KIMThe amount of shrinkage and the scanning electron microscope appearance of specimens of rat tongue, soft palate and small intestine, dehydrated by air drying , the critical point technique, the camphene technique and freeze drying were compare

Cleaton-Jones, P.

Wilton-Cox, H.

English

Wits Institutional Repository on DSPACE

Journal of the Dental Association of South Africa 32, 141 - 145
Soft-tissue specimen shrinkage during the preparation 
for scanning electron microscopy
Helen Wilton-Cox and P Cleaton-Jones
Dental Research Unit of the South African Medical Research Council and the University of the Witwatersrand, 
Johannesburg, 2001
Keywords: Electron microscopy; scanning electron microscopy; 
techniques in electron microscopy
SUMMARY
The amount of shrinkage and the scanning electron 
microscope appearance of specimens of rat tongue, 
soft palate and small intestine, dehydrated by air­
drying, the critical point technique, the camphene 
technique and freeze drying were compared. In the 
specimens of tongue the area shrinkage ranged be­
tween 11,8 and 30,4 per cent, in the soft palate from 
19,8 to 37,4 per cent and in the small intestine from 
26,0 to 51,4 per cent. The least amount of shrinkage 
was in tissues dehydrated using freeze-drying. 
Shrinkage due to the critical point technique itself 
was tongue 17,4 per cent, soft palate 15 per cent and 
small intestine 14,4 per cent. This was half the total 
shrinkage seen in the tongue and soft palate and a 
quarter of that in the small intestine. It was not possi­
ble to distinguish between the scanning electron 
microscope appearances of comparable samples dehy­
drated with the various techniques.
OPSOMMING
Die hoeveelheid inkrimping sowel as die aftaselektron- 
mikroskopiese beeld van tong, sagteverhemelte en 
kleinderm monsters van rotte wat deur middel van 
lugdroging, die kritiesepunttegniek, die kamfeenteg- 
niek en vriesdroging ontwater is, word vergelyk. Die 
inkrimping het in tongweefsel tussen 11,8 - 30,4 per- 
sent, in sagteverhemelteweefsel tussen 19, 19,8 - 37,4
8.37.4 persent, en in kleindermweefsel tussen 26,0 -
51.4 persent gewissel. Die minste inkrimping het in 
weefsel wat deur vriesdroging ontwater is plaasgevind. 
Inkrimping as gevolg van die kritiesepunttegniek self 
was soos volg: tong 17,4 persent, sagteverhemelte 15 
persent en kleinderm 14,4 persent. Dit staangelyk aan 
helfte van die totale inkrimping van tong- en sagte­
verhemelteweefsel en ’n kwart van die wat in die klein­
dermweefsel plaasgevind het. Dit was onmoontlik om 
die aftaselektronmikroskopiese beelde van die verge- 
lykbare monsters wat volgens die verskillende tegnie- 
ke ontwater is, van mekaar te onderskei.
When a soft specimen dries it shrinks producing dis­
tortions of two types, namely volume changes and sur­
face changes (Cohen 1974).
In the early days of scanning electron microscopy soft 
specimens were dehydrated by air drying but later 
other techniques namely freeze drying, sublimation 
and the critical point technique were felt to produce 
less distortion (Watters and Buck 1971, Waterman 
1972, Hollenberg and Erickson 1973). These conclu­
sions seem, for the most part, to be based on sub­
jective observations.
Only two studies appear to have included quantita­
tion of tissue shrinkage. Waterman (1972) reported 
that linear measurements showed that embryological 
specimens dried by the critical point technique retain­
ed approximately 80 per cent of their original 
measurements. In contrast, air dried samples were 
reduced to approximately 45 per cent of their original 
size. He noted that most of the shrinkage occurred 
during drying since fixation and dehydration through
either 100 per cent acetone or isoamyl acetate produc­
ed only 6-8 per cent shrinkage.
