Preparation and Results from Ushiki and Hoshi Studies ................................................15

4. 1 – Draft 100330
Atomic Force Microscopy of Biological Samples under Dry and Liquid Conditions
Abstract: The purpose of this study was to image biological samples by atomic force microscopy (AFM)
in dry and liquid conditions. One of the biological samples imaged was of chromosomes in the metaphase
stage of mitosis from normal lymphocytes. In metaphase, chromosomes are particularly condensed and
recognizable [1], which enables easy identification of the structures. The second sample type was of HeLa
whole cells and metaphase chromosomes. Image optimization was tested by varying AFM parameters,
including the imaging mode, probe type, and gain. Imaging was performed using contact mode and
intermittent contact mode. While liquid imaging increased the image resolution in contact mode, high
resolution images were also attained using a form of dry imaging in intermittent contact mode. Similarly,
while a low resonant frequency and low spring constant probe is considered preferable for AFM imaging of
soft, biological samples, both probe types in this study produced high resolution images with no apparent
sample damage. Adjustments to the gain were shown to affect the sample features and image quality. The
buffer type and sample preparation were also found to affect the image quality. Background, protocols,
results, and future directions are provided, and comparisons are made with previous studies [2][3][4][5][6].
1.0 Introduction
The purpose of this study was to obtain AFM images of biological samples. The biological samples used
were chromosomes extracted from the lymphocytes of a normal, male donor and dried HeLa cells and
chromosomes from a female donor. Chromosomes were of particular interest in this study because of the
nano-packaging mechanisms of chromosomes and the potential of these mechanisms in future technologies.
The lymphocyte chromosome samples were purchased from Abbott and came prepared as chromosome
spreads on glass specimen slides. The HeLa cell and chromosome samples were provided by the San José
State University (SJSU) Biology Department on glass cover slips. Image optimization was tested by
varying the imaging mode, probe type, and gain. Effects of the buffer type and sample preparation were
observed. The imaging modes used were contact and intermittent contact modes in dry, liquid and “just
dry” conditions, where “just dry” conditions involved brief exposure of the sample to a liquid buffer, where
the liquid was carefully wicked from the surface prior to imaging. Liquid conditions were used to mimic
the native biological environment to preserve as much of the original sample as possible and to reduce
adhesion forces, which are a type of loading force that can affect image resolution. Loading forces occur in
ambient conditions due to water condensation and other contamination that forms on the tip and sample
[7][8], where changes in pH, ion type, and ion concentration of the liquid buffer can be used to further
reduce loading forces [8]. However, for this study, DI water was used as the buffer as ionized solutions
such as phosphate buffered saline (PBS) reacted adversely with the probe. Two probe types were used.
The first probe had a low resonant frequency, low spring constant that is considered preferable for soft,
biological samples. The second type was a stiffer, high frequency, high spring constant probe. Effects on
image quality by adjusting the gain were studied, where a high gain increases the probe tip sensitivity.
Following is a description of AFM basics, background on the biological samples, the results of the study,
comparisons with previous studies, and future directions.
1.1 AFM Basics
AFM is a type of imaging under the umbrella of Scanning Probe Microscopy (SPM). SPM enables
imaging of the surface of materials and can also be used to analyze “adhesion, elasticity, electrostatic
charge [9],” magnetic spin, and more. In AFM, as the tip approaches the sample, the tip first experiences
attractive forces between the atoms of the tip and the sample. Non-contact mode operates in this attractive
region. As the tip further approaches the sample surface, the attractive forces gradually diminish and van
der Waals forces occur. As the tip continues towards the sample surface to a distance of less than a few
Ångstroms, the atoms begin to repulse each other [9]. Intermittent contact mode takes advantage of both

5. 2 – Draft 100330
the attractive and repulsive forces, which causes the tip to tap along the surface. Contact mode operates in
the repulsive region, where the AFM voltage is optimally set to overcome the repulsive forces to push the
tip onto the surface to maintain constant contact. Contact mode is ideal for imaging hard, smooth surfaces.
Non-contact and intermittent contact modes are used for imaging soft surfaces.
A simplified AFM probe operation is shown in Figure 1. The probe tip is located at the end of a cantilever.
A laser beam is aligned on top of the cantilever in the area above the tip. The laser reflects from the
cantilever onto a photodetector, where the voltage produced is fed back to a piezoelectric scanner to correct
the signal. The scanner is a cylinder that is made of a piezo-electric material that is moving in x, y, and z
directions and is able to respond to very small error signals. For the Agilent 5500 AFM system, voltages as
small as 10 millivolts are possible to control, which corresponds to 0.1 Ångstrom resolution [10]. The
voltage applied to the scanner is translated in displacements by the system to create a 3-D image. The
sharpness, or radius of curvature, of the probe tip, and the response of the piezo-electric scanner determines
the image resolution [9][10]. The tips used in this study had a radius of curvature less than
10 nanometers (nm).
Figure 1. AFM probe operation [9].
The setpoint is a voltage setting that controls the amount of force between the tip and sample. The setpoint
must be optimized to achieve the optimal image resolution and dimensions while maintaining the minimum
force possible to protect the sample from damage or deformation [11]. Alternately, the setpoint voltage can
be used to apply force to purposely cause controlled sample deformation, such as for indenting.
Advantages of AFM as an imaging technique are that the samples typically require minimal preparation
and that the samples can be imaged at atmospheric pressure or in liquid to mimic the native environment.
However, some additional sample preparation may be required to identify a target feature. If a specific
component, such as a protein, is to be imaged, use of a functionalized AFM probe tip may be necessary to
target the molecule [7][4]. If dry imaging is performed, care should be taken to determine a sample
preparation method to preserve as much of the original sample state as possible

