Membrane deformation of unfixed erythrocytes in air with time lapse
investigated by tapping mode atomic force microscopy
Yong Chen a,b, Jiye Cai a,1,*
a Department of Chemistry, Jinan University, Guangzhou 510632, People’s Republic of Chinab Department of Microbiology and Immunology, University of Illinois, 835 S. Wolcott Avenue, RE704, Chicago, IL 60612, USA
Received 5 May 2005; received in revised form 19 November 2005; accepted 19 November 2005
Abstract
Estimation of the time of death is one of the most important problems for forensic medicine and law. Physical and chemical postmortem
changes are evaluated together while estimating the time of death. The pattern analysis of antemortem and postmortem bloodstains is one of the
important parameters for forensic science, and cellular changes of blood cells can be useful for the quantitative assessment of the time of death. In
this study, by successively investigating erythrocytes exposed in air on mica for 5 days using tapping mode atomic force microscopy (TM-AFM),
we observed deformation of whole cell and membrane surface of unfixed erythrocytes with time lapse. We found that the time-dependent cellular
changes occurred after exposure of erythrocytes in air for several days. At 0.5 days of exposure, fissures and cell shrinkage were observed. At
2.5 days of exposure, the emergence of nanometer-scale protuberances were observed and these protuberances increased in number with
increasing time. The changes of cell shape and cell membrane surface ultrastructure can be used to estimate the time of death. Futhermore, smear-
induced abnormal erythrocytes and immunostained erythrocytes were observed here. The need for more precise research is indicated, such as the
correlation of membrane changes to intervals of less than 0.5 day of air exposure, and use of various substrates in addition to mica, including glass,
metals, fabrics, among others, on which the bloodstains appear in crime scenes. The results of this research demonstrate the efficacy of AFM as a
potentially powerful analytical tool in forensic science.
q 2005 Elsevier Ltd. All rights reserved.
PACS: 07.79.L
Keywords: Atomic force microscopy (AFM); Tapping mode; Erythrocyte; Forensic examination; Bloodstains
1. Introduction
Until now, the most widely used methods for the forensic
examination of evidence in crime scenes were optical and
electron microscopy. The recent development of atomic force
microscopy (Friedbacher and Harald, 1999; Dufrene, 2003;
Frederix et al., 2003; Horber and Miles, 2003; Ikai and Afrin,
2003; Rounsevell et al., 2004; Santos and Castanho, 2004;
Hansma et al., 2004; Chen et al., 2005) gives an opportunity to
complement or even replace the classical instruments used in
this field. For example, Smith quantitatively analyzed the outer
surfaces of human hair by AFM (Smith, 1998), and more
0968-4328/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2005.11.011
* Corresponding author. Tel.: C1 312 413 7892; fax: C1 312 996 5725.
E-mail addresses: [email protected] (Y. Chen), [email protected].
cn (J. Cai).1 Tel.: C86 20 8522 3569; fax: C86 20 8522 3569.
recently Gurden et al. classified the AFM images of human hair
and even presented an algorithm for the potential automatic
analysis of them (Gurden et al., 2004). In order to validate the
documentary evidence in many different types of litigation,
forensic examination of line crossings in documents by AFM
have been made by Kasas and co-workers (Kasas et al., 2001).
Bersellini and collaborators (Bersellini et al., 2001) have used
AFM in the research on latent fingerprints on metallic surfaces.
For humans, the size and shape of their erythrocytes are
important indicators of well being. Therefore, erythrocytes
have been extensively studied by AFM over the years because
of their relatively simple structure and ease of isolation. Much
research has been done on erythrocytes with AFM, including
normal erythrocytes (Nowakowski et al., 2001; O’Reilly et al.,
2001), pathological erythrocytes (Zachee et al., 1994),
tumorous erythrocytes (Chen et al., 2002), infected erythro-
cytes (Nagao et al., 2000), traumatized erythrocytes (Girasole
et al., 2001; Ohta et al., 2002), and the membrane cytoskeleton
of erythrocytes (Swihart et al., 2001; Liu et al., 2005). All this
research indicates that AFM is a powerful and mature
Micron 37 (2006) 339–346
www.elsevier.com/locate/micron
Y. Chen, J. Cai / Micron 37 (2006) 339–346340
technique to study cellular changes and membrane surface
ultrastructure of erythrocytes. The pattern analysis of ante-
mortem and postmortem bloodstains is one of the important
parameters for forensic science, and cellular changes of
erythrocytes can be useful for the quantitative assessment of
the time of death. AFM can be utilized to study antemortem
and postmortem bloodstains. Using a calibration curve,
investigators can pin down the age of a bloodstain with much
greater precision than simple visual examination affords. Even
so, there are few reports on the application of AFM in
postmortem bloodstains, and on the variation processes of cells
in postmortem bloodstains.
