Atomic force microscopy used in image mode

Suhyung Park, 2nd Biomedical Engineering, Universitat de Barcelona

Abstract
In this lab work, we used WSxM software and topographical images of different A549
cells to calculate the volume of lung alveolar epithelial cells with AFM. Using AFM
imaging, four images of the cell were obtained. We used a cantilever with a spring
constant k = 0.01 nN/nm and a pyramidal tip. We had to consider the volume that had
been squashed due to the force of the cantilever in order to determine the real volume
of the cell. Using WSxM, we were able to create 3D model of the cell.

Introduction
Cell shape is one of the fundamental biophysical properties of cells. Many experiments
has shown that cell shape regulates a variety of cellular processes including proliferation,
apoptosis, metabolism, differentiation and even stem cell commitment.
A major cell shape related property is cellular volume. Previous studies assessed the
volume of groups of cells by optical methods. However, an alternative method is based
on processing high-resolution images of the topography of cells obtained with the
nanotechnique Atomic Force Microscopy (AFM). Unlike traditional optical-based methods,
AFM imaging enables calculating the whole cell volume with unprecedented high spatial
resolution. Moreover, it allows examining local details of the topography of cells. However,
the application of AFM to assess the volume of small cells such as lung epithelial cells
has been very limited.
Goal and experimental approach
The goal of this study is to determine the volume of lung alveolar epithelial cells with
AFM.

Material and Methods

Sample preparation
Cells from the human lung alveolar epithelial line A549 were cultured following details
provided by the manufacturer (ATCC). For AFM experiments, A549 cells were seeded on a
glass surface for 3 days.
Instrumentation
A commercial AFM adapted to an optical microscope was used to image the topography
of A549 cells. The cantilever used had a spring constant k = 0.01 nN/nm, and a
pyramidal tip.
AFM imaging
Cell samples were imaged by raster scanning the tip in contact with the cells applying
low force and using low scanning speed (0.2 Hz). Different scan sizes were used (30-60
m).
Data analysis
Using WSxM software and the images obtained previously, experimental measurements
were processed. The average volume and maximum height per cell were calculated as
the average for all images. The relationship between the loading force F and the
indentation induced by a pyramidal tip is given by

where is the opening angle of the pyramidal tip (35), is the Poisson ration (which
was assumed to be 0.5) and E is the Youngs modulus of the cell. If E of A549 cells is
~500 Pa, and the force applied during imaging was ~0.5 nN, this allowed to calculat how
much (in %) was the maximum height underestimated. Using this information it was
possible to assess a correction factor for the cell volume by taking into account the
underestimation due to cell indentation. Later, a 3D representation of a selected A549
image was generated and also a dERI function (derivative) was applied to the same
image.

Results

Figure 1. Topographical images of different A549 cells.

From the images of Figure 1, numbering 1 to 4, from left to right, we have assessed the
maximum height, total volume and the average volume per cell, which can be seen in
Table 1. The image 3, has three cells, so we have taken values for those three cells.
Table 1. Maximum height, total volume, number of cells and average volume per cell for
each image. Average maximum height and volume per cell of the images.

Using the formula shown before, the correction factor could be found, so we added it up
to the max height of the Table 1. With those values, we calculated again other values
dependent of the max height of the cell. The results can be seen in Table 2.

Table 2. From table 1, fixed the values using the correction factor. Maximum real height,
area and real volume of each cell.

From the values of Table 1 and Table 2, we can see that the maximum height
underestimated is 22% and the volume underestimated is 45%.

The first image of Figure 2 shows the 3D image of the cell 2, and the second
image expresses a derivate function applied image, in which we can clearly see
the slope of the cell, despite being a 2D image. This is possible thanks to the
shadow-effect of the image. Our eyes feel a sensation of a slope when we see a
shadow.
Figure 2. 3D image and the result of the derivate function application of the cell2. (from
left to right)