More recently, Wheeler, Gavin and Seelye (1975) in­
vestigated the scanning electron microscope 
appearance and dimensional changes of endocardium 
prepared by freeze drying from water or tertiary 
butanol and the critical point technique. The per 
cent reduction in surface area with the three tech­
niques was: freeze-drying from water 6,8 per cent, 
freeze-drying from t-butanol 15,4 per cent and critical 
point drying 22,0 per cent.
The present investigation was carried out
a. to determine the degree of shrinkage in three varie­
ties of soft tissues using air-drying, the critical 
point technique, a modified form of the camphene 
technique of Watters and Buck (1971) and three 
methods of freeze drying, and
b. to compare the scanning electron microscope 
appearances of the tissues prepared with the 
various techniques.
Journal of the D.A.S.A. -  March 1977 141
Helen Wilton-Cox and P Cleaton-Jones
Fig. 1. Double bladed scalpel used to cut standard specimens.
MATERIALS AND METHODS
Specimens of Wistar strain albino rat soft palates, 
tongue and small intestine were excised and fixed for 
one week in 10 per cent neutral buffered formol saline. 
Standard sized specimens of each tissue were then cut 
using scalpel blades held 5 mm apart (Fig. 1). A total 
of 5 specimens of each tissue was used for each of the 
methods being investigated.
The specimens were removed from the fixative, lightly 
blotted and, together with an engineer’s scale, photo­
graphed using standardized lighting and a Canon FI 
35 mm camera with a bellows attachment set at cons­
tant magnification to which was attached a 65 mm 
macrolens. Illumination by direct lighting from above, 
to obviate shadows, was obtained through the use of a 
mirror attachment attached to the lens (Fig. 2). 
Photographs of all dimensions were taken of the 
tongue and small intestine specimens but, because of 
its thickness of 1 mm, only the flat surface of the soft 
palate. Once dehydration had been completed the 
specimens were rephotographed. Using the image of 
the engineer’s scale as an aid, photographs were 
printed at x 10 enlargement. A cut surface of the speci­
men was carefully outlined with ink and the area 
within this outline was measured using a planimeter. 
The length of the specimens was measured on the 
photographs using an engineer’s scale.
Area shrinkage was calculated directly from the plani­
meter area recordings. Volume shrinkage was deter­
mined after calculating the specimen volume as fol­
lows: Volume of specimen = area of cut surface x 
specimen height.
The sequence of events in the dehydrations, which 
made use of a short series of alcohols (Brain 1966, Cul­
ling 1974, Drury and Wallington 1976, Nunn 1970, 
Pease 1964), was as follows:
(i) Air Drying The specimens were passed through 
two changes of each of 70 per cent ethanol (90 
min), 96 per cent ethanol (90 min), then three 
changes of 100 per cent ethanol (60 min) and 
finally two changes of ether (60 min). The ether 
was allowed to evaporate in air at about 22°C.
(ii) Critical Point Technique Two changes of 70 per 
cent ethanol (90 min) and 96 per cent ethanol (90
Fig. 2. Macrophotography system used to photograph speci­
mens and a photograph of a specimen of tongue together with 
the 1 mm scale rule.
min) were followed by three of 100 per cent 
ethanol (60 min) and two of amyl acetate (60 
min). In this section of the study 4 of the 5 speci­
mens of each tissue were also photographed at the 
end of the amyl acetate stage. The fifth speci­
men served as a control. The specimens were 
finally dried in a Polaron E2000 Critical Point 
Apparatus using liquid C 02 at 1200 psi and 
41.0°C.
(iii) A Camphene Technique based on that of Wat­
ters and Buck (1971) Following two changes of 
70 per cent (90 min) and 96 per cent (90 min) 
ethanol the specimens were passed through two 
changes of acetone (30 min) and benzene (30 
min), one of each of benzene + 1,2-epoxypropane 
(20 min), 1,2-epoxypropane (20 min at 45°C) and 
camphene (20 min at 45°C). After cooling to 
room temperature (about 22 °C) they were kept 
under vacuum overnight at 20 psi.