In this study, the morphological changes of unfixed
erythrocytes in air over the lapse of time, after introduction
to substrate, were observed by TM-AFM. The aim is to
establish if this new technique can be a useful tool for the
examination of antemortem or postmortem bloodstains and for
the estimation of the time of death.
2. Materials and methods
A fresh human blood sample was obtained and mixed with
an anticoagulant (EDTA). The freshly extracted human blood
was diluted in phosphate buffered saline (PBS: 0.2 mg/ml
KH2PO4, 0.2 mg/ml KCl, 2.9 mg/ml Na2HPO4$12H2O,
8.0 mg/ml NaCl, pH 7.4; 80 times). Five microliters aliquots
of this solution and erythrocytes immunolabelled with
R-phycoerythrin (R-PE)—conjugated IgG to human glyco-
phorin A (Becton Dinckison Biosciences Pharmingen, San
Diego, CA) were deposited on the freshly cleaved mica
surfaces and were then air-fixed rapidly by vigorous manual
gesticulation. The film which was produced was examined
under the optical microscope.
Atomic force microscopy was performed in air on the blood
film using a commercial AFM (AutoProbe CP, Thermomicro-
scopes) in tapping mode. The mica carrying the blood film was
mounted onto the XY stage of the AFM and the integral video
camera was used to locate the regions of interest. Micro-
fabricated silicon cantilevers (Park Scientific Instruments) with
a force constant of approximately 2.8 N/m were used. Repeated
scanning of the same red blood cells confirmed that no physical
damage occurred during imaging. The blood film was kept on
the XY stage of AFM for 5 days (humidity: 75%, temperature:
25 8C. Humidity was externally controlled via two dehumidi-
fiers and monitored with a hygrometer.), and the same cell was
scanned every 0.5 day. For high resolution purposes 15 mm!15 mm area AFM images were obtained at a line scan rate of
0.5 Hz. These images were stable and reproducible during
repeated scanning.
Before quantification was carried out, the AFM images were
planar leveled using the software (Thermomicroscopes Proscan
Image Processing Software Version 2.1) provided with the
instrument. Using the line analysis function of the software, the
average width and the average height (i.e. thickness) of the red
blood cells and protuberances on cell were determined. Using
the particle analysis function of the software, the surface area
and the volume of red blood cell were determined.
3. Results
Fig. 1 is a relatively low-resolution image of single and
multiple erythrocytes. Both optical microscopy and electron
microscopy can observe a single, whole cell which is
approximately several microns in size, but AFM has distinct
advantages, such as clearer images, easy sample preparation,
extensive environment (in air or liquid) of sample to escape from
the damage of reagents, low-temperature, and ultrahigh vacuum
in electron microscopy, and so on. Smearing is a general
technique used to make cell films for optical microscopic
observation. However, it is not a good method for AFM
observation, for smearing will flatten erythrocytes (Fig. 1b and
e) and even convert them into echinocytes (Fig. 1c and f).
Erythrocytes from patients with various diseases have been
studied by AFM in our previous publication (Chen et al., 2002).
Normal red blood cells (RBCs) are biconcave in shape with an
approximate thickness of 2–3 mm and a diameter of around
7 mm. Fig. 1a is the AFM image ofmultiple normal erythrocytes
in 90 mm!90 mm scanning area. In Fig. 1d, the RBC is standard
biconcave in shapewith an approximate thickness of 1 mmand a
diameter of 8 mm.Because erythrocytes were on a solid support,
the thickness measurement was less than that of regular RBCs.
The size and shape of the central depression is variable, and
depending on its deformation. In Fig. 1d, the central depression
is 516 nm according to line analysis. As observed in a
conventional SEM, the blood cells have a tendency to stick to
each other or form stacks. In Fig. 1g, a biconcave shaped red
blood cell sticks to a spherical blood cell.
Figs. 2–10 illustrate the AFM three-dimentional topo-
graphic images of the same erythrocyte on mica at 0.5 day
exposure interval in air. After approximately 5 days exposure
in air and scanning many times by tip, the erythrocyte still kept
its biconcave shape, and the conditions of the cell environment
did not change, but the shapes were different at various
exposure intervals. After 0.5 days of exposure, a fissure
appeared in the erythrocyte (Fig. 3), another fissure appeared
after 1 day later (Fig. 4). After 1.5 days, there were more
fissures. These fissures were around 350 nm wide and 90 nm
deep, which are larger than the sizes of needle of the cantilever.