Discussion
A. Discussion of the experimental approach and data analysis
We used WSxM to asses the maximum height and volume from AFM images, however,
this tool has some possible sources of error such as :
Firstly, the Profile tool cannot be used randomly, every region in the cell must be
checked and the limits that determine the change in height must be well chosen.
Secondly, while using the Flooding tool, each hill must be isolated from the others, and
so one must rise the height the volume starts to be counted from sometimes, and so the
non-corrected volume is underestimated. The area of each cell has same problem as the
volume. On top of that, the corrected value for volume is likely to be mistaken since the
cell does not have a constant area, which we neglect in the correction.
AFM is commonly used to analyse morphological aspects of cells thanks to its ability of
imaging cells with nanometric resolution. However, the main problem with this technique
is that the cantilever tip has a determinate shape and size that it cannot differentiate a
space smaller than its tips, and this may induce structural modifications to the cell
surface. These modifications can lead to important miscalculations in morphological
characteristics such as the ones meant to be assessed in this work. The perturbations can
be minimized by exerting lower forces on cells.
Images available for analysis with WSxM were not really varied. Most of the cells showed
to share similar heights and volumes. 3 out of 4 images showed a cell fully appearing.
When we applied the derivative filter, we could see that the previous 2D image had
gained some effects that it looked like a 3D image without being so. The dark color
(shadow-like) appeared darker where the derivatives were higher, so where the derivative
was small, seemed like to be a plain. The 3D sensation is due to these cliff-like textures
and the fact that a source of light seems to be present at the left side of the image,
making that side brighter while the right side is darkened. Our eyes detect volumical
shape of an object when there is a contrast in light, and this is the reason that this
image looked like a 3D image to us.
The cell A549 cells from AFM images appear to have an oval shape, a height of 5.41
0.38 m and a volume of 748 37 m3. However, some cells seem more rounded and
spread whereas others have little elongations. Morphological details could have been
observed simply using optical methods, but as AFM images provide enough information
themselves and since AFM is one of most accurate techniques, small details observed by
AFM images cannot be observed using other techniques.

B. Contextualization of the results

Scanning Ion Conductance Microscopy (SICM)1 is a useful tool for obtaining non-invasive
images of cell surface topography, and It has been used for imaging live cells in culture
medium2. SICM can provide accurate volume measurement of cells due to its ability to
acquire quantitative dimension information. Furthermore, it can calculate cell volume
changes over time caused by physiological environment or its cell status changes, which
is a great advantage, since cell status changes can be observed while the time is passed
or some environmental conditions changes. The advantage of AFM compared to SICM is
that AFM, apart from measuring the volume, it can measure physical properties of the
cell, as it applies a mechanical force to the cell.

Figure 3. Sequential HEK 293T live cell images (SICM).3

1
P. K. Hansma, Science 243, 641 (1989).
2
Y. E. Korchev, Biophys. J. 73, 653 (1997).
3
Myunghoon Choi, Cell Volume Measurement of Ion Conductance Microscopy, 2011
Type I alveolar epithelial cells have diameter 50 to 100 microns and volume 2000 to 3000
m3, whileas type II alveolar epithelial cells have diameter approx. 10 microns and
volume 450 to 900 m3.4 Maximum height that we have is almost 5 m, which means
that the cell can have approx. 10 m of diameter and the volume was approx. 750m3.
As A549 cells are type II cells, our results are perfectly reasonable quantitatively.
Comparing with results reported in the literature by Jiang RD5, which claims to have used
inverted microscopy and transmission electron microscopy to measure the size, we can
clearly see that both results are similar. The diameter of A549 cells from inverted
microscopy and TEM images was 14.93 m and 10.59 m whileas our diameter was
approx. 10 m.

C. Discussion of the biological significance of the results

Physiological processes of cells that may be dependent or affected by their size or
shape.
The biconcave shape and corresponding deformability of the human red blood cell (RBC)
is an essential feature of its biological function. This feature of RBCs can be critically
affected by genetic or acquired pathological conditions. Many hereditary diseases can
cause deformation of RBC, causing the reduction of haemoglobines, which will decrease
the concentration of oxygen in the blood.6
Cell division is affected by the cell size. The growth and division rates of a cell are co-
ordinated to ensure that cell size is maintained. This coordination is achieved because
cell growth is rate-limiting for cell-cycle progression. Cell growth rate alone does not
determine the rate of cell-cycle progression and that cell size at division is variable and
depends on the concentrations of extracellular signal proteins that stimulate cell-cycle
progression, cell growth, or both.7
Diseases characterized by changes in cell size
Macrocytic anemia with large cell size can be found in B12 and/or folate deficiencies
such as pernicious anemia, poor dietary intake, vegan diets, excessive alcohol
consumption, use of erythrocyte-stimulating agens (ESAs), drugs affecting DNA synthesis,