6. 3 – Draft 100330
1.2 Biological Sample Background
1.2.1 Chromosomes
The discovery of chromosomes is reported to have occurred in the 1870’s [12]. At that time, rod-shaped
bodies were observed during cell division in animal and plant cells. These bodies were named
chromosomes, which are “colored bodies” in Greek [12]. In each human cell, there are 22 pairs of
chromosomes, called autosomes, and one pair of sex chromosomes for a total of 23 pairs of
chromosomes [1]. Each chromosome consists of scaffolding proteins, histone proteins, and DNA [13].
DNA resides in two places in animal cells, in the nucleus of the cells and in cellular organelles called the
mitochondria (Figure 2a). In the formation of chromosomes (Figure 2b), a DNA double helix molecule
wraps tightly around groups of eight histone proteins, which form DNA/protein complexes that are
generally known as chromatin. Upon initiation of cell division, the chromatin in the nucleus begins to
condense by coiling. Through a series of stages, called meiosis for the sex cells and mitosis for the non-sex
cells, the chromatin remodels itself to form the 23 pairs of metaphase chromosomes. Chromosomes are the
“most condensed and easiest to identify in dividing cells, particularly in the metaphase stage of cell
division [1].” Each metaphase chromosome is made up of left and right arms called chromatids. The DNA
in the chromosomes contains the genes that code for proteins, which are molecules that have a variety of
functions in living systems, including acting as molecular switches that turn processes on and off,
receptors, and gates and carriers that allow substances to pass in and out of the cells. The number of genes
in the human genome was once thought to be a one-to-one ratio with the estimated 200,000 proteins in the
body [13]. However, only a portion of the DNA in the chromosomes contains active gene sequences,
where one gene can produce many different proteins, and one chromosome can have many copies of a
single gene sequence [13]. As of 2003, there are an estimated 20,000 genes in the human genome [13][14].
Figure 2. (a) An artist’s rendition of an animal cell and (b) formation of a chromosome
[adapted from 16]. The DNA/protein complex, generally called chromatin, that forms the
11-nm and 30-nm fiber of the chromosomes initially exists dispersed in the nucleus. During
cell division, the chromosomes coil and condense into the familiar “X”- or “Y”-shaped
metaphase chromosomes.
1
st
stage “11-nm
fiber” chromatin
2
nd
stage “30-nm fiber“
(a)
(b)

7. 4 – Draft 100330
Due to its appearance, chromatin in the first stage of coiling is 11 nm wide and takes on the shape of
“beads-on-a-string [13]” (Figures 1 and 3). The second stage of coiling results in what has been called the
30-nm fiber or “30-nm chromatin fiber [13]” (Figures 1 and 3).
Figure 3. Stages of coiling from DNA to a metaphase chromosome [15].
Animal cells range in size from approximately 10-30 µm in diameter, with the exception of the human
egg cell, which is 100 µm in diameter [1]. The length of human DNA in one cell is estimated to be
1.8 meters [16] and must be able to fit inside the nucleus of the cell. As an example, the approximate size
of a dry HeLa cell [17] used in this study was 35 µm on one edge of the characteristically triangular-shaped
cell with a 20-µm-diameter nucleus. According to Lima-de-Faria, after cell division begins, the chromatin
“contracts heavily [13”] to form the highly-condensed chromosomes, where, in some species, one
chromosome can change its length “from 107 to 7 microns – a 15 times decrease in size [13].” In the
process of cell division, the DNA is thought to undergo a “10,000-fold linear compaction [18].” The genes
that make up the DNA in the chromosomes are able to perform genetic functions while in this compacted
state. As groups of genes are activated and inactivated, the “new gene situation [13]” is “locked with the
help of histone proteins [13].” The efficiency in chromosomal packaging and functional processes could be
of interest to industry in terms of micro- and nano-packaging, assembly, and delivery mechanisms in the
electronics, energy, bio-medical devices, and drug delivery industries.
1.2.2 HeLa Cells
HeLa cells are cervical tumor cells from a human female, Henrietta Lacks, and have been used for cancer
research since the 1950’s [17].

8. 5 – Draft 100330
2.0 Materials and Methods
2.1 Samples and Sample Preparation
The samples imaged were Vysis Normal Male Metaphase CGH Target slides (Abbott Molecular,
DesPlaines, IL, #30-806010) [19] and HeLa cell samples (ATCC, Manassas, VA, #CCL-2) in the form of
dried, whole cells and chromosome spreads prepared by the SJSU Biology Department. Contact and
intermittent contact modes were tested. For contact mode, imaging in dry and liquid conditions was
performed. For intermittent contact mode, imaging “just dry” conditions was performed.
“Just dry” conditions consisted of the application of DI water to hydrate the sample surface and careful
blotting of the surface prior to imaging.
Abbott chromosome spreads. The Abbott slides were received in frozen conditions as chromosome spreads
on positively-charged glass specimen slides. According to Abbott, the normal male metaphase
chromosomes were manufactured using standard cytogenetic slide preparation methods that are optimized
for comparative genomic hybridization (CGH) [19]. The slides came prepared as phytohemagglutanin-
(PHA)-stimulated lymphocytes cultured for 48 to 72 hours. PHA is a protein purified from beans, such as
red kidney beans, and is used to stimulate the production of lymphocytes [20]. Thymidine was used by
Abbott to synchronize the cells [19]. The length of the chromosomes was reported as 400-500 bands [19].
Until use, the slides were stored at -20°C. An Olympus IX51 inverted phase contrast microscope (Center
Valley, PA) was used to identify the chromosome locations. A permanent marker was used to mark the
locations on the back of the slides. Prior to imaging, the sample was allowed to thaw for 15 minutes.
HeLa whole cell samples. HeLa whole cell samples were prepared using a modified version of the
procedure in the Hoshi, et al [3], study as follows. The cells were cultivated Dulbecco Modified Eagle’s
Medium (DMEM) supplemented with 10% Bovine Growth Serum and 1% Antibiotic-Antimycotic (all
purchased from HyClone/Thermo Fischer Scientific, Waltham, MA) for 72 h at 37°C under 5% CO2 and
95% air. Colcemid was added to the culture medium at a final concentration of 0.05 µg/ml for 1 h. The
cell suspension was then exposed to 75 mM KCl for 30 min at room temperature and fixed with a mixture
of methanol and acetic acid (3:1). The cells were made by dropping the cell suspension onto glass slides
perpendicular to the slides, followed by air-drying in a humid condition for 10 min. The cell suspensions
should have produced chromosome spreads but instead produced whole cells as they were dropped
perpendicular to the slides.
HeLa chromosome spreads. HeLa chromosome spreads were prepared by the SJSU Biology Department.
Colcemid (1:1000 KaryoMAX) was added directly to culture dish, swirled, and incubated 30 min to two
hours. Metaphases can be prepared without colcemid. Colcemid should increase the number of metaphase
chromosomes but longer incubation times will result in shorter, more compact chromosomes. Cells were
trypsinized as normal and washed 1X in 10 mL phosphate buffered saline (PBS). At this point, it was no
longer necessary to be sterile. As much PBS was removed as possible and the cells were gently
re-suspended in the residual. 0.075 M KCl was added dropwise to 10 mL. 1-2 drops were added and the
tube inverted. After 3 mL of KCl in the tube, addition can become faster. Cells were incubated at 37ºC in
a water bath for exactly 6 minutes and centrifuged at 900rpm for 5 minutes. As much KCl was removed as
possible and cells were gently re-suspended in the residual. 5 mL of fixative (3:1 methanol-acetic acid,
prepared fresh) were added dropwise and carefully mixed the whole time. Adding fixative too quickly will
result in clumping. Cells were cetrifuged at 900rpm for 5 minutes and fixative removed. Two mL of
fixative were added dropwise. Cells were centrifuged at 900 rpm 5 minutes and all but 200-500 µL of
fixative were removed. Cells are stable for extended times in the fixative. If desired, cells can be stored at
4ºC. A few drops were dropped from about 18 inches high onto angled, humidified microscope coverslips
and the slides were initially dried by blowing on them gently. The drop angle is a critical step. The