In AFM technique, a needle with a sharp ultralever tip is
scanned over the cell surface. The typical radius of curvature of
tip used in our experiment is w10 nm, and the diameter of the
needle where it is 90 nm away from the top end of the tip,
is less than 90 nm. So it could be concluded that these fissures,
as well as those in Fig. 1g, were not caused by the AFM needle.
It must be mentioned that the parallel lines (shown by
arrowheads) in Fig. 1g, 2, 4, and others were not caused by
scratching of the AFM needle, but by artifacts of the imaging
system since they dont’t appear in Figs. 3 and 6–10.
Another of the time dependent cellular changes of the
erythrocyte was shrinkage of cell. For example, the part of the
cell indicated by the arrow in Fig. 3 sank after 0.5 day, also
shown by the arrow in Fig. 4. Fig. 5 illustrates four large
fissures in the membrane surface of the erythrocyte after
1.5 days of exposure in air, but only two large fissures remained
3 days later. The other two large fissures shown by the two
Fig. 1. AFM images of single and multiple normal erythrocytes, and abnormal erythrocytes from smear samples. (a) AFM image of multiple normal erythrocytes,
scanning area: 90 mm!90 mm; (b and c) AFM images of multiple abnormal erythrocytes of various degrees, scanning area: 90 mm!90 mm; (d) AFM images and
surface profiles obtained for biconcave shaped erythrocyte; scanning area: 15 mm!15 mm, max. height 1 mm, width about 8 mm; (e and f) AFM images and surface
profiles of flatter and flatter erythrocytes caused by smear technique; scanning area: 15 mm!15 mm; (g) three-dimensional AFM image of two blood cells sticking to
each other; scanning area: 17 mm!17 mm.
Y. Chen, J. Cai / Micron 37 (2006) 339–346 341
arrows in Fig. 5 disappeared. That is, the part of the erythrocyte
with the other two large fissures has sunk.
Fig. 11 indicates the changes of AFM-measured volume and
surface area of the same erythrocyte based on Figs. 2–10. It is
clear that the AFM-measured surface area decreased but the
AFM-measured volume did not change much within the time
lapse period of 5 days. It is understandable that the sinkage of
the cell may be the main reason for the results, that is,
Fig. 2. AFM image of an erythrocyte introduced to the mica for 0 day. Fig. 4. AFM image of the same erythrocyte introduced to the mica for 1 day.
Y. Chen, J. Cai / Micron 37 (2006) 339–346342
additional cell surface were not detectable by AFM once they
were beneath the cell.
Figs. 7–10 also show that, after a 2.5-days exposure of
erythrocytes in air, many protuberances appeared in the
membrane. Fig. 12a is the three-dimensional topographic
AFM image of the concave part of the erythrocyte 2 days after
exposure in air, and Fig. 12b is the three-dimensional
topographic AFM image of the same part of the cell 2.5 days
Fig. 3. AFM image of the same erythrocyte introduced to the mica for 0.5 day.
after exposure in air. In the center of the two figures, there are
some large particles with a diameter of approximately several
100 nmwhichmay be dustwhichwas not seen on the surfaces of
other erythrocytes. The difference between the two figures is
the appearance ofmany protuberances in Fig. 12b. These images
of the protuberances were enlarged in order to be seen more
clearly in Fig. 12c. There are large protuberances with an
approximate height of 8–10 nmand a diameter of approximately
Fig. 5. AFM image of the same erythrocyte introduced to the mica for 1.5 day.
Fig. 6. AFM image of the same erythrocyte introduced to the mica for 2 day. Fig. 8. AFM image of the same erythrocyte introduced to the mica for 3 day.
Y. Chen, J. Cai / Micron 37 (2006) 339–346 343
50 nm, and some small protuberances with an approximate
height of 2–3 nm and a diameter of approximately 20 nm. There
are w47 protuberances in 1 mm!1 mm surface area. It is very
clear that the cell surface become more rough from Fig. 2 to
Fig. 10, especially in Fig. 10, where the protuberances are
obvious, implying that the appearance of these protuberances is
time-dependent.
Another phenomenon worth paying attention to is the
change of fluidity of the erythrocyte. In the first 2 days
Fig. 7. AFM image of the same erythrocyte introduced to the mica for 2.5 day.
