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Visualization of internal in situ cell structure by atomic force microscopy

Article  in  Histochemie · September 2018

DOI: 10.1007/s00418-018-1721-6

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Histochemistry and Cell Biology
https://doi.org/10.1007/s00418-018-1721-6

REVIEW

Visualization of internal in situ cell structure by atomic force

microscopy
María L. Segura‑Valdez1 · Lourdes T. Agredano‑Moreno1 · Alma L. Zamora‑Cura1 · Reyna Lara‑Martínez1 ·
Luis F. Jiménez‑García1 

Accepted: 4 September 2018

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Light and electron microscopy have been used to study cell structure for many years, but atomic force microscopy is a more
recent technique used to analyze cells, mainly due to the absence of techniques to prepare the samples. Isolated molecules or
organelles, whole cells, and to a lesser extent in situ cell structure have been observed by different atomic force microscopy
imaging modes. Here, we review efforts intended to analyze in situ the cell structures using approaches involving imaging of
the surface of semithin sections of samples embedded in resin and sections prepared with an ultramicrotome. The results of
such studies are discussed in relation to their implications to analyze the fine structure of organelles at the nanoscale in situ
at enhanced resolution compared to light microscopy.

Keywords  Atomic force microscopy · Cell · Nucleus · Nucleolus · RNA · Ultramicrotome

Introduction microscope. Since specimen preparation is often simplified

Traditionally, cell structure and composition have been
studied by light and electron microscopy using different
approaches including histochemical and cytochemical tech-
niques. However, studies using scanning probe microscopes,
such as the atomic force microscope to visualize and analyze
cell structure are less abundant. In this brief review, we dis-
cuss various efforts to visualize internal cell structures by
atomic force microscopy.
The atomic force microscope (AFM) was invented in
1986 as an instrument derived from its predecessor, the
scanning tunneling microscope (STM) (Binnig et al. 1986).
For imaging, STM requires conductive samples, whereas,
the AFM was invented to analyze non conductive materi-
als. This feature presented AFM as a better tool to analyze
biological samples that characteristically are non conductive.
In biological studies, samples usually are soft and need to be
chemically processed to be observed by the light or electron
* Luis F. Jiménez‑García
luisfelipe_jimenez@ciencias.unam.mx
1
Department of Cell Biology, Faculty of Sciences, National
Autonomous University of Mexico (UNAM), Circuito
Exterior, Ciudad Universitaria, Cd. Mx., Coyoacán,
04510 Mexico City, Mexico
for AFM, initially preferred samples consisted of isolated
molecules because the resolution of the microscope is very
high, reaching nanometers dimensions in X, Y and Z axis
working under room pressure and even under liquid condi-
tions. In addition studies analyzing whole cells have been
conducted to explore cellular properties including adhesion,
displacement or migration. However, less work has been
published on the observation of internal cell structure in situ.
The scanning probe microscopes (SPM) are a mechano-
optical systems developed to characterize surfaces with
nanometric resolution. This analysis is done through the
measurement of the interaction between an extremely thin
tip and the surface of the sample. Figure 1 shows the system
of the AFM. The tip is attached to the end of a flexible can-
tilever. The cantilever or sample holder can be mounted on a
device of piezoelectric material (PIEZO) to ensure continu-
ous and reproducible displacements at the nanoscale level.
The interaction between the tip and the surface of the sample
results in bending of the cantilever in response to repulsive
or attractive forces (van der Waals forces) between tip and
sample’s surface. The measure of bends senses the changes
in the forces. The scanning tunneling microscope (STM)
was the first device developed to measure the bending of
the cantilever at the nanometer level, by the tunnel current
that depends on the tip–surface separation. However, this

