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Novel approaches for Scanning Near Field Optical Microscopy
Imaging of Oligodendrocytes in culture
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Trevisan E., ²Fabbretti E., ³Medic N.,4Troian B., 4Prato S., ³Vita F., ²Nistri A., ³Zabucchi G. and
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Zweyer M.
Clinical Department of Biomedicine, University of Trieste, Trieste, 34138
²Neurobiology Sector, SISSA, Trieste, 34014
³Department of Life Science, University of Trieste, Trieste, 34127
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A.P.E. Research S.r.l., Area Science Park, Basovizza, Trieste, 34012
Correspondence:
Marina Zweyer, Associate Professor
Clinical Department of Biomedicine
University of Trieste
Via Manzoni 16
Trieste, 34138
Phone: 00390405586010
Fax: 00390405586016
Email: zweyer@units.it
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ABSTRACT
Newborn rat oligodendrocyte cultures were investigated by Scanning Near Field Optical Microscope
(SNOM), a versatile new tool to map cell membranes in 3 D and to simultaneously obtain cytoplasm images.
Topography/3D, error, transmission and reflection signals were acquired to describe cell morphology with
nanometer-scale resolution. Oligodendrocytes were studied as a model because their extensive membrane
processes (typical of their physiological role in myelination) made them particularly suitable to test the
sensitivity of the new method. Furthermore, we combined a classical histochemical method with SNOM, to
identify specific intracellular proteins at high definition. In particular, with this technique, cytoskeleton
elements of olygodendrocytes, such as microtubules, were observed with tubulin antibodies. Images obtained
with SNOM were also compared with those from conventional scanning electron microscopy (SEM) and
optical microscopy. Our results showed that SNOM allowed observing cell nano-structures otherwise
undetectable with other microscopy methods. In conclusion, SNOM, combined with rapid and non-invasive
methods of specimen preparation, appears to be a powerful tool that can offer new possibilities in the field of
neuroscience imaging at nano-scale level.
Keywords: scanning near-field optical microscopy (SNOM); oligodendrocytes; scanning electron
microscopy (SEM); microtubules.
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INTRODUCTION
Exploration of cell surface at nanoscopic level is an important issue of cell biology, since the
morphology of cell membrane and its molecules distribution may provide important clues to cell physiology
and pathology. Traditionally, conventional electron microscopy techniques, like scanning electron
microscopy (SEM), are currently considered the best tools to investigate cell surfaces. The major limit of this
approach is, however, due to invasive protocols like strong dehydration methods and metal-coating of
biological specimens. These procedures may disrupt fragile structures and nano-scale membrane details.
Furthermore, SEM analysis does not allow detection and localisation of specific structures using
immunolabelling approaches, and cannot, in any case, provide information on the cell interior. Confocal
scanning microscopy has been developed in the last years providing high-resolution optical images useful for
cell research. However, as for all optical conventional microscopes, the resolution power of confocal
microscope is limited by the diffraction limit (De Lange et al., 2001).
Several advanced non-conventional microscopy techniques like scanning near-field optical microscopy
(SNOM) and atomic force microscopy (AFM) have been recently developed and might be used to explore
the surface of biological membranes at high resolution. These novel instruments should supply important
details of cell topography (Rasmussen et al., 2005). SNOM in particular is highly attractive because it
combines scanning probe techniques (SPM) with optical microscopy, providing a lateral spatial resolution
beyond the traditional diffraction limit using a point light source coming from a subwavelength-aperture (De
Serio et al., 2003; Rasmussen et al., 2005). SNOM imaging may, thus, offer a unique technology to
simultaneously map cell surface topography and investigate the local optical properties (near-field) of the
inner cell structures. In the last decade, various studies have been carried out with SNOM analysis of cell
surfaces, using fluorescent or non-fluorescence samples (De Lange et al., 2001; Höppener et al., 2005;
Enderle et al., 1997; Enderle et al., 1998; Nagy et al., 1999; Rieti et al., 2004; Ianoul et al., 2004 and Ianoul
et al., 2005) or using thin tissue sections (Zweyer et al., 2008). Hence, SNOM may represent a synergistic
link between the numerous micro- and nano-scale tools available today and is potentially helpful to obtain
novel details of cell membrane surfaces involved in various processes of cell biology (Lewis et al., 2003).