(Figs. 3–6), the shape of the whole erythrocyte changed
dramatically, and even projected to a great extent at some
positions (as shown by the asterisks in Figs. 4 and 5). However,
after 2 days, the overall shape of the erythrocyte didn’t change
any more (Figs. 7–10), implying that the fluidity of the
erythrocyte became weaker with the loss of water. Further-
more, the period (2–2.5 days) when the cell stopped projecting
dramatically is consistent with the period (2–2.5 days) when
the protuberances on cell surface started appearing, implying
Fig. 9. AFM image of the same erythrocyte introduced to the mica for 4 day.
Fig. 10. AFM image of the same erythrocyte introduced to the mica for 4.5 day.
Y. Chen, J. Cai / Micron 37 (2006) 339–346344
that most of the water in the erythrocyte was lost by this point
and the cell membrane had attached to the cytoskeleton
network allowing transmembrane and peripheral proteins to
form those protuberances.
It is notable that the protuberances appearing on the
membrane surface of the erythrocyte look like antibodies that
were used to label the membrane surface (Fig. 12). It was very
difficult for AFM to detect single antibodies on the membrane
surface because they are similar in size to membrane proteins.
Therefore, R-PE proteins were used to increase the size of the
antibodies. In Fig. 12e, the R-PE—conjugated IgG antibodies
to human glycophorin A antigen, with a diameter of 40–60 nm
which is consistent with our previous study (Chen et al., 2004),
are shown clearly.
In the large scan area, the overall morphology of the cells is
visible, but detailed structural features on the cell surface are
Fig. 11. AFM-measured volumes and surface areas of the erythrocyte in Figs.
2–10 extracted from the images by the AFM software. It is clear that the AFM-
measured surface area of the erythrocyte decreased with time lapse although
the AFM-measured volume had little change.
not resolved. As depicted in Fig. 12, by zooming in on the
surface of the erythrocyte to a smaller scan size, 3 mm!3 mmand 0.6 mm!0.6 mm, the fine structure of membrane surface of
the erythrocyte can often be resolved. With a 0.6 mm!0.6 mmscan size, the smallest features on the erythrocyte that can be
resolved are approximately 3 nm.
4. Discussion
In certain situations, bloodstain analysis can be considered a
supplemental tool for determining the postmortem interval
(PMI). Bloodstain pattern analysis is often useful in establish-
ing and reconstructing the sequence of events or mechanisms
that caused blood flow. It is important to remember, however,
that a body can release blood either while alive or after death,
due to gravity. Analysis of this ‘static aftermath’ may assist in
determining some time factors, such as exposure to air, heat
and humidity, when making any time of death estimations
using bloodstain analysis.
Like other tissue cells, blood cells also lose their normal
morphology because of postmortem autolysis and putrefaction,
and are unidentifiable in the last period. The changing process
through normal morphology to the unidentifiable period can be
a useful criterion for estimating postmortem interval. In our
paper, after exposure in air for several days, some morpho-
logical changes of an erythrocyte could be observed.
Firstly, fissures on the membrane surface of the erythrocyte
appeared after 0.5 day of exposure (Fig. 3), and more fissures
appeared with the lapse of time (Figs. 4–10). The difference of
local microenvironments may be one of the reasons, which
may result in repeated dehydration in some areas of the
erythrocyte but in relatively stable conditions in other areas,
just as the fissions of earth surface and human skin. On the
other hand, a progressive stiffening of the membrane due to
oxidation of the cell may occur and promote formation of the
fissions. Second, after exposure in air, because of gravity of
itself, the erythrocyte stabilized on mica would sink step by
step in different degrees correlated to exposure times. The
arrows in Figs. 3–5 show sinking parts of the cell. Thirdly,
protuberances at the nanometer scale emerged after 2.5 days
exposure (Fig. 12), and the number of the emerging
protuberances was time-dependent. There are at least two
possible causes to explain these protuberances. The hemo-
globin in cytoplasm flows out through dehydration-induced
holes in the membrane. With the water in the membrane (about
20% of the total membrane weight) evaporated, the membrane
becomes thinner and exposes these integral membrane
proteins, such as band three protein, glycophorin A, and
others, or the cytoskeleton proteins. In fact, there has been
some literature reporting AFM imaging of the intracellular
membrane skeleton from the extracellular surface of cells
(Takeuchi et al., 1998; Christian et al., 1997). In Takeuchi’s
paper, a sunken membrane model was given, which stated that,
‘during drying, the membrane sinks with the loss of water into
the cell where there is no support by the membrane skeleton,
leaving behind the membrane skeleton and the membrane
attached to it’ (Takeuchi et al., 1998). Because scanning was
Fig. 12. Comparison of the three-dimensional AFM images of the concave center part of the erythrocyte in (a) Fig. 2 and (b) Fig. 7; scanning area: 3 mm!3 mm. In b,
the white circles indicate some protuberances. These large particles in center maybe dust; (c) enlargements of the same part of b, left two-dimensional image, right
three-dimensional image; scanning area: 0.6 mm!0.6 mm. The central parts of erythrocytes labeled without (d) and with (e) R-PE—conjugated mouse monoclonal
antibodies to human glycophorin A antigen; insets: the enlargements of the parts of the boxed parts in d and e; scanning size: 3 mm!3 mm (d and e); 1 mm!1 mm
(insets).
