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Observations of the intestinal mucosa using environmental scanning
electron microscopy (ESEM); comparison with conventional scanning
electron microscopy (CSEM)
Caroline Habold, Suzanne Dunel-Erb, Claudine Chevalier, Pierre Laurent, Yvon Le Maho,Jean-Herve Lignot*
Centre National de la Recherche Scientifique, Centre d’Ecologie et de Physiologie Energetiques, 23 rue Becquerel, F-67087 Strasbourg cedex 2, France
Received 23 June 2003; revised 23 June 2003; accepted 26 June 2003
Abstract
In order to evaluate the potential use of environmental scanning electron microscopy (ESEM) in biology, structural changes of the jejunal
villi of rats were studied after periods of fasting and refeeding, using a conventional scanning electron microscope (CSEM) and ESEM. While
observation using the CSEM, involves chemical fixation, drying and coating, observation of fresh, unprepared materials can be directly
realized with the ESEM. Environmental microscopy provides a relatively new technology for imaging hydrated materials without specimen
preparation and conductive coating. Direct observation of biological samples in their native state is therefore possible with an ESEM.
After fasting, the jejunal mucosa is dramatically reduced in size, splits and holes appearing at the tip of the villi. These changes were
observed whatever the type of technique used. Artifacts due to the sample preparation for CSEM observation (drying, coating) can therefore
be excluded. However, CSEM and ESEM must be used jointly. While, CSEM must be preferred for surface analysis involving high
magnifications, ESEM observation, on the other hand, can prove valuable for determining the living aspect of the samples.
q 2003 Elsevier Ltd. All rights reserved.
Keywords: Fasting; Refeeding; Jejunum; ESEM; CSEM
1. Introduction
Biological samples, containing water and exhibiting low
conductivity, cannot be observed directly in a conventional
scanning electron microscope (CSEM) which requires a
high vacuum sample environment. Samples have to be
fixed, dehydrated, dried and coated to be electrically
conductive before they can be observed. The environmen-
tal scanning electron microscope (ESEM), owing to
technical modifications (Danilatos, 1981, 1988), permits
to vary the sample chamber environment through a range
of pressure, temperature and gas composition. Wet, non-
conductive, oily samples may therefore be examined
without preparation. This technology associates two
technical modifications compared to a CSEM: a separation
of the high vacuum column from the low vacuum sample
chamber using pressure limiting apertures, and a new type
of electrical current detector: the patented gaseous
secondary electron detector (GSED). The secondary
electrons emitted by the sample collide with gas molecules
in the chamber. The resulting ionization produces positive
ions and additional secondary electrons. The positive ions
are attracted to the sample surface and suppress the effects
of negative charges on the surface of non-conductive
samples.
Only few ESEM observations have been performed on
biological samples. These include plant material (Danilatos,
1981; Uwins et al., 1993), microorganisms (Collins et al.,
1993), teeth (Habib et al., 1998), and bone (Forster and
Fisher, 1999). More recently, other biological samples have
been tested such as soft animal tissues, microorganisms,
leaving cells (Djano et al., 1999; Martinez-Alvarez et al.,
2000; Tai and Tang, 2001) as well as soft condensed matter
and delicate material (Stokes, 2001). However, a thorough
study using some of the ESEM capabilities, on a whole
organ such as the small intestine subjected to external
conditions (feeding, fasting for example) is still lacking.
0968-4328/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-4328(03)00080-5
Micron 34 (2003) 373–379
www.elsevier.com/locate/micron
* Corresponding author. Tel.: þ33-388-106938; fax: þ33-388-106906.
E-mail address: [email protected] (J.-H. Lignot).
The small intestine shows an extraordinary phenotypic
flexibility, typical of vertebrates living in variable environ-
ments (Piersma and Lindstrom, 1997). The intestinal
mucosa exhibits a tremendous capacity to adapt in response
to a new set of nutritional demands (starvation, fasting,
malnutrition, etc.) (Altmann, 1972; Carey, 1990; Secor et al.,
1994; Nunez et al., 1996). The induced modifications occur
in diverse physiological states and are associated with a
characteristic array of changes in structure and function
(Galluser et al., 1988; Carey, 1992; Secor and Diamond,
1995).