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In the present study we have analysed, with different microscopy approaches, membrane and intracellular
details of rat oligodendrocytes (ODC) in culture. Samples were analysed with electron microscopy and
fluorescence to validate the SNOM resolution level and the quality of the information acquired.
Oligodendrocytes are glial cells that develop thin, highly specialised cell membranes (myelin sheaths)
surrounding neuronal processes. They have a fundamental role in central nervous system physiology, as
documented by severe phenotypes of disease caused by their loss, like multiple sclerosis (Wood and Bunge,
1984; Mastronardi and Moscatello, 2005). In vitro differentiated ODC are useful to characterize their
peculiar, thin membrane extensions (Barry, 1996; Wood and Bunge, 1984).
In our study different acquisition signals of SNOM (like topography/3D, error, transmission and reflection)
were compared to obtain original observations of cell surface topography and sub-membrane structures of
ODC with resolution below the conventional diffraction limit.
Importantly, using specific antibodies-based immunolabelling and diaminobenzidine staining, we originally
developed a novel, technically-feasible method to explore (at nano-scale level) specific cellular elements
(microtubules) with SNOM. In conclusion, this study is the first comparative analysis of ODC cells in
culture with SNOM.
MATERIALS AND METHODS
Animals
All experimental procedures involving the use of rats were reviewed and approved by the Trieste University
Animal Care and Use Committee. All animals were housed under specific pathogen-free conditions with
light–dark cycles, fed standard animal food and given free access to autoclaved distilled water from bottles.
Cell Culture
Oligodendrocyte primary cultures were obtained from neonatal rat brain. After decapitation of neonates (P0
or P1) telencephala were dissected and the meninges removed. This first step permitted to obtain a mixed
primary culture (Barbarese et al., 1991). Pooled tissue was treated with trypsine solution and cells were
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separated from debris on 3% BSA cushion. The cells were growth in complete DMEM (containing 10% FCS
and 1% penicillin/streptomycin) on poly-D-lysine (0.025%, Sigma Chemical Co, St. Louis, MO, USA)
coated flasks for two weeks as mixed culture of microglia, oligodendrocytes, astrocytes, fibroblasts and
neurons. After this period, we performed purification of oligodendrocytes. Hence, cells (5 x 10 4) were
seeded into 24-well flat-bottom plates coated with poly-ornithine (50µg/ml, Sigma Chemical Co, St. Louis,
MO, US). The cells were cultured in N2 modified medium (100 ml of DMEM, 11 mg of pyruvate, 5 mg
transferrin, 1 ml 4% BSA, 100 μl sodium selenite (0.28 mg/ml), 100 μl insulin (5 mg/ml), 100 μl biotin (0.1
mg/ml), 100 μl triiodo-L-thyronine sodium salt (10 μg/ml), 100 μl penicillin/ streptomycin and 100 μl
hydrocortisone (10 μM). Oligodendrocytes were differentiated in 12-14 days. To inhibit microtubule
polymerization, cells were treated in culture with colchicine (10-6M, Sigma Chemical Co, St. Louis, MO,
USA) for 60 minutes.
Optical microscopy
Surface membrane staining was obtained with in vivo labelling of ODC culture with wheat germ agglutinin
(WGA) AlexaFluor488-conjugated (1:200, Invitrogen, S.Giuliano Milanese, Italy) for 15 min at 37C. After
paraformaldehyde (PFA 4%, Sigma Chemical Co, St. Louis, MO, USA) fixation, specimens were observed
with immunofluorescence microscope (Axio Scope, Zeiss, Germany). Image analysis was obtained with
MetaMorph software (Molecular Devices, Downingtown, PA).