9. 6 – Draft 100330
samples were air dried at least 10 minutes. It was indicated that the samples should have a long storage
time of at least two weeks. The chromosome spreads were visible in the AFM CCD camera view and
required no further preparation for imaging by AFM.
2.2 AFM Equipment
An Agilent 5500 Atomic Force Microscope (Santa Clara, CA) was used in contact and Acoustic
Alternating Contact (AAC) mode, also known as intermittent contact mode. Intermittent contact was
achieved by setting the non-contact mode Stop At field to 90%. A 100 µm scanner (Agilent Technologies,
Santa Clara, CA) was used. Two probe types were used: 1) PPP-NCHR silicon probe from Nanosensors
(Neuchâtel, Switzerland), tip radius of curvature <7 nm; single 125 µm silicon cantilever with aluminum
reflex coating: 42 N/m spring constant, 330 kHz resonant frequency; and 2) PNP-DB pyrex nitride probe
from NanoWorld (Neuchâtel, Switzerland), tip radius of curvature <10 nm, silicon nitride dual cantilevers
with gold reflex coating: 100 µm cantilever: 0.48 N/m spring constant, 67 kHz resonant frequency, 200 µm
cantilever: 0.06 N/m spring constant, 17 kHz resonant frequency. It was only possible to use the 100 µm
cantilever in this study, and future work should include experiments to test the 200 µm cantilever. For the
PPP-NCHR probe, a non-contact nose amplifier was used (Agilent Technologies, Santa Clara, CA).
A known sample, Ultrasharp TGZ02 (MikroMasch, Wilsonville, OR), was imaged to test the condition of
the PPP-NCHR probe. For the PNP-DB probe, a contact nose amplifier was used (Agilent Technologies,
Santa Clara, CA). Agilent’s PicoView 1.4.8 software was used to control the AFM and to capture and
analyze the images. Further analysis was performed using Gwyddion 2.1.4 open source data visualization
and analysis software.
3.0 AFM Imaging Procedures
All samples were imaged in ambient conditions. The following procedures describe the steps taken to
perform AFM imaging in dry, liquid, and “just dry” conditions. Dry means that the sample was imaged
after removal from refrigerated (4ºC) or frozen (-20ºC) conditions. Liquid means that the sample was
imaged while the sample was immersed in 200 µL of DI water buffer. “Just dry” means that 100 µL of
DI water was pipetted onto the sample and the sample was left in the DI water buffer in ambient conditions
for 15 minutes; the sample was then tilted and the moisture wicked from one side of the sample with the tip
of a paper towel and prior to AFM imaging. The samples were acquired according to the sample
preparation section.
3.1 Contact Mode in Dry Conditions with the PNP-DB Probe
The Abbott and HeLa chromosomes were imaged in contact mode in dry conditions with the PNP-DB
probe. The chromosome samples were removed from refrigerated or frozen conditions and left in ambient
conditions for 15 minutes. During this time, a known sample, Ultrasharp TGZ02, was successfully imaged
to ensure proper function of the AFM system. As the Abbott chromosomes were not visible in the AFM
CCD camera view, the approximate locations of the chromosomes were viewed by phase contrast
microscopy, and a pen mark was made on the back of the sample to identify the locations. The pen marks
could then be viewed in the AFM CCD camera view. Preparation and imaging are described in detail in a
separate protocol designed for this study [21]. The Abbott specimen slides were cut into 2 cm x 2 cm
samples in order to fit them into the AFM liquid cell. The HeLa chromosomes were readily visible in the
AFM CCD camera view and came prepared on 2-cm diameter glass coverslips that fit in the AFM liquid
cell without modification. Two tests were run, one with the Abbott chromosomes and one with the HeLa
chromosomes. The appropriate sample was placed in the AFM liquid cell and installed under the AFM
stage. Prior to each approach of the AFM probe tip to the sample, a recommended 1 V differential voltage
and an initial gain of 5-10 % were set per the Agilent 5500 User’s Guide [9]. The probe tip was positioned