Our field of research deals with the study of the effects of
fasting and refeeding on the jejunal structure and involves
morphological observations, using various microscopy
techniques (Dunel-Erb et al., 2001). The aim of this present
study was to compare observations made on the jejunal
mucosa of rats, under high vacuum conditions (CSEM) and
environmental conditions (ESEM). Possible artifacts due to
sample treatment before observation were determined.
Finally, the performances of both microscopy techniques
for detecting structural modifications occurring in a soft
epithelium such as the intestinal mucosa were assessed and
discussed.
2. Materials and methods
2.1. Animals
Male Wistar rats were purchased from IFFA-CREDO
(L’Arbresle, France) and housed individually in leucite
cages with a wire mesh floor to minimize coprophagia. Rats
were maintained in a temperature-controlled room
(23 ^ 1 8C; humidity 50–60%) under a 12/12 hr light–
dark cycle (light from 8 a.m. to 8 p.m.), and fed ad libitum a
laboratory powdered diet (AO3 from UAR, Epinay-sur-
Orge, France) consisting of 23% (by mass) protein, 51.1%
carbohydrates, 4.3% fat, 4% cellulose, 5.6% minerals, and
12% water. The rats had free access to water throughout the
experiments.
Our experimental protocol followed the CNRS (Centre
National de la Recherche Scientifique) guide for care and
use of laboratory animals.
2.2. Experimental procedures
When body mass of rats averaged 350 g, rats were either
sacrificed as control animals or fasted for a determined
period of time. After 5 days of fasting, a group of rats was
sacrificed. Others were provided with food during 2, 6, or
24 h and then sacrificed. Another group of rats continued
fasting until 8–10 days when the daily rate of body mass
loss increased rapidly.
Five rats of each group were sacrificed between 9 and 10
a.m. and proximal segment of their jejunum was removed.
2.3. Microscopy techniques
The jejunal segment was cut longitudinally and pinned
flat in a box coated with paraffin wax. The mucosal surface
facing upward, was gently cleaned and was then cut in small
pieces (about 5 mm). Samples, either placed fresh in a drop
of saline or plunged in 1% glutaraldehyde in buffered saline
for 30 s (for blocking mucous secretion) were viewed in an
environmental mode with a Philips XL30 ESEM (FEI
Company) equipped with a lanthanum hexaboride (LaB6)
electron gun and a GSED. The pressure chamber and the
temperature of the specimen were regulated to maintain a
relative humidity from 90 to 75%. A Peltier-cooled
specimen stage allowed to regulate specimen temperature.
A pressure of 4–5 Torr and a temperature of 4 8C were the
optimal conditions to maintain a relative humidity of 85%.
For CSEM, pieces of tissues from the same jejunal
portion were fixed for 2 h at 4 8C in 5% glutaraldehyde in
0.05 M cacodylate buffer at pH 7.4, rinsed in cacodylate
buffer, postfixed in 1% osmium tetroxide, and then
dehydrated in a series of increasing alcohol baths.
Dehydrated samples were air-dried following two baths of
1,1,1,3,3,3-hexamethyldisilazan (Aldrich), glued on speci-
men stubs with carbon adhesive tabs or with silver paint,
gold coated (Edwards Sputter Coater), and observed with
the Philips XL30 ESEM used in high vacuum mode with the
Thornley-Everhart secondary detector.
Some of the ESEM and CSEM numerized micrographs
were enriched by using a differential hysteresis image-
processing software (Lucise, Image Content Technology
LLC, New Britain, CT).
3. Results
Figs. 1–14 correspond to rat jejunal samples and show
intestinal villi either observed in a high vacuum mode
(Figs. 1–6) or in an environmental mode (Figs. 7–14). Villi
are projecting above surface and possess a mucosa made of
absorptive tall columnar cells (enterocytes), goblet cells,
Fig. 1. CSEM micrograph of the rat jejunal mucosa. The specimen was
fixed, dehydrated and coated, and observed under high vacuum conditions
with the SE detector. Control rat (fed); observation at 20 kV.