Scanning Electron Microscopy (SEM)
Briefly, cells were dehydrated in a graded 15 to 100% ethanol series, dried in a CO 2 apparatus at critical
point (Bal-Tec; EM Technology and Application, Furstentum, Liechtenstein), sputter coated with gold in a
Edwards S150A apparatus (Edwards High Vacuum, Crawley, West Sussex, UK), and examined with a Leica
Stereoscan 430i scanning electron microscope (Leica Cambridge Ltd., Cambridge, United Kingdom).
Specimens preparation for SNOM or SNOM-DAB
For SNOM analysis, cells grown on glass coverslips were fixed with 4% PFA in phosphate buffered saline
(PBS) for 10 min at room temperature, washed three times in PBS, 2 times in water, partially dehydrated (up
to 70%) and allowed to dry. For the SNOM-DAB method, after fixation, cells were immunolabelled with a
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rat monoclonal antibody against α-tubulin (Yol-34, AbD Serotec, Germany). Immunoreaction was visualized
using a secondary antibody anti-rat biotin-conjugated (Dako, Glostrup, Denmark), amplified with ABC kit
(Vector
Laboratories,
Burlingame,
CA)
and
finally
detected
with
3-3,3-diaminobenzidine
tethrahydrochloride (DAB). DAB labelling Kit (Vector) was utilized according to manufacturer instructions.
Scanning Near Field Optical Microscopy (SNOM)
For topographic characterization and near-field studies cellular samples were analysed using a TriA-SNOM
microscope (A.P.E. Research, Trieste, Italy), equipped with a flexure scanning stage with a maximum xyz
scan range of 100µm x 100µm x 10µm (high voltage mode), with a strain gauge sensors to provide an
absolute positioning of the probe. The TriA-SNOM set-up was provided with interchangeable laser sources,
coupled with a single optical fibre. In this work a laser wavelength of 473 nm was used because it was
optimal to emphasize the contrast of the sample treated with DAB (Gordon, 1988). SNOM optical reflection
and optical transmission signals were acquired simultaneously together with topography and detected with
two photomultipliers (type R74000, Hamamatsu Photonics Italia S.r.l., Milano, Italy) with narrow band-pass
interference filters at 473nm wavelength (full width-half maximum = 10nm ± 2nm). SNOM probe was an
aluminium-coated tapered pulled optical fiber with a tip aperture of 50 nm (Lovalite, Troyes, France).
Two optical vision systems were integrated within the SNOM head to control the probe position and to select
the scan area. An upper optical vision system was used to monitor probe approach to the sample, while one
transmission camera (with interchangeable achromatic objectives) was utilized for a bottom view of the
sample and to select the scan area.
Even if SNOM-based imaging does not require staining procedure, some cell specimens prepared for SNOM
observations were immuno-stained with DAB.
Data analysis
SNOM images were processed using A.P.E. Research SPM control software (A.P.E. Research, Trieste,
Italy), SPIP (Image Metrology, Denmark), and Gwyddion (free software).
RESULTS
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Different SNOM signals and related imaging (move to methods??there are no results here)
TriA-SNOM enabled us to acquire different kinds of signal simultaneously.
a) SNOM topography. The image obtained is the result of acquisition of signals in xyz axis that allows to
measure detailed surface morphology and nanostructures on a nanometric scale.
b) Error: it is due to the residual signals derived from the feedback circuit. It can be used as a complement of
the topography and 3D signals to emphasise graphically the borders and some structures on the sample
surface.
c) 3D topography: it is an accurate three-dimensional image of the sample. Unlike conventional microscopy
(2D techniques), SNOM produces real 3D images (like for all scanning probe microscopies) since z data are
directly measured point by point on the vertical axis.
d) SNOM optical transmission: the image results from the light transmitted through the whole thickness of
the sample. While in conventional optical microscope all the sample is illuminated, SNOM near-field light
interacts only locally producing signals point by point. SNOM optical transmission images are comparable
with conventional optical images but their lateral resolution is more than 10 times higher.
e) SNOM optical reflection: image is obtained with near-field light that interacts locally with a superficial
layer of the sample, giving information on structures confined within 30-100 nm (depending on the tip
aperture) below sample surface.