13
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Fig. 1  Schematic view of
components and principle of
an atomic force microscope.
The tip of the microscope is
mounted at the end of a flex-
ible cantilever attached to a
piezoelectric device (PIEZO).
A photodetector registers the
oscillation of a reflected laser
beam to produce an image on a
computer monitor
mechanism is restricted to electrically conductive samples.
Maybe this is the reason why the most used system to meas-
ure this interaction is based on optics using a laser beam
directed to the end of the cantilever, where the tip is attached
and reflected to a photodetector in a classical closed loop
feedback system as is performed in the atomic force micro-
scope (Fig. 1).
Due to the short distances between point and surface,
during the AFM operation, the van der Waals interactions
predominate over other forces involved. There are three
modes of general operation of the AFM, namely, (1) con-
tact mode (in which repulsive forces predominate), (2) non-
contact mode (in which attractive forces predominate), and
(3) intermittent contact (tapping mode). In the contact mode,
the tip scans directly the surface of the sample and the can-
tilever flexes in response to its topographic irregularities.
A laser beam is directed to the end of the cantilever, where
the tip is attached and reflected to a photodetector (Fig. 1).
The elasticity or level of cantilever flexibility (Hooke’s law)
influences the sensitivity of the feedback and detection
system. The system records the lengths of the deviation of
the reflection of a laser beam in the reference quadrant of a
photodetector (Error = deflection-setpoint). With these data,
a computer graphs point by point and line by line the com-
plete image of the surface with quantitative information in
three dimensions (x, y and z) for a further digital processing
and projection of the resulting image. The process can be
performed even on air or under liquid conditions (i.e., physi-
ological environment). In contact mode, when the image is
produced with a sample in liquid (physiological buffer for
example) the capillarity becomes a problem produced by the
forces of attraction. This problem can be solved by using the
AFM in non-contact mode that takes advantage of the attrac-
tive forces to monitor the tip–surface interactions. The forces
of attraction are weaker in the contact mode. This technique
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Histochemistry and Cell Biology
has the disadvantage of offering lower resolution. Intermit-
tent contact is another way of measuring cantilever flexions
and the image produced by the interactions between the tip
and the surface of the sample is similar to that achieved in
the contact mode. In non-contact mode, the cantilever oscil-
lates in a specific resonance frequency (given by the charac-
teristics of the tip cantilever complex) and scans the surface
without damaging it, depending on both the repulsive and
attractive forces (Morris et al. 1999; Hinterdorfer 2002; Jena
and Cho 2002; Braga and Ricci 2004).
Whole cell studies
Many studies have been devoted to the analysis of whole
cells, either, growing in culture or adhered to different sub-
strates. These studies have revealed several aspects of the
structure of cell membranes. For example, AFM was used to
investigate the formation of membrane elements involved in
exocytosis in unfixed pancreatic acinar cells (Schneider et al.
1997). As a result of these studies revealing the nanoscale
topography of biological membranes, new models of cell
membranes have emerged, such as proposing a semi-mosaic
model for red blood cells (Shan and Wang 2015). Other
authors also studied viruses in living cells by imaging the
cell surface (Ohnesorge et al. 1997).
Additional studies present images of cells both, under
dried conditions or immersed in physiological buffers. As an
example, Butt and colleagues (1990) imaged archaebacteria
and stomata, as well as blood cells, showing structural fea-
tures between 8 and 10 nm. Figure 2 shows AFM images of
red and white blood cells smeared onto a glass slide. Force
measurements between interacting cells have also been con-
ducted by AFM (see Dufrêne et al. 2017; Gavara 2017).
Such approaches involve the quantitative determination of
Histochemistry and Cell Biology
Fig. 2  Atomic force micrograph of whole human red blood cells.
Sample was prepared as a smear onto glass slide. Front view (a) and
projected image (b) show the cells (arrow), but no organelles are
observed. Scale bar: 10 µm
the forces acting between cells and their substrate, provid-
ing information on mechanosensitive phenomena (Gavara
2017).
To acquire images of cells and their interactions with the
substrate, along with limited compositional analysis of the
sample, a combination of AFM with other microscopy tech-
niques including epifluorescence light microscopy, confocal
microscopy or super resolution microscopy, have been per-
formed. In such investigations, a correlative image of struc-
tural elements at the nanoscale level together with detection
of proteins by immunolocalization procedures is provided
(Dufrêne et al. 2017).
Using an immuno-AFM concept, high-resolution imaging
of molecules such as bacteriorhodopsin in the cell membrane
and the force required to remove bound antibodies were pre-
sented (Müller et al. 1996). Cryo-AFM has been performed
Fig. 3  Schematic representation of the AFM scanning the surface of
semithin sections of resin-embedded tissue mounted on a glass slide.
The tip mounted on the cantilever of the microscope scans the sec-
tion, producing images of the surface face
in multiple studies, producing images of molecules such as
myosin and whole red blood cells (Shao and Zhang 1996).
More recent innovations in the field include high-speed
AFM, providing results such as the movement of F1-ATPase
or nuclear pore transport (see Dufrêne et al. 2017).
Internal in situ cell structure visualization
Several studies have been performed intended to visualize
internal cell structure by AFM. Various approaches have
been attempted, including microtome sectioning of paraf-
fin or plastic resin embedded samples to expose the inner
aspect of the cell.
Initial observations using this approach were made on
tissue sections from plastic-embedded material. Resulting
images were similar to those obtained by electron micros-
copy (Ushiki et al. 1996). Later, plant cells processed for
standard transmission electron microscopy were imaged by
AFM. Flowers of the plant Lacandonia schismatica were
prepared and semithin sections were mounted on glass
slides. In this approach, the samples are produced by a pro-
cedure for standard transmission electron microscopy, fixed
with aldehydes, with or without post-fixation with osmium
tetroxide and then embedded in epoxy resin (Jiménez-García
and Segura-Valdez 2004) or prepared for immunoelectron
microscopy by embedding in a hydrophilic resin such as
Lowicryl K4M resin (Roth 1986, 1989). Semithin sections
(ca. 100 nm) are mounted onto a glass slide and the section
surface is scanned by the AFM tip. A schematic view of this
approach is depicted in Fig. 3.
Cells from the teguments were scanned by AFM imag-
ing in contact mode. Cell walls, nuclei, nucleoli, compact
chromatin, and vacuoles were recognized. The recognition
of compact chromatin by AFM was then used as an addi-
tional parameter to identify this type of chromatin in plants,
where reticulated patterns of distribution had been identified
13