10. 7 – Draft 100330
over a chromosome spread with an AFM CCD camera zoom of 3.8X. An approach was performed and the
setpoint and gain were optimized during imaging to maximize the resolution and feature size of the sample.
Images were obtained down to 1 µm resolution for the Abbott sample and to 500 nm for the HeLa sample.
PicoView images were saved for later analysis.
3.2 Contact Mode in Liquid Conditions with the PNP-DB Probe
Immediately following dry imaging without removing the sample from the AFM stage, the Abbott
chromosome samples were prepared for imaging in liquid conditions using the same materials as
previously described. Tests were performed on chromosomes of interest. During dry imaging, a
chromosome was identified at an image size of 20 µm. Withdraw was performed to 800 µm. The liquid
cell was removed from the AFM stage. With the sample still in the liquid cell, 200 µL of DI water buffer
were pipetted onto the sample to completely immerse the sample. Still at a tip-to-sample distance of
800 µm, the liquid cell with sample was returned to the AFM stage. The photodetector was removed and
re-adjusted due to the change in refractive index of the laser beam through the liquid buffer. The tip-to-
sample distance of 800 µm ensured direct immersion of the probe tip in the buffer, which was found to
ensure a stable signal from the laser beam to the photodetector. The photodetector was returned to the
AFM system and imaging was performed according to the Agilent User’s Guide protocols for liquid
imaging, this time with an initial gain of 2% [9]. Images were obtained down to 20 µm resolution.
PicoView images were saved for later analysis.
3.3 Intermittent Contact Mode in “Just Dry” Conditions with PPP-NCHR Probe
The liquid cell and samples from the previous tests were removed and stored appropriately. The Agilent
5500 was set up with the 100 µm scanner with a non-contact nose amplifier and the PPP-NCHR probe.
The cantilever on this probe is coated with an aluminum reflex coating, and exposure of the coating to the
buffer would cause a reaction with the aluminum that would change the reflective properties of the
cantilever surface and make AFM imaging impossible. “Just dry” conditions were used to avoid
immersing the PPP-NCHR cantilever in the liquid buffer. Dried HeLa cells were prepared one week
previously according to the protocol in the sample preparation section. A 2-cm diameter glass coverslip
containing dried HeLa cells was removed from 4ºC conditions. The sample was held in ambient conditions
for 30 minutes. During this time, a known sample, Ultrasharp TGZ02, was imaged to ensure proper
function of the AFM system. “Just dry” conditions were achieved by pipetting 100 µL of DI water onto the
sample in the liquid cell for and leaving the sample in ambient conditions for 15 minutes. The DI water
buffer was removed from the sample by tilting the liquid cell with sample and wicking the water from the
edge of the sample using the tip of a paper towel. The HeLa coverslip was mounted on the AFM liquid cell
plate and the liquid cell with sample was placed under the AFM stage. A group of three HeLa cells were
identified using the AFM CCD camera at maximum zoom of 3.8X. The initial image size was set to 100
µm and AFM imaging was performed according to the Agilent 5500 User’s Guide [9], with a Stop At of
90% to ensure intermittent contact. The setpoint and gain were re-adjusted after approach to optimize
resolution. The tip of one of the cells was targeted. Images were obtained down to 2.5 µm resolution. The
PicoView images were saved for later analysis.

11. 8 – Draft 100330
4.0 Data and Analysis
Table I summarizes the results of the tests that were performed. The applicable figures are also provided.
Table I. Sample Preparation and Final Feature Sizes
Test Test / Cell
Type
Reagents Sample
Type
Dry or
Liquid /
Buffer
Mode /
Probe
Resolution Height
(nm)
Fig.
1 “Abbott”
Human
lymphocytes
(male,
normal)
Prepared by
Abbott: PHA /
thymidine
Chromosome
spreads on
specimen
slides, drying
method
unknown
(performed
by vendor)
Dry and
liquid in
DI water
Contact
/ PNP-
DB
To 30-100
nm fiber
level
Dry:
30 nm
Liquid:
170-
230 nm
4, 6,
9
3 “HeLa”
Cervical cells
(female,
tumor)
Prepared by
SJSU:
Colcemid /
trypsin / PBS /
KCl / 3:1
methanol-
acetic acid
Chromosome
spreads,
air-dried,
humid on
glass
coverslip
rounds
Dry Contact
/ PNP-
DB
To 30-70
nm fiber
level
140 nm 5
2 “HeLa”
Cervical cells
(female,
tumor)
Prepared by
SJSU:
Colcemid /
trypsin / PBS /
KCl / 3:1
methanol-
acetic acid
Whole cells,
air-dried,
humid on
glass
coverslip
rounds
“Just
dry”
using DI
water
buffer
Intermit
tent
contact
/ PPP-
NCHR
Unknown
structures
30-70 nm
80 nm 7, 8

12. 9 – Draft 100330
4.1 Dry AFM Imaging in Contact Mode with the PNP-DB Probe
AFM images were obtained in ambient, dry conditions of Abbott normal male metaphase chromosomes.
Imaging was performed in contact mode. The probe used was the PNP-DB probe with the 100 µm
cantilever. This cantilever has a low resonant frequency of 67 kHz and low spring constant of 0.48 N/m.
A low resonant frequency and low spring constant were used to minimize sample damage. At an image
size of 20 µm, uncharacteristic ridges appeared between the chromatids, presumably due to reagent salts
from the sample preparation and drying process (Figures 4a-b). The average dimensions of the
chromosomes were 4 µm width x 29 nm height, where the width of one chromatid was 2 µm. The range of
lengths was 5 µm to 14 µm. At an image size of 1 µm, chromatin fiber was successfully imaged (Figures
4c and d), which measured 30-100 nm. As previously indicated, chromosomes have been reported to
contain 11-nm and 30-nm fiber (Figures 2 and 3). However, Hirano, et al, reported evidence of 70- to 80-
nm granular fibers as well [4], which appears to be supported by the 30-100 nm range observed in the
present study. Repeated imaging of the sample using the PNP-DB probe in dry conditions did not appear to
degrade the sample.
Figure 4. AFM images of Abbott chromosomes and 30-100 nm chromatin fiber in dry conditions.
(a-b) 20 µm Gwyddion image 090716-3 of Abbott normal male metaphase chromosomes in (a) two and
(b) three dimensions. (c-d) 1 µm Gwyddion image 090716-4 of 30-100 nm chromatin fiber in (c) two
and (d) three dimensions.
(a)
(b)
(c)
(d)
5 µm
100 nm