C. Habold et al. / Micron 34 (2003) 373–379374
and scattered endocrine cells. Only the apical surface of the
enterocytes (covered with microvilli) and goblet cells can be
seen using a scanning electron microscope.
Fixed, dehydrated, and coated jejunal villi of fed rat
observed with CSEM appear large and closely packed
(Figs. 1 and 2). The network of furrows and the apical
grooves are well marked. Fig. 3 shows mucosa of rat that
fasted for 5 days. The tips of the villi are irregular and
folded, often with a deep transversal split clearly visible
(see also Fig. 16). After 24 h of refeeding, the folds have
resumed and the network of furrows reappears (Fig. 4).
Fig. 3. CSEM micrograph of the rat jejunal mucosa. The specimen was
fixed, dehydrated and coated, and observed under high vacuum conditions
with the SE detector. Rat fasted for 5 days; observation at 30 kV. Note the
numerous folds at the tip of the villus. Compare with Figs. 2 and 4.
Fig. 4. CSEM micrograph of the rat jejunal mucosa. The specimen was
fixed, dehydrated and coated, and observed under high vacuum conditions
with the SE detector. Rat refed 24 h after 5 days fasting; observation at
30 kV. Lucise treatment.
Fig. 5. CSEM micrograph of the rat jejunal mucosa. The specimen was
fixed, dehydrated and coated, and observed under high vacuum conditions
with the SE detector. Rat fasted for 10 days; observation at 15 kV. Lucise
treatment. Note the short size of the villi and the spaces between them,
the crypt apertures are visible (arrows).
Fig. 6. CSEM micrograph of the rat jejunal mucosa. The specimen was
fixed, dehydrated and coated, and observed under high vacuum conditions
with the SE detector. Rat fasted for 10 days; observation at 20 kV. Lucise
treatment. Note the split at the tip of the villus (arrows). Compare with Fig. 1.
Fig. 2. CSEM micrograph of the rat jejunal mucosa. The specimen was
fixed, dehydrated and coated, and observed under high vacuum conditions
with the SE detector. Control rat (fed); observation at 15 kV. Lucise
treatment.
Fig. 7. ESEM micrograph of the rat jejunal mucosa. The specimen was
hydrated and uncoated. It was observed at a pressure of 4–5 Torr and a
temperature between 2 and 5 8C with a GSE detector. The relative humidity
was 80–85%. Observations were done at 10–15 kV. Lucise treatment.
Control rat (fed).
C. Habold et al. / Micron 34 (2003) 373–379 375
Fig. 9. ESEM micrograph of the rat jejunal mucosa. The specimen was
hydrated and uncoated. It was observed at a pressure of 4–5 Torr and a
temperature between 2 and 5 8C with a GSE detector. The relative humidity
was 80–85%. Observations were done at 10–15 kV. Lucise treatment. Rat
fasted for 5 days. Note the aspect of the villi that are retracted and folded. A
hole is visible at the tip of a villus (arrow) Mucus is still present between the
villi (asterisk).
Fig. 8. ESEM micrograph of the rat jejunal mucosa. The specimen was
hydrated and uncoated. It was observed at a pressure of 4–5 Torr and a
temperature between 2 and 5 8C with a GSE detector. The relative humidity
was 80–85%. Observations were done at 10–15 kV. Lucise treatment.
Control rat (fed).
Fig. 10. ESEM micrograph of the rat jejunal mucosa. The specimen was
hydrated and uncoated. It was observed at a pressure of 4–5 Torr and a
temperature between 2 and 5 8C with a GSE detector. The relative humidity
was 80–85%. Observations were done at 10–15 kV. Lucise treatment. Rat
fasted for 5 days. Note the aspect of the villi that are retracted and folded. A
hole is visible at the tip of a villus (arrow). Mucus is still present between the
villi (asterisk).
Fig. 11. ESEM micrograph of the rat jejunal mucosa. The specimen was
hydrated and uncoated. It was observed at a pressure of 4–5 Torr and a
temperature between 2 and 5 8C with a GSE detector. The relative humidity
was 80–85%. Observations were done at 10–15 kV. Lucise treatment. Rat
refed 2 h after 5 days fasting.