from light and electron micrographs (Jiménez-Ramírez et al.
2002). Figure 4 shows images of plant cells produced with
the protocols just described, whereby organelles including
the nucleus, nucleolus and vacuoles can be recognized. By
using a similar approach, i.e., serial sections of samples
prepared for standard transmission electron microscopy
mounted onto glass slides, AFM was used to reconstruct
internal in situ cell structures of mouse embryonic stem cells
(Chen et al. 2005). Mitochondria, nuclei and chromatin were
observed, and a model of mitochondria was constructed. The
Fig. 4  Atomic force micrograph of a semithin section of epoxy resin-
embedded plant cell. a Front and b the projected image of the same
cells. Internal cell structures including the nucleus (N), cytoplasm
(C), vacuoles (V), and nucleolus (arrow) are seen. Scale bar: 10 µm
13
Histochemistry and Cell Biology
authors propose using this methodology for 3-D reconstruc-
tion of cellular structure at the nanoscale.
Another interesting approach used samples of Caeno-
rhabditis elegans and Otodectes cynotis prepared for trans-
mission electron microscopy using high-pressure freezing
and freeze substitution followed by embedding in epoxy
resin, in conjunction with the NTEGRA-Tomo technique.
The NTEGRA-Tomo is an AFM coupled to an ultrami-
crotome, whereby the tip scans the surface of the face of
the freshly trimmed specimen block, instead of using sec-
tions mounted on glass slides (Efimov et al. 2007). This pro-
posed approach to nanotomography measurements and 3D
reconstruction from serial ultrathin sections, has been used
in materials science and has been proposed for the measure-
ment of biological materials (Efimov et al. 2017). Similar
images were obtained by AFM and transmission electron
microscopy. Moreover, the authors combined the ultrami-
crotome capabilities of the microscope scanning to produce
reconstructions by nanotomography from serial sectioning.
Additional work centering on the microscope has been
carried out to study cells and tissues (Mariani et al. 1994).
The microscope included the possibility to scan a wider
area in such a way that human chromosomes can be imaged.
Authors analyzed isolated chromosomes and the effect of
trypsin during G-banding, concluding that chromatin col-
lapses during digestion. Moreover, the authors also men-
tioned that the microscope is capable of imaging cell struc-
ture using carrot cells to demonstrate images of the nucleus
and microtubules.
Other work has focused on using AFM to extract mor-
phological details from the block face of epoxy embedded
biopolymers. In this method, ethanol treatment of the block
face of the sample after microtomy elutes non-cross-linked
polymer chains and allows the smallest details of the embed-
ded biomaterial amenable to detection. AFM (height and
phase contrast) examination of the block face of accordingly
prepared cells of Caenorhabditis elegans provided data com-
parable to transmission electron microscopy (Matsko and
Mueller 2004).
Semithin sections stained with a dye such as toluidine
blue or left unstained yield similar results when imaged by
AFM (Fragoso-Soriano et al. 2009, 2011). By using this
approach, the structure of the cell nucleus revealed nuclear
pores, nucleoli and compact chromatin (Jiménez-García and
Fragoso-Soriano 2000; Jiménez-García and Segura-Valdez
2004; Segura-Valdez et al. 2010). Moreover, by analyzing
the onion (Allium cepa) root meristem by AFM, the different
phases of mitosis have also been observed (Segura-Valdez
et al. 2010).
Tapping mode AFM imaging on semithin sections of
Hep2 cells resulted in observation of interchromatin granule
clusters within the cell nucleus, increasing the possibility of
analyzing ribonucleoproteins involved in gene expression
Histochemistry and Cell Biology
in situ by AFM (Zamora-Cura and Jiménez-García 2014).
Figure  5 shows AFM imaging of cultured HeLa cells,