13. 10 – Draft 100330
Also imaged in ambient, dry conditions were the HeLa chromosome samples (Figure 5). AFM imaging
was performed in contact mode. The probe used was the PNP-DB probe using the 100 µm silicon nitride
cantilever with a low resonant frequency of 67 kHz and low spring constant of 0.48 N/m. The shape of the
chromosomes (Figure 5a) differed from that of the Abbott samples (Figure 4a). The average size of the
chromosomes was measured as 1.2 µm width x 140 µm height, where one chromatid measured 0.6 µm.
The length ranged from 1.5 µm to 7 µm, where the longest HeLa chromosomes are 50% shorter than the
Abbott normal male metaphase chromosomes from Figure 4. The differences in shape and reduction in
size could be due to the difference in cell type, where the Abbott samples are from normal lymphocytes and
the HeLa samples are from cervical tumor cells. Per the sample preparation section, the use of colcemid in
the HeLa sample preparation could also have resulted in shorter, more compact chromosomes. At an image
size of 1 µm, chromatin ranging from 30-70 nm (Figure 5b) was imaged from the chromosome indicated
(Figure 5a). The same chromatin area was also imaged at 500 nm image size (Figure 5c). Repeated
imaging of the sample using the PNP-DB probe in dry conditions did not appear to damage the sample.
Figure 5. AFM images of HeLa chromosomes and 30-70 nm fiber in dry conditions.
(a) 50 µm PicoView image 090710-2 of HeLa chromosomes. The average size of the
chromosomes is 1.2 µm width x 140 µm height, where one chromatid measured 0.6 µm.
The length ranged from 1.5 µm to 7 µm, where the longest chromosomes were 50%
shorter than the Abbott normal male metaphase chromosomes from Figure 4. (b) 1 µm
PicoView image 090710-5 of chromatin in the chromosome indicated in Figure 4.
(c) 3-Dimensional image of the same area, PicoView image 090710-5.
10 µm (a)
(b) (c)
100 nm

14. 11 – Draft 100330
4.2 Comparison of Dry and Liquid Imaging with the PNP-DB Probe in Contact Mode
Abbott normal male metaphase chromosomes were imaged in dry and liquid conditions (Figure 6).
Imaging was performed in contact mode using the PNP-DB probe and 100 µm cantilever with a low
frequency of 67 kHz and low spring constant of 0.48 N/m. After dry imaging, the liquid cell was removed
from the AFM, and 200 µL of DI water buffer was added to the sample in the liquid cell and returned to the
AFM stage after fifteen minutes. Imaging in liquid resulted in a higher resolution image and caused a
separation of the chromatids, presumably due to dissolution of reagent salts that were initially observed.
The addition of the liquid buffer caused an 80% increase in the chromosome height, from approximately
30 nm to 150 nm (Table II), where the maximum measured value was 173 nm. The chromatin fiber could
not be imaged in liquid conditions due to sample degradation.
Figure 6. 20 µm AFM images of Abbott normal male metaphase chromosomes in (1) dry
conditions (Gwyddion image 090716-3) and (2) liquid conditions (Gwyddion image
090716-5).
Table II. Feature Sizes for Contact Mode Imaging in Dry and Liquid conditions with PNP-DB Probe
Chromo
-some
Image Setpoint
(V)
Gain
(%)
Scan
Size
(µm)
Speed
(ln/s)
Scan
Angle
(degrees)
Feature
Length
(µm)
Feature
Width
(µm)
Feature
Height
(nm)
Fig.
A 090716-3 0.75 2 20 2 0 14 4 30 6
B 090716-3 0.75 2 20 2 0 12 3.4 28 6
C 090716-3 0.75 2 20 2 0 5 4 29 6
Average width and height: 4 29
D 090716-5 0.89 2 20 3 0 11.1 3.0 120 6
E 090716-5 0.89 2 20 3 0 10.9 2.8 168 6
F 090716-5 0.89 2 20 3 0 9.6 3.9 173 6
Average width and height: 3 154
(1) (2)
(A)
(B)
(C)
(D)
(E)
(F)

15. 12 – Draft 100330
4.3 “Just Dry” Imaging with the PPP-NCHR Probe in Intermittent Contact Mode
Imaging of HeLa cells was performed in “just dry” conditions in intermittent contact mode (Figure 7a).
The probe used was the PPP-NCHR probe with a high resonant frequency of 330 kHz and a high spring
constant of 42 N/m. The ultimate goal of imaging biological samples by AFM is to image the sample in the
native environment, namely, in liquid. Liquid imaging was not possible in this part of the study due to the
aluminum reflex coating on the cantilever of the PPP-NCHR probe as described in the materials and
methods section. To avoid immersion of the cantilever in the liquid buffer, the sample was partially
re-hydrated by exposing the sample to DI water, and the sample slide was carefully blotted to remove
excess liquid.
The upper tip of HeLa cell 1 was imaged at a 10 µm image size (Figure 7b). A chromosome-like structure
with dimensions 1.8 µm length x 1.1 µm width x 114 nm height was observed at the top of the image as
indicated. The structure was further imaged at a 2.5 µm image size (Figure 7c), and comparable
dimensions were found. While this structure could not be definitively identified in the scope of this study,
the dimensions are consistent with the dimensions of metaphase chromosomes previously reported in this
study and in other studies [2][3]. A three-dimensional image of this structure was generated using
Gwyddion image and analysis software (Figure 7d) to show the additional capability of AFM.
Figure 7. (a) 100 µm AFM PicoView image 090702-22 of three HeLa cells initially dried and partially
re-hydrated by immersing the sample in DI water. (b) 10 µm AFM PicoView image 090702-25 of the
area indicated from HeLa cell 1 with a chromosome-like structure that appears at the top of the image.
(c) 2.5 µm AFM Gwyddion image 090702-39 of the indicated structures. (d) A three-dimensional version
of the 090702-39 image that was generated using Gwyddion image and analysis software.
35 µm
(a)
(b) (c)
(d)
1 µm 100 nm