Y. Chen, J. Cai / Micron 37 (2006) 339–346 345
done in contact mode in their study, the depression of the AFM
tip on the membrane during scanning should be considered
more or less one of the reasons why they obtained such
resolution of the cytoskeleton network. Besides the reasons
mentioned above for interpretations of the fissions and
protuberances, other factors shall be considered, most
important two of which are pH value and salt concentration.
Actually, with repeated dehydration of the cell membrane, the
pH value and salt concentration will continually change, and
this will affect cell morphology to some extent.
All these changes in cell shape and cell membrane surface
ultrastructure will be useful for estimating the time of death.
But more precise research is indicated, such as the correlation
of membrane changes to shorter intervals of less than 0.5 day
exposure in air, and the use of various substrates in addition
to mica, including glass, metals, fabrics, among others, on
which the bloodstains appear in crime scenes.
In addition, the speed of all postmortem changes varies
widely in dependence of the conditions of storage during the
postmortem interval. Because of many factors, it is quite
difficult to report the exact time of death. Although, there are
many methods to estimate the time of death in forensic
medicine, none of them is reliable enough to be used alone.
Therefore, many of the available methods are applied together
and the most plausible time of death is estimated. Optical
microscopy is the traditional method to observe shape changes
of cells, and only the whole cell, not membrane surface
ultrastructure, can be observed. Because of its high resolution
and fast imaging, AFM cannot only investigate promptly
a multitude of cells (Fig. 1a–c) and a single whole cell
Y. Chen, J. Cai / Micron 37 (2006) 339–346346
(Figs. 1d–g–10), but also membrane surface ultrastructure
(Fig. 12), such as the protuberances at the nanometer scale
emerging on membrane surfaces. Observation of the mem-
brane surface ultrastructure may be useful for more accurate
estimation of the time of death. Therefore, AFM technique will
be a potentially powerful assistant tool in forensic science.
5. Conclusions
Morphological changes in a whole erythrocyte and of the
erythrocyte membrane surface ultrastructure, unfixed on mica
substrate, exposed in air over a 5-day period, were observed by
tapping mode atomic force microscopy (TM-AFM). The
changes include the appearance of fissures, shrinkage of
cells, and especially emergence of protuberances at the
nanometer scale on cell membrane surfaces. All the changes
of cell shape and cell membrane surface ultrastructure were
time-dependent and could help to improve estimation of the
time of death. However, this present study is quite preliminary,
more in-depth research will be needed to provide a calibration
curve for some kind of morphological marker of time lapsing in
order to realize as precise estimation of the time of death as
possible.
Acknowledgements
This research project is supported by national 973 program
of China (No. 2001CB510101), national natural science
foundation of China (No. 62078014) and key program of
national natural science foundation of China (No. 30230350).
We’d like to give many thanks to Zahida Ali, Department of
Microbiology and Immunology, University of Illinois at
Chicago, for her helpful corrections of the revised paper.
References
Bersellini, C., Garofano, L., Giannetto, M., Lusardi, F., Mori, G., 2001.
Development of latent fingerprints on metallic surfaces using electro-
polymerization processes. J. Forensic Sci. 46, 871–877.
Chen, Y., Cai, J., Zhao, J., 2002. Diseased red blood cells studied by atomic
force microscopy. Int. J. Nanosci. 1, 683–688.
Chen, Y., Cai, J., Xu, Q., Chen, Z.W., 2004. Atomic force bio-analytics of
polymerization and aggregation of phycoerythrin-conjugated immuno-
globulin G molecules. Mol. Immunol. 41, 1247–1252.