4
Qutayba Hamid, Physiologic Basis of Respiratory Disease. PMPH-USA, 2005.
5
Jiang RD, et al., The morphometrical analysis on the ultrastructure of A549 cell, 2010.
6
Monica Diez-Silva, et al., Shape and Biomechanical Characteristics of Human Red Blood Cells in
Health and Disease, 2010.
7
Conlon, I.J., et al., Extracellular control of cell size. Nat Cell Biol, 2001.
or liver disease etiologies.8
Establishing and Maintaining Cell Size
Cell growth rate depends on the concentrations of extracellular signal proteins that
stimulate cell growth. 9
The best-characterized example of which is the
IGF/PI3K/AKT/mTORC1 pathway. This evolutionary conserved pathway has been shown to
be a major regulator of cell growth and thus a key determinant of cell size; moreover,
artificial activation of this pathway can promote additional growth in most cell types
tested (Edgar, 2006, Laplante and Sabatini, 2012 and Tumaneng et al., 2012a).
The size of an adult organism is determined by both intrinsic developmental programs
and by extracellular signals, which integrate to control cell number and cell size.
Differences in animal size are mostly genetically determined and primarily reflect
differences in cell number rather than differences in cell size. However, despite the more
or less fixed target size of most organisms, external signals can still impinge on this
genetic program. One clear example of this is the effects that nutrient levels can have
during development. When in excess, nutrient levels do not appreciably affect maximal
organismal size, but when limited, they can have a dramatic effect. For example, it has
been shown that extreme nutrient deprivation during development can decrease a flys
size to 15% of normal.10

Conclusion
We have accomplished our goal, which was to determine the volume of lung alveolar
epithelial cells with AFM. During the process, we had found the values with some errors
due to the inaccurateness of the imaging process because of the cantilever and the
mechanical force applied, but after some calculations, we could fix the error and assess
the real volume of the cells. The average volume of the cells was 748.32m3

8
Joanne K., Primary Care, Second Edition: An Interprofessional Perspective, 2014.
9
Conlon, I.J., et al., Extracellular control of cell size. Nat Cell Biol, 2001.
10
Alison C. Lloyd, The Regulation of Cell Size, Cell, 2013
References
- Introduction, Materials and Methods from Campus Virtual.
1. P. K. Hansma, B. Drake, O. Marti, S. A. Gould, C. B. Prater, Science 243, 641 (1989).
2. Y. E. Korchev, C. L. Bashford, M. Milovanovic, I. Vodyanoy, M. J. Lab, Biophys. J. 73, 653
(1997).
3. Myunghoon Choi, Cell Volume Measurement of Ion Conductance Microscopy, Park
Systems Application Note, 2011
4. Qutayba Hamid,Joanne Shannon,James Martin, Physiologic Basis of Respiratory Disease.
PMPH-USA, 2005.
5. Jiang RD, et al., The morphometrical analysis on the ultrastructure of A549 cells. Rom J
Morphol Embryol. 2010;54(4):663-7.
6. Monica Diez-Silva, Ming Dao, Jongyoon Han, et al., Shape and Biomechanical
Characteristics of Human Red Blood Cells in Health and Disease, MRS Bull. 2010 May;
35(5): 382-388.
7. Conlon, I.J., et al., Extracellular control of cell size. Nat Cell Biol, 2001. 3(10): p. 918-21.
8. Joanne K. Singleton PhD, RN, FNP-BC, FNAP, FNYAM, Robert V. DiGregorio PharmD,
BCACP, Primary Care, Second Edition: An Interprofessional Perspective , Springer
Publishing Company, 2014. 11. 12
9. Conlon, I.J., et al., Extracellular control of cell size. Nat Cell Biol, 2001. 3(10): p. 918-21.
10. Alison C. Lloyd, The Regulation of Cell Size, Cell, Volume 154, Issue6, 12 September 2013