Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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Visualization of cell structure in situ by atomic force microscopy
María de L. Segura-Valdez1, Alma Zamora-Cura1, Nadia Gutiérrez-Quintanar1,2, Ernesto VillalobosNájera3, Jennifer Berenice Rodríguez-Vázquez1, Tania Citlalli Galván-Arrieta1, Diana JiménezRodríguez1, Lourdes T. Agredano-Moreno1, Reyna Lara-Martínez1 and Luis F. Jiménez-García1
1
Department of Cell Biology, Faculty of Sciences, National Autonomous University of Mexico (UNAM), Circuito
Exterior, Ciudad Universitaria, Coyoacán 04510, México D.F., Mexico
2
Exchange program from Universidad Autónoma del Estado de México (UAEMéx), Mexico
3
Present adress: Universidad Autónoma de la Ciudad de México, México D.F., Mexico
Since its invention, atomic force microscopy has been used for the study of life sciences, in addition to its application in
materials science. Although most work has focused on the study of isolated molecules, some research also has been
performed on entire cells growing in culture. Here we revise some efforts made to study in situ internal cell structure with
the atomic force microscope using sections of biological samples prepared for transmission electron microscopy. The
texture of every section reveals details of cell organization similar to the ultrastructure obtained with the electron
microscope. Visualization of cell structure during the cell cycle including interphase and mitosis, suggests the possibility
to approach further investigation taking advantage of the vertical resolution of the microscope. Particularly, the analysis of
some aspects of gene expression may also be possible at the nanoscale range.
Keywords atomic force microscopy; cell; nucleolus; nucleus
1. Study of isolated molecules and entire cells in culture by atomic force microscopy
The cell structure has been analyzed over the years mainly by light and electron microscopy. The invention of scanning
probe microscopes specially the atomic force microscope in 1986 [1] made possible to consider the analysis of
biological samples at the nanoscale without the restriction of working either under vacuum conditions characteristic of
the transmission electron microscopy [2] or the need of conductivity of samples for scanning tunneling microscope [3].
Moreover, atomic force microscopy also offers a very high resolution in the vertical z axis, allowing the possibility of
analyzing living cells at the nanoscale spatial resolution. Many attempts have been devoted to the analysis of biological
samples and particularly the study of isolated molecules such as nucleic acids such as DNA or proteins [4-11]. In
addition, the analysis of cell structure has focused on either living or fixed entire cells. In such kind of studies, for
example, cells from smears or cell culture placed onto a glass slide or other surface may be fixed by air-drying or by the
use of chemical reagents known as chemical fixatives. Also they can be observed by atomic force microscopy even
under physiological conditions as buffer solutions. Moreover, cells growing in culture can be observed with the atomic
force microscope while they are still alive. This approach produces high resolution images that however, do not reveal
internal aspects due to the presence of the cell membrane that faces the tip of the microscope.
Human red blood cells or lung fibroblasts may be tested under this protocol. In human red blood cells placed onto a
glass slide, the characteristic concavity in every mature erythrocyte due to the absence of nucleus is observed either by
using two dimensional or three dimensional displaying (Figure 1). Some other cell types present in the smear can also
be observed. Morphologically, these elements correspond to authentic red blood cells because the phenotype including
the size of the cells.
Observations of human fibroblasts growing as monolayers in cell culture conditions and attached to glass coverslips
were possible after rinsing the cells and fixing them either with 2% glutaraldehyde, 10% formaldehyde or 4%
paraformaldehyde buffered with several solutions as phosphate buffer saline (PBS). Cell then may be air-dried or
maintained under solution and monitor them using the atomic force microscope on contact or intermittent modes. The
use of a liquid chamber allows the observation under liquid. Several cell components as the interphase nucleus and the
cytoplasm -or even the nucleolus- can be recognized under these conditions because the cell membrane is attached
above the cell organelles (Figure 2), preventing the observation of the structure of the organelles themselves. No
internal structures are seen in any of these examples.
This approach also make possible to study several properties of many cell types including cancer cells [12]. Also, it is
possible to study some elements of the extracellular matrix such as those characterizing the bone [13]. The structure of
the protein collagen may be compared to that obtained by the transmission electron microscope as a banded regular
pattern.
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Méndez-Vilas and J. Díaz (Eds.)
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10 µm
a
b
Figure 1. Atomic force micrographs of entire human red blood cells from a smear: a) two dimension display of sample. b) three
dimensional display. Both images show the concavity of the red blood cells (arrows) whose size is about 10 µm. The images show
only the surface but not the interior of the cells which is defined by the presence of the cell membrane.
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Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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C
N
n
20 µm
a
C
N
n
b
Figure 2. Atomic force micrograph of a human lung fibroblast growing in culture fixed with glutaraldehyde and air-dried: a) two
dimension display. b) three dimensional display. Nucleus (N), nucleolus (n) and cytoplasm (C) are also shown.