whereby the nucleus, nucleolus and cytoplasm are seen at
low magnification. In addition to visualization of the cells,
a topographic height profile from different regions of the
cell was obtained.
Recent approaches combining cryosectioning and AFM
have been used that produced high-resolution results. These
studies combine tissue cryosectioning with non-destructive
AFM imaging for development of a methodology to both
preserve and visualize bio-molecular structures (in par-
ticular extracellular matrix assemblies) in situ. This tissue
section AFM technique is capable of: (1) resolving nm–µm
Fig. 5  Atomic force micrograph of a semithin section of epoxy resin
embedded HeLa cells. a Front and the corresponding projected image
(b) of the same cells. Nuclei (N), nucleolus (n), compact chromatin
(arrow), and cytoplasm (C) are identifiable. Scale bar: 10 µm
scale features of intra- and extra-cellular structures in tissue
cryosections; (2) imaging the same tissue region before and
after experimental interventions; and (3) combining ultra-
structural imaging with complimentary microscopical and
micromechanical methods. These types of work allow the
visualization of macro-molecular structures of unstained and
unfixed fibrillar collagens (in skin, cartilage and interverte-
bral disc), elastic fibers (in aorta and lung), desmosomes (in
nasal epithelium) and mitochondria (in heart), to quantify
the ultrastructural effects of sequential collagenase digestion
on a single elastic fiber, and to correlate optical (autofluo-
rescent) with ultrastructural (AFM) images of aortic elastic
lamellae. The technique involves the immobilization of tis-
sue samples by freezing in an aqueous embedding medium
prior to cryosectioning. Freshly cut 5-µm-thick sections were
adsorbed directly onto 13-mm-diameter glass coverslips,
allowed to thaw, air-dried, washed with water to remove
the support medium, and air-dried again prior to imaging
(Graham et al. 2010). A very interesting recent technical
approach uses an “unroofing method”, which amounts to
breaking the cell membrane, to visualize the cell interior.
When the cell membrane is broken, the intracellular region
is exposed by removing the cytoplasmic soluble compo-
nents, which allows the AFM cantilever to access intracel-
lular structures such as the cytoskeleton and organelles.
This preparation technique, which uses sonication, was
originally developed to observe membrane undercoats in
freeze-etching electron microscopy. Although rupture of the
apical cell membrane is caused by the collision of micro-air
bubbles (cavitation) induced by sonication, the specimen
manipulation for this technique requires meticulous care to
prevent the detachment of whole cells from the coverslips.
This study presents a new unroofing apparatus consisting
of an improved sonicator and light stereomicroscope that
improves reproducibility and facilitates sample handling.
The improved (custom-made) sonicator is able to generate
cavitation at a much lower power (0.3–1 W) than that of the
commonly used sonicator (50 W or higher). The authors
used this method for the AFM analysis of the intracellu-
lar cytoskeleton in phosphate-buffered saline at a molecu-
lar resolution. Furthermore, to improve the signal-to-noise
(S/N) ratio and thereby refine the images obtained with the
AFM, serial-scanned images of a target organelle were aver-
aged using mathematical calculations. This method allowed
the AFM cantilever to reach directly into a cell to visualize
the intracellular cytoskeletal actin filaments, microtubules,
clathrin coats, and caveolae at a higher resolution than that
afforded by conventional electron microscopy. Thus, the
unroofing approach allowed intracellular AFM imaging in a
liquid environment with a level of quality equivalent or supe-
rior to that of transmission electron microscopy (Usukura
et al. 2016). Other internal cellular structures such as the
nucleus, however, were not observed. Similar studies were
13