16. 13 – Draft 100330
Intermittent contact mode provides three types of images that are produced simultaneously during AFM
imaging: topography (Figure 8a), phase (Figure 8b), and amplitude (not pictured). Differences in materials
in the topography and phase images can clearly be seen (Figure 8), where the (b) phase image shows
obvious distinctions between two types of materials and provides higher resolution compared to the
(a) topography image. Particles not immediately observed in the topography image become clearly visible
in the phase image. Previous reports indicate that phase imaging has “been shown to be sensitive to
material surface properties, such as stiffness, viscoelasticity, and chemical composition [22].” Although
identification and characterization of the materials was not in the scope of this study, an observation was
made that the particles surrounding the chromosome-like structure were 90-250 nm in diameter. Compared
to the measured 30-100 nm chromatin fiber measured in the Abbott and HeLa chromosome samples, these
particles are up to 150% larger and could be the subject of future study.
Holland and Marchant reported that a stiff, high resonant frequency, high spring constant probe can be used
to “distinguish individual protein molecules from [an] underlying polymer surface [23].” Successful use of
a stiff probe was shown in this part of the study as evidenced by the high resolution and potential use of the
phase image in characterizing the materials. However, use of a stiff probe may be limited to dry and “just
dry” imaging as it is more commonly found that flexible probes with a lower resonant frequency and lower
spring constant are recommended for liquid imaging of biological samples.
Figure 8. (a-b) 10 µm AFM PicoView image 090702-25 of internal HeLa cell structures
showing a comparison of the (a) topography image based on the error signal fed to the AFM
system and (b) the phase image based on the lag in phase fed back to the AFM system [22].
(c-d) 2.5 µm AFM PicoView image 090702-39 showing additional resolution achieved when
comparing the (c) topography and (d) phase images.
(a) (b)
(c) (d)
1 µm 1 µm
100 nm 100 nm

17. 14 – Draft 100330
4.4 Effects of Gain with the PNP-DB Probe in Liquid using Contact Mode
Effects of gain adjustments on image quality and feature height were observed (Figure 9). Imaging was
performed in liquid conditions in contact mode using the PNP-DB silicon nitride probe and 100 µm
cantilever with a low resonant frequency of 67 kHz and a low spring constant of 0.48 N/m. Parameters and
resulting feature sizes appear in Table III. Gain is known to affect the tip sensitivity and can cause ringing
or noise that can cause a blurred image. While the overall quality of these images is low, most likely due to
sample degradation in the liquid buffer over time, visually inspecting the images shows that images (b) or
(c) have the optimal resolution. A setting between 1-5% provided the optimal image quality with optimal
feature height. An increase in gain to 5% in image (c) produced the greatest height at 231 nm, a 35%
overall increase.
Table III. Effects of Gain on Image Quality and Feature Height in Liquid using Contact Mode
Image Gain % Image Quality Profile Quality
Feature
Height (nm)
% Change
(a) 090716-21 0.2 Poor Poor 150 -
(b) 090716-9 1 Fair Fair 200 25
(c) 090716-10 5 Poor Fair 231 13
(d) 090716-11 10 Poor Fair 187 23
Figure 8. Effects of gain adjustments on image quality and feature height on the Abbott
chromosome samples. Left-to-right: 20 µm AFM Picoview deflection images and profiles for
gain settings of 0.2, 1, 5, and 10%, respectively.
(a) (b) (c) (d)

18. 15 – Draft 100330
4.5 Comparison with Previous Studies
As stated earlier, Hirano, et al, has reported evidence of 70- to 80-nm granular fibers in addition to the
known 30-nm fibers [4], which is consistent with the findings in our study, where fibers the Abbott and
HeLa chromosome samples ranged from approximately 30-100 nm. In the HeLa whole cell samples in
Figures 7 and 8, it was found that particles near the unidentified chromosome-like structure were closer to
90-250 nm in diameter and could be the subject of further study.
In a study by Eltsov, et al, cryo-electron microscopy (cryoSEM) was used to image chromosomes.
No evidence of the 30-nm fiber in situ [5] was found. However, this method involved a rapid-freeze
technique, which is known to cause cell damage, and it is unlikely that the evidence in this study is valid.
In a review of SEM and AFM of fungal cells by Kaminskyj and Dahms, it was reported that, while
cryoSEM can be used to image cells in a frozen hydrated state, “most fungal cryoSEM specimens are
destroyed upon thawing due to their water content and the damaging effect of cytoplasmic ice crystal
formation on membrane integrity [6].” It is presumed that most cells would also be affected by CryoSEM
in a similar fashion, including detrimental effects to chromosomes and 30-nm fiber. Kaminskyj and Dahms
further state that internal structures are not viably imaged using CryoSEM because, although there have
been “excellent images of cell interfaces [6],” there “is a problem controlling the fracture plane [6],” which
makes the imaging of internal structures difficult. Kaminskyj and Dahms indicate that critical point drying
(CPD) and lyophilization (freeze drying) are preferable sample preparation methods and that AFM and
environmental/variable pressure SEM are preferable biological imaging techniques.
In a 2008 study by Ushiki and Hoshi [2] and a 2004 study by Hoshi, et al [3], chromosomes and 30-nm
chromatin fiber from human lymphocytes and leukemia cells were imaged by AFM (Figures 9 and 10).
Comparing these studies with the present study, it is clear that sample preparation, buffer type, and imaging
conditions affect imaging results. A summary of the imaging studies appears in Table IV. In these studies,
intermittent contact mode was used in dry and liquid conditions. The probe types used for the Ushiki and
Hoshi images were silicon with a nominal spring constant of 42 N/m for dry conditions and a silicon nitride
probe with a triangular tip and a nominal spring constant of 0.37 N/m. For liquid imaging, the buffers used
were phosphate buffered saline (PBS) and hexylene glycol.
Table IV. Sample Preparation and Results from Ushiki and Hoshi Studies
Cell Type Reagents Used
in Protocol
Final
Sample
Type
Dry or
Liquid /
Buffer
Resolution Dimensions Reference Fig.
Human
lympho-
cytes
Colcemid / 3:1
methanol-acetic
acid/KCl
Chromosome
spreads,
air-dried,
humid
Both dry and
liquid in
phosphate
buffered
saline (PBS)
Dry and wet:
chromosome
level down to
1 µm
Dry:
1.3 µm x 40
nm
Liquid:
1.3 µm x
300-600
[2][3] Not
pictured
Human
lympho-
cytes
(male,
healthy)
Colcemid / 3:1
methanol-acetic
acid/KCl/ PBS/
tannic acid/OsO4,
ethanol/CO2
Chromosome
spreads, air-
dried,
PBS, CO2
critical-point
dried [24]
Dry Reported to
30-nm fiber
level
Dry height:
200-350 nm
[2] 10
Human B
Cell
Leukemia
Colcemid/KCl/
hexylene glycol/
silane glass slide
Isolated
chromosomes
in hexylene
glycol
Liquid in
hexylene
glycol
Reported to
30-nm fiber
level
Liquid:
1 µm x
400-800 nm
[2] 11