SNOM detection of cells in culture
Oligodendrocytes have a fundamental role in central nervous system since they cover neuronal processes
providing efficient and fast conduction of electrical signals (Wood and Bunge 1984). Primary ODC from
young rat brain differentiate in vitro by emitting thin and flat end-membrane processes (Barry 1996; Wood
and Bunge 1984). These features render these cells very suitable for validating the SNOM capabilities to
analyze biological samples. We have studied primary cell cultures of ODC to compare the images obtained
with SNOM with other microscopies, as SEM and fluorescence (Figure 1). There is no independent
demonstration (e.g. specific markers) that these cells are ODC and only ODC. SEM analysis of ODC (n= 50
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cells in at least 3 different preparation) revealed a large, flat cell surface (Figure 1A) without detectable
humps related to underlying intracellular organelles. Another way to observe cell surface was performed by
fluorescence labelling. In this case, glycoconjugates of the membrane outer side were identified by wheat
germ agglutinin (WGA) with conventional optical microscopy, but the image did not provide any particular
structural details (Figure 1B).
With SNOM, ODC images (n= 150 cells in 30 preparations) were analysed for error, topography and 3D,
respectively (Figure 1 C, D). These data clearly revealed specific surface details connected with nucleus,
nucleolus and organelles (see arrows, Figure 1). Additional information was also simultaneously obtained by
optical signals from the same area of investigation (Figure 1). These images demonstrated that intracellular
elements of ODC lie in correspondence with cells surface humps.
ODC observed with SNOM, indeed, showed flat expansions at the end of their long, slender processes and
the large lamellipodia-like structures sloping down from their lateral edge, confirming previous published
data (Etienne-Manneville, 2004). We, thus, decided to acquire SNOM images at higher magnification, to
explore the potential resolution of near-field technique comparing SNOM with SEM microscopy. The
morphology of ODC was similar in images acquired with both instruments (see Figure 2). Using SNOM
acquisition, we obtained important information of the Z axis of samples (Figure 2B, C, left panel).
Interestingly, at high resolution, we observed particular structures emerging from ODC membranes at
terminal endings with roundish shape of 100-150 nm diameter (Figure 2B, C, right panel). Figure 2 unclearly
labelled.
In conclusion the signals observed with SNOM provided accurate details like SEM images, despite intrinsic
differences of the two technologies. In comparison with fluorescence, SNOM demonstrates the high
performance of near field optical microscopy.
SNOM-based acquisition of cytoskeleton structures
Cell SNOM signals suggested the presence of long filamentous intracellular structures supporting the surface
membrane, and resembling cytoskeleton elements (see Figure 1). These observations suggested exploring the
cell cytoskeleton using SNOM. For this purpose, we analysed ODC specimens treated in culture with
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colchicine (10-6M, 60 min), a drug known to inhibit microtubule polymerization and cytoskeleton
arrangement (Figure 3). Conventional SEM analysis used as control, demonstrated the colchicine-induced
wide retraction of cytoskeleton processes and the dramatic reduction in the flat cell membranes sheet of
ODC with respect to untreated cells (Figure 3A, n = 48). Analogous experiments performed with SNOM
allowed us to observe similar changes in topography (Figure 3B) and in reflection (Figure 3C). The
topographic profiles (obtained along a line scan through the image) illustrated the effective cell extensions
along the Z dimension (Figure 3B). Profiles obtained from a cross section of a process confirmed the strong
retraction of the mantles after colchicine treatment (Figure 3C, left) with respect to normal morphology
(Figure 3C, right). Interestingly, peculiar inner cell structures were detected in optical reflection only (Figure
3C) and were particularly evident especially after cytoskeleton disruption (Figure 3C, rigth).