developed to observe intracellular structures combining
immunocytochemistry techniques with the AFM in tissues.
A recent study focused on cryosections prepared using a
method in which samples are fixed slightly with glutaral-
dehyde and embedded in sucrose. Sucrose was expected to
dissolve easily from the sample when soaked in buffer. This
technique was originally developed for effective immunola-
beling rather than ultrastructural analysis. The authors men-
tion that immunocytochemistry is also possible for cryosec-
tioned tissues and can be observed by using AFM combined
with confocal microscopy (Usukura et al. 2017).
Conclusion and perspectives
In this brief review, we have discussed efforts to analyze the
cellular internal structure by AFM in situ in both isolated
cells and tissues. In addition, organelles such as the Golgi
apparatus, mitochondria and cytoskeleton have been ana-
lyzed after isolation from a cell. For example, AFM imaging
of fractions of isolated Golgi membranes stably attached
onto an APTES-mica surface under quasi-native conditions,
revealed the asymmetrical structure of the Golgi apparatus
membranes. The isolated Golgi apparatus membrane frac-
tions were first confirmed by Western blot analysis using an
anti-beta-1,4-galactosyltransferase antibody. In addition, to
further verify the existence of the Golgi apparatus fractions
in isolated samples, the samples were treated with Golgi-
tracker Red (a specific fluorescent dye of the Golgi complex)
and imaged by fluorescence microscopy. The basic structure
of the Golgi apparatus - the Golgi stack, single Golgi cis-
terna, and associated tubules and vesicles were visualized.
AFM images suggest that the Golgi outer leaflet membrane
is smooth, while the inner leaflet membrane is rough with
dense proteins (Xu et al. 2013). Other studies have evalu-
ated heart mitochondria by AFM to estimate the degree of
myocardial injury (Lee et al. 2013). In this study, the authors
characterized the nanostructural changes in mitochondria
resulting from myocardial ischemia–reperfusion (IR), injury
and examined the relationship between the degree of swell-
ing and severity of injury by AFM topographic imaging.
They also simultaneously investigated the nano-mechani-
cal changes in rat heart mitochondria caused by IR using
force–distance curve measurements. The mitochondrial
solution was diluted with adsorption buffer and dropped
onto a fresh mica surface. The prepared samples were briefly
air-dried at room temperature and immediately imaged by
non-contact AFM. The latest development in AFM, i.e.,
the high-speed atomic force microscopy, has been used to
analyze molecular movements, such as ATPase, providing
new approaches for structural and dynamic studies at the
nanoscale level. High-speed AFM employs tapping mode
with a cantilever oscillating near its resonant frequency. The
13
Histochemistry and Cell Biology
system optimizes scanning at high speed and includes a new
feedback control technique. Therefore, very high resolution
is achieved in tandem with detection of very rapid nanoscale
phenomena (Ando 2014). In conclusion, although cell prop-
erties in whole cells can be studied using high-speed AFM,
in situ analysis of internal cell structures at nanoscale repre-
sents another approach that may be compatible with detec-
tion systems at high speed.
Acknowledgements  Support of PAPIIT-DGAPA-UNAM IN217917
and PAPIME PE213916 is acknowledged.
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