Observations of the intestinal mucosa using environmental scanning electron microscopy (ESEM); comparison with conventional scanning electron microscopy (CSEM)

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).

http://www.elsevier.com/locate/micron

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).



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.



with the SE detector. Rat refed 24 h after 5 days fasting; observation at

30 kV. Lucise treatment.



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).



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.



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




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).




was 80–85%. Observations were done at 10–15 kV. Lucise treatment.

Control rat (fed).





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).





refed 2 h after 5 days fasting.


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).





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).


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).





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).


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.


(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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Observations of the intestinal mucosa using environmental scanning electron microscopy (ESEM); comparison with conventional scanning electron microscopy (CSEM)