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2.
In situ cell structure studies
Few studies have been carried out to analyze cell structure and function. They used entire cells to know more about
some physiological properties.
Since the atomic force microscope explores the surface of materials, it is also necessary to make the interior of the
sample available for scanning. Sectioning samples is useful since every section has a surface that can be scanned in
order to reveal details of internal animal tissue cell structure or cell samples prepared for histology [14] or plant material
processed for transmission electron microscopy [15]. It is a regular observation that working under the ultramicrotome,
that semithin or thin sections may allow us to place the sample in a useful direction to choosing the area to show under
the electron microscope. First studies using this approach produced images from unstained samples displaying a texture
correlating to cell structures [15]. Therefore, further analyses were devoted to improve this technique and increase the
resolution of the microscope.
2.1 A technique to analyze internal in situ cell structure by atomic force microscopy
In spite of several protocols were used in the recent past for the study of cell structure, a protocol has been described
recently that produces reproducible results [15-21]. The method may be briefly described as follows. First, samples are
prepared as for standard transmission electron microscopy. Semithin sections are mounted on glass slides and air-dried.
Then the slides are observed under an atomic force microscope working in contact or tapping modes, with a resonance
frequency of 150-350 kHz. A scan rate of 1-2 hertz and a contact force of 10 nN were regularly used. The scan size
used has been 100 or 60 mm. Images have been obtained by using the Nanoscope IIIa control system. Silicon nitride
tips with a curvature radius of 20-50 nm have been used. Figure 3 shows the approach to analyze internal cell structure.
AFM
glass slide
semithin sections
light microscopy
trasmission electron microscopy
atomic force microscopy
Figure 3. Schematic view showing the classical approach the study of in situ cell structure on semithin sections. The tip of the
microscope scans the sections placed onto a glass slide.
2.2 Visualization of cell structure in situ by atomic force microscopy
Using this technique and approach, it has been possible to analyze some aspects of the cell structure, specially that one
related to the structure of the interphase cell nucleus. In fact, the presence of chromatin strands and nucleolus as well as
nuclear pores were identified in the cells of the plant Lacandonia schismatica [15-16]. These observations made
possible using an additional criterion of atomic force microscopy for the identification of reticulated chromatin in plants
[17-19]. Further studies allowed the visualization of interphase cell nucleus and the different phases of mitosis in onion
root tip cells [19-20]. More recently nuclear ribonucleoprotein particles (30 nm in diameter) known as Lacandonia
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Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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granules were visualized in the nucleoplasm of L. schismatica cell nuclei [21]. Figure 4 shows onion root tip interphase
cells where nucleus, cytoplasm, cell wall and vacuoles are distinguished. Figure 5 shows cells undergoing mitosis.
V
cw
N
10 µm
a
V
cw
N
b
Figure 4. Atomic force micrographs of cell structure of onion root tip cells from semithin sections: a) two-dimension and b) three
dimensional display of the same area. Cell wall (cw), nucleus (N), nucleolus (rounded structure within the nucleus); vacuole (V).
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I
M
25 µm
a
I
M
b
Figure 5. Atomic force micrographs of interphase and mitotic cell structure of onion root tip cells from semithin sections. a) Two
dimension and b) three dimension display of the same area. I, interphase cell; M, mitotic cell in prometaphase.
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Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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In a wide view of onion root tips interphase and mitotic cells are distinguished by the arrangement of the genome in the
form of compact chromatin or chromosomes, respectively. In Figure 5 a cell in prometaphase is observed close to
interphase cells.
The use of semithin sections of biological samples prepared for standard transmission electron microscopy is
producing images of cells by scanning the surface. The images are of similar resolution to those generated by
ultrastructural approaches. In fact, recent studies have shown that intranuclear 32 nm in diameter ribonucleoprotein
particles -known as Lacandonia granules because they were first described in the plant Lacandonia schismatica-, can be
visualized using this method [21]. On the other hand, the use of serial sections and atomic force microscopy was
proposed few years ago to analyze cell structure [22-23]. Future work will surely focus on trying to obtain nanometer
resolution of the surface of a section embedded in different resins or prepared as cryosections. Additional efforts may
also be useful on resinless material.
Acknowledgements The support by DGAPA-UNAM PAPIME PE205607 and PAPIIT IN227810 is gratefully acknowledged.
Alma Zamora-Cura is a graduate student from the program on Biomedical Sciences-Universidad Nacional Autónoma de México
(UNAM), supported by CONACyT, México. Nadia Gutiérrez-Quintanar is an undergraduate exchange program student from the
Universidad Autónoma del Estado de México (UAEMex). We thank Martha Montaño and Carina Becerril from Instituto Nacional de
Enfermedades Respiratorias (INER), México, for providing us with human lung fibroblasts slides.
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