19. 16 – Draft 100330
Images from the Ushiki and Hoshi 2008 study are shown (Figure 9), where a height of 200-350 nm was
reported for critical-point-dried samples. Comparing this result to the present study, our sample height of
air-dried chromosomes was 30 nm, a decrease of one order of magnitude. Similar to our results, however,
were the results of the study by Hoshi, et al, where a height of 40 nm for air-dried chromosomes was
reported [3]. The difference in height of the samples is explained by the difference in sample preparation,
where critical-point drying yielded a greater sample height. The Ushiki and Hoshi study indicates that a
pre-treatment using 1% OsO4 hardens the sample to preserve the shape and that a critical-point drying
method [24] in liquid CO2 prevents shrinking of the sample during the drying process.
Figure 9. Images of the Ushiki and Hoshi dry sample prepared by critical-point drying
using a silicon probe with high resonant frequency and high spring constant [2]. Image
(a) shows a chromosome from a human lymphocyte with a height of 200-350 nm.
The images in (b) and (c) show chromatin fiber reported as 30-nm chromatin.
In the Ushiki and Hoshi 2008 study [2], another experiment was conducted using a hexylene glycol buffer
(Figure 10). This change resulted in an increase in height to 400-800 nm [2]. The sample was primarily
kept in the buffer and had only brief contact with air. In our study, the samples were air dried and then
immersed in DI water. The height of the chromosomes in the present study in liquid conditions ranged
from approximately 170-230 nm, which is an approximate 50% reduction in height due to effects of the
buffer and the sample preparation process. The cell type in the Ushiki and Hoshi study was a leukemia cell
as opposed to our sample that was from normal lymphocytes. It is possible that the cell type resulted in
differences in feature size. In addition, in image (e) Ushiki and Hoshi observed an unfolding of the 30-nm
fiber. It is presumed that the hexylene glycol buffer caused the unfolding and could be used for future
work in using AFM to characterize the chromosomal packaging mechanism.
Figure 10. Ushiki and Hoshi chromosomes from leukemia cells imaged in hexylene glycol buffer [2].
d e

20. 17 – Draft 100330
5.0 Future Work
In a report by Richmond and Davey, the theories behind the functional coiling that produces the compacted
chromatin is described [25] and could be the subject of further AFM investigation into the chromosome
packaging mechanism for use in micro- and nano-packaging applications such as electronics, energy
mechanisms, bio-medical devices, and drug delivery. The self assembly mechanisms of RNA, which are
copies of specific DNA gene sequences in and around the chromosomes are of interest in the study of
“self-assembling, programmable biomaterials [26],” including nanobiomaterials [26].
6.0 Conclusion
The purpose of this study was to use AFM to image biological samples. The samples were chromosomes
from normal male lymphocytes, cellular structures from whole, dried HeLa cells, and chromosomes from
HeLa cells. Chromosomes were successfully imaged in dry, liquid, and “just dry” conditions using contact
and intermittent contact mode and two probe types with an approximate cantilever length of 100 µm.
30-100 nm chromatin fiber was successfully imaged in dry conditions. It was found that the primary
factors that affected the image resolution and feature size were the imaging conditions, mode, gain, buffer
type, and sample preparation. Future work was described in terms of chromosomes as potential
nano-packaging mechanisms and the potential of these mechanisms in future technologies.
Dry imaging conditions in contact mode with the lower resonant frequency, lower spring constant PNP-DB
probe with the 100 µm cantilever produced high resolution images but a low sample height of 30 nm.
Liquid imaging of the Abbott samples using the same probe and cantilever in contact mode produced
chromosomes with distinct, separated chromatids, higher resolution, and an 80% increase in height. In
liquid conditions, however, it was not possible to obtain successful images of the 30-nm chromatin fiber
due to sample degradation. In “just dry” conditions, high resolution phase images of chromosome-like
structures in whole HeLa cells were achieved with a stiff, high resonant frequency, high spring constant
probe. Sample degradation was expected with this stiffer probe, but no sample degradation was observed
after several scans. A chromosome-like structure was observed with a height of 114 nm. If this structure is
indeed a chromosome, this indicates that “just dry” conditions do not yield full-height dimensions, which
should be closer to 400-800 nm according to previous studies [2][3]. Observing the higher resolution phase
images from “just dry” conditions and the lower resolution images in liquid conditions, it is possible that
the increase in resolution was due to use of intermittent contact mode as opposed to contact mode, where
contact mode may have caused sample damage because of the constant force of the tip on the sample. Use
of intermittent contact mode also presented the potential use of the phase image to characterize future
samples.
In terms of the effects of gain on image quality and on dimensions, it was clearly shown that gain must be
optimized to produce an image with both high resolution and appropriate dimensions. For soft, biological
samples, the user can adjust the gain by testing gain settings using a progression such as 0.5, 1, 2, to 4 % to
find the optimal combination of resolution and feature size.
It was initially expected that DI water was not the ideal buffer due to ionization effects and potential
sample dissolution; DI water, however was the only alternative possible during the study due to adverse
effects of ionized buffers on the probe cantilevers. Based on previous studies [2][3], in which phosphate
buffered saline and hexylene glycol were used as buffers, hexylene glycol yielded the maximum height at
400-800 nm and is a potentially a preferable buffer for future work. For dry samples, it appears that
critical-point drying was the preferable sample preparation method over air drying. Critical-point drying
preserved the shape and yielded a feature height of 200-350 nm in intermittent contact mode, an order of