Fig. 12. ESEM micrograph of the rat jejunal mucosa. The specimen was
hydrated and uncoated. It was observed at pressure of 4–5 Torr and a
temperature between 2 and 5 8C with a GSE detector. The relative
humidity was 80–85%. Observations were done at 10–15 kV. Lucise
treatment. Rat refed 24 h after 5 days fasting. The villi look like the
villi of control rat (Fig. 8).
Fig. 13. ESEM micrograph of the rat jejunal mucosa. The specimen was
hydrated and uncoated. It was observed at a pressure of 4–5 Torr and a
temperature between 2 and 5 8C with a GSE detector. The relative humidity
was 80–85%. Observations were done at 10–15 kV. Lucise treatment. Rat
fasted for 10 days. The villi are short, narrow, and bent; large spaces are
visible at their bases and several crypts open in this space (arrows). Mucus is
still present (asterisk).
C. Habold et al. / Micron 34 (2003) 373–379376
Figs. 5 and 6 show jejunal mucosa of rats that fasted for
about 10 days. The villi are dramatically reduced in size.
Numerous longitudinal folds due to the loss of turgidity
can also be observed on the wall of the villi and a
longitudinal split is generally present at the tip of the
villi (Fig. 6). Between the villi, large spaces are visible
with numerous crypt apertures (Fig. 5).
Figs. 7–14 show ESEM images of fresh jejunal samples
directly observed without long fixation, dehydration and
coating. The flexible villi are often bent and bridges of
mucus are present between the villi. In control animals
(Figs. 7 and 8), the network of furrows is visible but the
ornamentation at the surface is not as visible as on CSEM
micrographs because of the presence of water and mucus
(compared to Figs. 1 and 2). In 5 days fasting individuals
(Figs. 9 and 10), the villi are retracted and present numerous
folds. The surface is irregular with, from time to time, some
holes at the tip of the villi (Fig. 10). Figs. 11 and 12 show
two successive moments after refeeding, 2 and 24 h,
respectively. The villi have straightened back and the
shape of their tips is more regular. After 24 h of refeeding
(Fig. 12), the villi look similar to the controls.
After 10 days of fasting, the villi observed with ESEM are
reduced in size and bent, largely spaced apart with mucus
between them (Fig. 13). The villus tip appears smooth. Only a
longitudinal split, which is already visible with CSEM (Fig.
6) can be observed at the villus tip (Fig. 14).
Comparison between CSEM micrographs and ESEM
micrographs often shows a similarity of structure. For
example, the large transversal splits present across the villus
can either be observed on CSEM (Figs. 15 and 16) or ESEM
(Fig. 17) images. However, some observations at high
magnification can only be possible with CSEM. For
instance, details of the microvilli of the brush border of
the enterocytes (Figs. 18 and 19) cannot be observed with
ESEM because of the presence of water and mucus.
An image-processing software such as Lucise, was also
used in order to optimize image contrast and to enhance
details of some of the numerized images obtained with
ESEM and CSEM. An example of the effect of such a
processing on a poorly contrasted ESEM micrograph is
given and shows enhanced details that would not be visible
otherwise (Fig. 20a and b). Similarly, Fig. 21a and b show
the difference between a non-treated CSEM micrograph
(Fig. 21a) and a processed micrograph (Fig. 21b).
Fig. 14. ESEM micrograph of the rat jejunal mucosa. The specimen was
hydrated and uncoated. It was observed at a pressure of 4–5 Torr and a
temperature between 2 and 5 8C with a GSE detector. The relative humidity
was 80–85%. Observations were done at 10–15 kV. Lucise treatment. Rat
fasted for 10 days. Detail of the villus tip that is smooth with only a
longitudinal split (arrows). Compare with Fig. 6 obtained with CSEM.
Fig. 15. CSEM micrograph of a villus. Two hours of refeeding after 5 days
fasting. The villus shows a large transversal split (arrow).
Fig. 16. CSEM micrograph of a villus. Detail of the split at the villus tip
(arrow). Five days fasting.