The use of colchicine allowed us to demonstrate that SNOM topographic and reflection signals provided
complementary information.
SNOM-DAB, a novel method of nanoscopic investigation
In order to better distinguish intracellular elements and, at the same time, to evaluate the capability and
versatility of SNOM, ODC were analysed with SNOM after immunolabelling with a monoclonal antibody
against -tubulin followed by DAB staining (SNOM-DAB). This approach allowed the nanoscopic detection
of ODC immunolabelled microtubules. This tool was indeed useful since SNOM optical transmission and
reflection signals were coupled to detect specific ODC elements, located below the cell surface (Figure 4B,
C). SNOM-DAB staining of ODC showed a homogeneous tubulin distribution (Figure 4, left panels) in
accordance with previous data (Wilson and Brophy, 1989). Topography allowed us to control the effect of
colchicine on microtubule polymerization (see Figure 4 A). Line scan profiles (Figure 4 A) and optical
images (Figure 4B, C) clearly showed the different distribution of microtubule bundles in control and in
colchicine treated cells with dramatic disorganization of cytoskeleton elements (Figure 4, right panels).
When we analyzed the ODC flat enlarged terminal expansions, a certain number of tubulin bundles
remained, perhaps, resistant to colchicine treatment, and were found particularly in the periphery of the flat
endings (Figure A, right profile and Figure B and C, right panels). In conclusion, these results indicated that
the SNOM-DAB technique was an efficient method to identify/localize inner cell structures both in
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transmission and reflection, increasing the image contrast. Furthermore, SNOM-DAB appeared to be a novel
and versatile method to simultaneously study of cell-surface and intracellular nanostructural details.
Untreated ODC labelled with DAB technique for α tubulins in topography, reflection and transmission
signals.
With the SNOM-DAB approach, the simultaneous acquisition of topographical and optical (transmission and
reflection) signals and their comparison, allowed us to better characterize the origin and significance of the
signals acquired with this instrument. In particular, we compared surface structures with their relative
underlining structures obtained in transmission and reflection. Thus, we used SNOM-DAB technique to
evaluate the spatial position of labelled tubulin elements below the cell surface and the corresponding
membrane humps (Figure 5). We have analysed in transmission certain structures (Figure 5A, transmission),
corresponding to long, thin crests in 3D-topography (see rectangle in Figure 5A, 3D-topography).
We expect to see that DAB labelled structures appear dark, that is: the dark area should correspond to greater
absorbance (optical density), while clear area should correspond to less absorbance. Actually we can see in
transmission a dark cord-shaped structure, probably related to immunolabelled microtubules (see arrows
Figure 5A). The same details observed in reflection (Figure 5A) exhibit a white zone in correspondence of
that overmentioned dark one. This should testify that microtubules are not present in the layer immediately
underlying the membrane, but rather in depth (Figure 5C, model). These data demonstrated that transmission
and reflection signals of the same area may generate different and additional information. This should be a
further proof of the specificity of reflection in the view of the SNOM capabilities. This paragraph is
confused and unclear.
Moreover, when we analysed at higher magnification the specimen, we identified many details (see arrows
Figure 5B, optical reflection) beyond the conventional diffraction limit. This is a peculiar aspect of scanning
near field optical microscopy.