21. 18 – Draft 100330
magnitude greater than our 30 nm dry sample in contact mode. However, liquid imaging in the native state
should be the focus of future imaging studies to image the samples with as little sample preparation as
possible to minimize changes to the sample characteristics. With liquid imaging, however, care should be
taken to select the probe, taking into consideration the stiffness of the probe.
7.0 Acknowledgments
This project would not have been possible without the guidance and support of Dr. E. Carr from Agilent
Technology, who supplied the initial samples and the technical advice about the optimization of the AFM
conditions. We would also like to thank Dr. M. Sneary and Dr. B. White from the San José State
University Biology Department for their support and sample preparation during the second part of this
project. Lastly, a special thank you is given to Dr. M. Goedert for the use of the AFM lab and equipment at
San José State University and for his expertise and guidance.
8.0 References
1
C. Starr and R. Taggart, 7th
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2
T. Ushiki and O. Hoshi, “Atomic Force Microscopy for Imaging Human Metaphase Chromosomes,”
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3
O. Hoshi, R. Owen, M. Miles, and T. Ushiki, “Imaging of Human Metaphase Chromosomes by Atomic
Force Microscopy in Liquid,” Cytogenet. Genome Res., 107, pp. 28–31 (2004).
4
Y. Hirano, H. Takahashi, M. Kumeta, K. Hizume, Y. Hirai, S. Otsuka, S.H. Yoshimura, K. Takeyasu,
“Nuclear Architecture and Chromatin Dynamics Revealed by Atomic Force Microscopy in
Combination with Biochemistry and Cell Biology,” Pflugers Arch – Eur J Physiol 456, pp. 139-153
(2008).
5
M. Eltsov, K.M. MacLellan, K. Maeshima, A.S. Frangakis, and J. Dubochet, “Analysis of Cryo-
Electron Microscopy Images does not Support the Existence of 30-nm Chromatin Fibers in Mitotic
Chromosomes in situ,” Proceedings of the National Academy of Sciences of the United States of
America, 105 (50), pp. 19732-19737 (2008).
6
S.G.W. Kaminskyj and T.E.S. Dahms, “High Spatial Resolution Surface Imaging and Analysis of
Fungal Cells using SEM and AFM,” Micron, 39, pp. 349-361 (2008).
7
Y.F. Dufrene, “Atomic Force Microscopy and Chemical Force Microscopy of Microbial Cells,” Nature
Physics, Corrections: Nature Protocols, 3 (7), pp. 1132-1138 (2008).
8
K.O. van der Werf, C.A.J. Putman, B.G. de Grooth, and J. Greve, “Adhesion Force Imaging in Air and
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9
“Agilent Technologies 5500 Scanning Probe Microscope.” User’s Guide. Rev. B, 2008.
10
M. Goedert, Lecture 2 Presentation: AFM Fundamental Components, Materials
Engineering 145, San José State University, Spring 2009.
11
Y. Jiao and T.E. Schaffer, “Accurate Height and Volume Measurements on Soft Samples with the
Atomic Force Microscope,” Langmuir, 29, pp. 10038-10045 (2004)
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E.J. DuPraw, DNA and Chromosomes. (Holt, Rinehart and Winston, Inc., 1970), pg. 1.
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(World Scientific, 2008), pp. xvii, 41, 42, 41, 46, 17, 13, 232.
14
Human Genome Project Information [Online], Available at:
http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml (accessed 24 July 2009).

22. 19 – Draft 100330
15
B. Alberts, Molecular Biology of the Cell (Garland Publishing, Inc. New York, USA, 1994) as appears
in A. Lima-de-Faria, Praise of Chromosome “Folly,” Confessions of an Untamed Molecular Structure,
(World Scientific, 2008), pg 46.
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17 (4), pp. 380-386 (1998).
18
J. Widom, “Chromosome Structure and Gene Regulation.” Physica A, 244, pp. 497-509 (1997).
19
Abbott Datasheet. Comparative Genomic Hybridization (CGH*) Reagents for Laboratory Use. Date
not given.
20
Roche Diagnostics Datasheet. Phytohemagglutinin-M (PHA-M), 0706.11352938001 (2006).
21
L. Clark, “Protocol for AFM Imaging of Biological Samples in Liquid Conditions,” Unpublished
Report (2009).
22
D. Raghavan, X. Gu, T. Nguyen, M. VanLandingham, and A. Karim, “Mapping Polymer
Heterogeneity Using Atomic Force Microscopy Phase Imaging and Nanoscale Indentation,”
Macromolecules, 33, pp. 2573-2583 (2000).
23
N.B. Holland and R.E. Marchant, “Individual Plasma Proteins Detected on Rough Biomaterials by
Phase Imaging AFM,” 51, 3, pp. 307-315 (1999).
24
G.D. Cagle, “Critical-Point Drying: Rapid Method for the Determination of Bacterial Extracellular
Polymer and Surface Structures,” Applied Microbiology, 28 (20), pp. 312-316 (1974).
25
T.J. Richmond and C.A. Davey, “The Structure of DNA in the Nucleosome Core,” Nature, 423,
pp. 145-150 (2003).
26
H.G. Hansma, E. Oroudjev, S. Baudrey, and L . Jaeger, “TectoRNA and ‘Kissing-Loop’ RNA: Atomic
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