(iv) Freeze-Drying Three types of freeze drying ap­
paratus were used but the initial preparative tech­
nique was identical for each. After removal from 
the fixative the specimens were washed over­
night. They were then placed into one change of 
chloroform in water (30 min) to nucleate ice 
crystal formation (Boyde 1972), before being 
quenched in isopentane pre-cooled in liquid nitro­
gen.
The three freeze-drying apparatuses were an 
Edwards-Pearse tissue dryer, an Edwards E12E4 
vacuum coating unit (Edwards Ltd., Crawley, 
Kent, U.K.) and a Gallenkamp freeze drying ap­
paratus (A. Gallenkamp, London, U.K.). The 
specimens were dehydrated in each apparatus for 
8 hours.
Phosphorous pentoxide was used in the Edwards- 
Pearse tissue dryer while in the Edwards E12E4 
vacuum coating unit the frozen specimens were 
placed into numbered compartments in a brass 
block pre-cooled in liquid nitrogen.
All the dehydrated specimens, irrespective of the 
technique used, were individually wrapped in 
metal foil and stored in a desiccator over silicon 
dioxide.
142 Tydskrif van die T.V.S.A. -  Maart 1977
Soft tissue shrinkage
fable I. Percentage reduction in surface area of blocks (mean + 
SD)
Tissue
Method of Tongue Small Intestine Soft Palate 
Dehydration
Critical point 
Ether-air drying 
Camphene 
Freeze Drying
(i) Edwards-Pearse
(ii) Edwards E12E4
(iii) Gallenkamp
30.4 + 1,5 51,4 ±
29,6 ± 1,5 50,0 ±
29.4 + 2,3 50,8 ±
17.4 ± 5,1** 27,8 ±
11,8 ± 2,2** 26,9 ±
20,0 ± 4,1* 43,0 ±
3,7 32,8 ± 1,6
6,2 35,2 + 4,4
3,7 37,4 ± 4,1
89* 19,8 + 1,5*
3,8* 21,4 ± 5,6"
5,4 29,6 ± 6,6
*P< 0,01 **P< 0,001
Table II. Percentage area shrinkage after amyl acetate and the 
critical point technique (mean ± SD)
Hssue After amyl After critical Difference i.e.
acetate point technique shrinkage during
critical point 
technique
Tongue 13,0 ± 1,8 30,4 ± 1,5 17,4
Small Intestine 37,0 + 10,1 51,4 + 3,7 14,4
Soft palate 17,8 + 2,4 32,8 + 1,6 15,0
Table III. Percentage reduction in volume of blocks (mean ± SD)
Tissue
Method of Tongue Sm all Intestine
Dehydration
Critical point 
Ether-air drying 
Camphene 
Freeze-drying
(i) Edwards-Pearse
(ii) Edwards E12E4
(iii) Gallenkamp
43,0 + 0,7
42.4 ± 2,6 
42,8 ± 2,2
29.4 + 6,5*
23.4 + 2,2* 
35,2 + 6,8
65.4 ± 2,3
64.4 ± 5,7 
65,8 ± 4,6
29.4 ± 8,1* 
34,2 ± 3,3*
52.4 ± 4,8
*P < 0,01
For the scanning electron microscopy part of the stu­
dy the specimens were mounted on aluminium stubs 
with colloidal graphite, coated with gold palladium 
and finally examined in a Cambridge S4 Stereoscan 
operated at 20 kV.
Statistical analysis was carried out using the analy­
sis of variance, Scheffe’s test and Student’s t test.
mens were similar except for those dehydrated in 
the Edwards E12E4 apparatus where the tongue 
shrank less than the soft palate (P<0,001). The 
small intestine specimens all shrank consider­
ably more than the tongue and soft palate 
(P<0,001). Comparing the effect of the different 
techniques on the same type of tissue the amount 
of shrinkage was similar in all the non-freeze dry­
ing techniques i.e. the critical point, camphene 
and air-drying. Likewise, with one exception, the 
freeze drying shrinkages were similar. The only 
exception was in the small intestine where the 
Gallenkamp shrinkage was significantly more 
than the Edwards-Pearse or the Edwards E12E4 
(P<0,001). In the tongue specimens the freeze 
drying techniques produced siginificantly less 
shrinkage than the other techniques (P<0,001). In 
the soft palate this was the case only with the air­
drying and camphene techniques and the 
Edwards-Pearse and Edwards E12E4 apparatus 
(P<0,001). Small intestine shrinkage using the 
Gallenkamp apparatus was similar to that ob­
tained with the non-freeze drying techniques. 