DISCUSSION
Oligodendrocytes (ODC) are non neuronal cells characterized by extended cell membrane processes which
allow myelination of neuronal axons in the central nervous system. This intrinsic characteristic of mature
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ODC is clearly linked to drastic changes of cytoskeleton rearrangement during cell process-membranes
growth. Indeed, microtubules are essential for architectural support and stability of cell membranes (Lunn, et
al., 1997; Richter-Landsberg, 2001) and act as a path for the transport of materials necessary for the
assembly and maintenance of the myelin sheaths (Bizzozero et at., 1982; Song et al., 1999). It has also been
demonstrated that the maintenance of myelin membrane sheaths requires continuous microtubules turnover
(Benjamins et al., 1994). Morphological studies have, in fact, shown that growing ODC processes contain
dense arrays of microtubules, tightly coalesced into continuous bundles (Wilson et al., 1989).
Thus, the ODC cytoskeleton seems highly necessary for their function and survival and, hence, for the
optimal interaction with neurons. Pharmacological inhibition of cytoskeleton polymerisation with drugs
strongly affect ODC morpho-physiology with active and fast demyelination (Lunn, et al., 1997). SNOM is
the most complete tool among scanning probe and electron microscopies, because is the only instrument
capable to map the surface and simultaneously the objects below. Out of context and repeat.
In our work we explored the ODC cell membrane and its underlying microtubules with the novel
non-conventional microscopy technique SNOM. To this aim, we set up a new method (namely SNOMDAB) to visualise peculiar elements of the ODC cytoskeleton, using α-tubulin antibodies and DAB-based
immunocytochemistry technique. This method, originally introduced for cytochemical localization of the
endogenous enzyme horseradish peroxidase (Graham et al., 1966), has been subsequently utilized in
combination with immunolabeling to identify virtually every proteins of the cell both in optical and in
electron microscopy (Essner E., 1974, Powley et al., 2007; Deng et al., 2006, Schikorski et al., 2007). The
present study developed, for the first time, the SNOM-DAB method and optimised SNOM optical set up
(laser source and filters) to increase the DAB reaction product contrast. This technique allowed us to label
cell antigens with standard SNOM equipment without resorting to fluorescence.
Thus, the study of these aspects of ODC is very important to understand the physiology of these cells and
their abnormalities in the disease.Out of context and there is no physiological investigation
Unlike other conventional microscopies, we observed ODC membrane and submembrane nanostructures
with different kinds of signal. Simultaneous sample acquisition of topography, error, transmission and
reflection images demonstrated the versatility of SNOM. Topography may compete with conventional SEM
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imaging and exceed its resolution in the Z axis, because of its direct-measurement. Reflection has been
described up to now mainly for non-biological materials (Kerimo et al., 2000, Cline et al., 1991; Durkan et
al., 1995, Wei et al.,1995 random order). Our study provided a novel description of the difference between
signals originated by reflection and those by transmission on the same cells. Our data suggest that this
method could give us new, so far unavailable data about the region 30-100 nm below the cell surface, with
particular reference to the presence (or the absence) of cytoskeleton structures. We could in fact identify and
localize specific intracellular antigens and correlate them, at nano-scale level, with the 3D structure of the
cell membrane. Despite the intrinsic difficulties in image interpretation which will certainly need future
investigations unclear meaning, we posit the relevance of this technique to disclose details of the space
immediately below the cell surface, where important cell activities occur like, for example, membrane
turnover, endo/exocytosis (Winterstein et al., 2008; Trajkovic et al., 2006), Ca2+ and protein translocation
(Mastronardi et al., 2006), membrane-cytoskeleton dynamics (Dyer, 1991) or vesciculation (Kramer-Albers,
2007).
Segregation/identification of the cell molecules is indeed one of the most important features to understand
cell physiology in response to environmental stimuli. A deeper knowledge and characterisation of the
molecules and/or organelles located or not located in this space appear an interesting employment of SNOM
for its use in basic cell biology applied to many aspects of biomedicine. So, the nanostructural study of the
ODC and its cytoskeleton is very important for monitoring the morpho-functional state of these cells in
normal conditions and following drug interaction.repeat
In conclusion, we used SNOM imaging to analyse, at high magnification, the morphology of cell membranes
and the microtubules distribution of rat ODC in culture. The use of SNOM avoided invasive procedures
required for SEM analysis such as total dehydration, critical point treatment and gold metal coating. Methods
in scanning near field optical microscopy are versatile and innovative and should be exploited for the study
and interpretation of biological processes in neuroscience.