Chen, Y., Cai, J., Zhao, T., Wang, C., Dong, S., Luo, S., Chen, Z.W., 2005.
Atomic force microscopy imaging and 3-D reconstructions of serial thin
sections of a single cell and its interior structures. Ultramicroscopy 103,
173–182.
Christian, L.G., Eric, L., Marie-Cecile, G., Eric, F., Jean-Pierre, G., 1997.
Simultaneous imaging of the surface and the submembraneous cytoskeleton
in living cells by tapping mode atomic force microscopy. Life Sci. 320,
637–643.
Dufrene, Y.F., 2003. Recent progress in the application of atomic force
microscopy imaging and force spectroscopy to microbiology. Curr. Opin.
Microbiol. 6, 317–323.
Frederix, P.L., Akiyama, T., Staufer, U., Gerber, C., Fotiadis, D., Muller, D.J.,
Engel, A., 2003. Atomic force bio-analytics. Curr. Opin. Chem. Biol. 7,
641–647.
Friedbacher, G.F., Harald, F., 1999. Classification of scanning probe
microscopies. Pure Appl. Chem. 71, 1337–1357.
Girasole, M., Cricenti, A., Generosi, R., Congiu-Castellano, A., Boumis, G.,
Amiconi, G., 2001. Artificially induced unusual shape of erythrocytes: an
atomic force microscopy study. J. Microsc. 204, 46–52.
Gurden, S.P., Monteiro, V.F., Longo, E., Ferreira, M.M., 2004. Quantitative
analysis and classification of AFM images of human hair. J. Microsc. 215,
13–23.
Hansma, H.G., Kasuya, K., Oroudjev, E., 2004. Atomic force microscopy
imaging and pulling of nucleic acids. Curr. Opin. Struct. Biol. 14, 380–385.
Horber, J.K., Miles, M.J., 2003. Scanning probe evolution in biology. Science
302, 1002–1005.
Ikai, A., Afrin, R., 2003. Toward mechanical manipulations of cell membranes
and membrane proteins using an atomic force microscope: an invited
review. Cell Biochem. Biophys. 39, 257–277.
Kasas, S., Khanmy-Vital, A., Dietler, G., 2001. Examination of line crossings
by atomic force microscopy. Forensic Sci. Int. 119, 290–298.
Liu, F., Mizukami, H., Sarnaik, S., Ostafin, A., 2005. Calcium-dependent
human erythrocyte cytoskeleton stability analysis through atomic force
microscopy. J. Struct. Biol. 150, 200–210.
Nagao, E., Nishijima, H., Akita, S., Nakayama, Y., Dvorak, J.A., 2000. The cell
biological application of carbon nanotube probes for atomic force
microscopy: comparative studies of malaria-infected erythrocytes.
J. Electron Microsc. (Tokyo) 49, 453–458.
Nowakowski, R., Luckham, P., Winlove, P., 2001. Imaging erythrocytes under
physiological conditions by atomic force microscopy. Biochim. Biophys.
Acta 1514, 170–176.
Ohta, Y., Okamoto, H., Kanno, M., Okuda, T., 2002. Atomic force microscopic
observation of mechanically traumatized erythrocytes. Artif. Organs 26,
10–17.
O’Reilly, M., McDonnell, L., O’Mullane, J., 2001. Quantification of red blood
cells using atomic force microscopy. Ultramicroscopy 86, 107–112.
Rounsevell, R., Forman, J.R., Clarke, J., 2004. Atomic force microscopy:
mechanical unfolding of proteins. Methods 34, 100–111.
Santos, N.C., Castanho, M.A., 2004. An overview of the biophysical
applications of atomic force microscopy. Biophys. Chem. 107, 133–149.
Smith, J.R., 1998. A quantitative method for analysing AFM images of the
outer surfaces of human hair. J. Microsc. 191, 223–228.
Swihart, A.H., Mikrut, J.M., Ketterson, J.B., Macdonald, R.C., 2001. Atomic
force microscopy of the erythrocyte membrane skeleton. J. Microsc. 204,
212–225.
Takeuchi, M., Miyamoto, H., Sako, Y., Komizu, H., Kusumi, A., 1998.
Structure of the erythrocyte membrane skeleton as observed by atomic
force microscopy. Biophys. J. 74, 2171–2183.
Zachee, P., Boogaerts, M., Snauwaert, J., Hellemans, L., 1994. Imaging uremic
red blood cells with the atomic force microscope. Am. J. Nephrol. 14,
197–200.