Fig. 17. ESEM micrograph of a villus. Six hours of refeeding after 5 days
fasting. Note the large transversal split in the villus still present in ESEM
micrograph (arrow) and showing it is not an artifact in CSEM micrograph
(Fig. 15).
C. Habold et al. / Micron 34 (2003) 373–379 377
4. Discussion
The obvious advantage of the ESEM for imaging
hydrated biological material, is the ability to view delicate
samples without prior preparation and without conductive
coating (Collins et al., 1993). In addition to saving time, the
absence of preparation avoids formation of possible
artifacts. Therefore, by comparing CSEM and ESEM
observations from the same biological samples, it is possible
to distinguish between possible artifacts due to the sample
preparation and the real structural effects of external events
such as food deprivation and refeeding. This is particularly
important when studying a soft tissue like the jejunal
mucosa, continuously renewing its absorptive cells.
The well-described structural changes in the small
intestine resulting from acute food deprivation include
destruction of the villus tips, fissuring of the basal portions
and marked shortening of the villi (Brown et al., 1963). The
tissue, however, is rapidly restored with refeeding (Cameron
and Cleffmann, 1964; Altmann, 1972; Dunel-Erb et al.,
2001).
Our study, along with previous CSEM observations of
the small intestine mucosa, confirms the shrinkage of the
villi during fasting. This phenomenon which could be due to
the sample preparation (dehydration and drying), however,
is not an artifact, as proved by the ESEM observation of the
same samples, but a loss of the villi turgidity due to fastingFig. 19. CSEM micrograph of the brush border of the enterocyte. Rat fasted
for 5 days.
Fig. 21. CSEM micrographs of the rat jejunal mucosa (rat refed 24 h after 5 days fasting). (a) Original micrograph, (b) image processed with Lucise. The
contrast is optimized and more details appear after treatment.
Fig. 20. ESEM micrographs of the rat jejunal mucosa (rat fasted for 5 days). (a) Original micrograph directly from microscope, (b) image treated with Lucise
in order to optimize the contrast.
Fig. 18. CSEM micrograph of the brush border of the enterocyte. Rat refed
24 h after 5 days fasting.
C. Habold et al. / Micron 34 (2003) 373–379378
(compare Fig. 3 with Figs. 9 and 10). Also, the large splits
observed by CSEM either at the villus tips (Fig. 6) or
transversally in the villi of fasting animals (Figs. 15 and 16)
were confirmed by ESEM observations (Figs. 14 and 17).
While the villi observed in a high vacuum mode were
straight and erected, they appeared flexible, bent, with
bridges of mucus and in a more natural state, when observed
in a low vacuum mode.
However, for samples such as intestinal mucosa whose
surfaces are covered by secretion, if fixation is omitted,
mucus secretion continues within the sample chamber and
prevents any observation. According to Tai and Tang (2001),
a rapid fixation must be done for these samples to stop mucus
secretion. Despite the fixation, tissues stay hydrated.
The environmental microscopy therefore, cannot comple-
tely take over the routine use of CSEM. Both techniques must
be used in parallel, especially for tissues such as intestine
whose surface is covered by secretion. Water and mucus
limiting surface observations, detailed studies at high
magnification cannot therefore be imaged in a low vacuum
mode. Even at low magnification, the presence of a watery
film on the sample alters the quality of the contrast of the
ESEM images. Computer software employing an image-
processing algorithm to filter out contrast variations, must
therefore be used in order to optimize image contrast and thus,
to extract image information that would otherwise not be
perceptible. This treatment is particularly useful to improve
ESEM images, as shown in Fig. 20 but can also be applied to
other techniques of microscopy such as CSEM (Fig. 21).
ESEM provides a particularly attractive alternative to
CSEM. ESEM enables non-conductive, moist or even liquid
specimens to be observed in their native states without any
preparation nor conductive coating. In addition, dynamic
experiments can be done on numerous materials and ESEM
may be a way to combine scanning electron microscopy and
immunogold labeling on fresh samples such as living cells
(Djano et al., 1999; McMenamin et al., 2002). Nevertheless,
the techniques must be adapted for each type of specimen
and CSEM stays a useful technique for studies involving
imaging at high resolution.
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