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Figure 1. ODC cells observed with SEM or SNOM. A Image realized by SEM analysis of ODC at low
magnification. The overall cell surface is flat and there are not present any relieves in correspondence of
underling nucleus and/or organelles. Bar 10 µm. B Image obtained with in vivo WGA fluorescent labelling
of the glyco-components located on the outer side of the membrane, analysed with fluorescence optical
microscopy. C and D, Images derived from simultaneous acquisition of SNOM signals representative of the
same cell scan area. Specimens are not stained. SNOM topography (C, topography on the left and 3D
topography on the right) and error (D, left panel) reveal surface details ascribable to nuclear (N), subnuclear
structures (Nu) and probably cytoskeleton (C). The image obtained with SNOM transmission is comparable
with images derived from conventional optical microscopes (C, right panel). Bar 20 µm.
Figure 2. Details of ODC cells observed with SEM or SNOM. Example of different images acquired with
SEM and SNOM at two different magnifications. A, image obtained with SEM analysis of a flat terminal
membrane expansion of ODC. Bars=10 µm and 2 µm, respectively. B, SNOM topography of ODC measured
in z axis. The Z axis measurements reveal large surface height differences in ODC: they are shown in
pseudocolors (relative pseudocolor scale bar, in nm). Bars = 10 µm and 4 µm, respectively. C, Images
obtained with SNOM error signals of ODC analysis. Note the large resolution of ODC details acquired with
SNOM respect to SEM. Bar 10 µm and 4 µm, respectively.
Figure 3. Morphology of ODC depend by intracellular colchicine-sensitive structures. A, images
obtained after SEM observation of ODC cells in control condition (left) and after pre-treatment with
colchicine (10-6M, 60 min; right). Note different overall surfaces of cell membranes in control conditions,
while colchicine treatment exhibit a dramatic reduction of membrane veils. B, At higher magnification,
SNOM observation of ODC in control condition and after colchicine treatment. Pseudo-colour topography
analysis and linear scan cross-section profiles (along the white line) provided quantitative information
(measured in nm) of Z axis of selected regions. The profiles shouded a higher volume of intracellular
components respect the colchicine-treated cell. C, images obtained from SNOM reflection signals of the
same area of B. Bar 10 µm.
Figure 4. Analysis of DAB-stained ODC with SNOM. Images obtained with SNOM analysis of ODC in
control condition (A, B, C) and after colchicine (10-6M, 60 min) treatment (A, B, C right) in topography,
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optical reflection and transmission. ODC were immuno-labelled with a monoclonal antibody against αtubulin and stained with DAB. A, Pseudo-colour topography analysis and linear scan cross-section profiles
(along the white lines) provide quantitative information (measured in nm) of Z axis of selected regions. Note
that higher volume regions correspond to DAB-immunostained tubulin, that sustain cellular membranes and
are highly reduced after colchicine treatment. Bar 10 µm and 20 µm.
Figure 5. Nanoscopy of ODC microtubules with SNOM-DAB approach. A SNOM 3D-topography,
reflection and transmission of ODC labelled with DAB. In these panels the rectangles evidence the same
interesting area. The arrows indicate in transmission a dark cord-shaped structure (greater absorbance)
probably related to tubulins, while in reflection the correspondent zone appears clear (less absorbance). B,
SNOM topography and reflection at higher magnification: we can detect, in the optical image, many details
beyond the conventional diffraction limit (see arrows). C, Proposed model of section perpendicular to the
surface to explain the phenomenon described in A.
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FIGURE 1
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FIGURE 2
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FIGURE 3
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FIGURE 4
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FIGURE 5