However, the two Edwards apparatuses produced 
significantly less shrinkage (P< 0.01).
During the dehydration using the critical point 
technique the amount of shrinkage after the amyl 
acetate stage, i.e. from the critical point appara­
tus itself was 14,4 per cent for the small intes­
tine, 15 per cent for the soft palate and 17,4 per 
cent for the tongue (Table II). Thus in the tongue 
and soft palate specimens about half the shrink­
age occurred in the critical point technique and 
about a quarter of the shrinkage in the small 
intestine specimens.
(ii) Volume Shrinkage The soft palate was not in­
cluded in this portion of the study. As a result of 
its thinness it was not possible to photograph the 
lateral aspect.
In the tongue specimens the shrinkage with the 
critical point, air drying and camphene tech­
niques were very similar (Table III). Of the freeze 
drying techniques the Gallenkamp apparatus 
resembled this. The Edwards-Pearse and 
Edwards E12E4 freeze drying apparatus showed 
significantly less shrinkage (P<0,01). The freeze 
drying techniques did not differ significantly 
from each other.
In the small intestine the volume reduction was 
greater than that of the tongue (except for the 
Edwards-Pearse technique) but the pattern of 
shrinkage was similar.
RESULTS
The amount of shrinkage seen depended both on the 
technique used and the tissue involved.
(i) Area Shrinkage The percentage area shrinkage 
in each tissue obtained with each dehydration 
method is shown in Table I. The amount of 
shrinkage in the tongue and soft palate speci-
(iii) Scanning electron microscopy Examination of 
the various tissues in this study revealed that it 
was not possible to say from the SEM image 
which technique had been used except that occa­
sional cracks in the specimen surface identified 
those that had been freeze-dried. Examples of 
scanning electron micrographs are shown in Figu­
res 3-10.
Journal of the D.A.S.A. -  March 1977 143
Helen Wilton-Cox and P Cleaton-Jones
Fig. 3. Filiform papillae of rat tongue. Critical point method. 
SEM  X  240.
Fig. 4. Fungiform-like papillae on surface of rat soft palate. 
Air dried. SEM  X  184.
Fig. 5. High magnification o f the surface o f a soft palate papilla. 
Air dried. SEM  X  880.
Fig. 6. Villi in rat small intestine. Camphene. SEM  X  115.
Fig. 7. Fungiform-like papillae in rat soft palate. Freeze dried in 
Edwards-Pearse apparatus. SEM  X  192.
Fig. 8. Filiform papillae of rat tongue. Freeze dried in Edwards 
E12E4 apparatus. SEM  X  216.
Fig. 9. Filiform papillae of rat tongue. Freeze dried in Gallen- 
kamp apparatus. SEM  X  232.
Fig. 10. High magnification of the surface of a soft palate 
papilla. Freeze dried in Edwards E12E4 apparatus. SEM  X  960.
144 Tydskrif van die T.V.S.A. -  Maart 1977
Soft tissue shrinkage
DISCUSSION
This study confirms quantitatively the observation of 
Boyde and Wood (1969) that there is less tissue shrink­
age when tissues are freeze-dried than with any of the 
other fundamental SEM preparative techniques used 
today.
The amount of shrinkage varied from tissue to tissue 
which could account for the differences between the 
area shrinkage results obtained in our study and those 
of Wheeler et al (1975) who used canine endocardium 
dehydrated with freeze drying and critical point tech­
niques. A similar explanation may apply to the mark­
ed differences in shrinkage results obtained in our 
study and by Waterman (1972) in his embryological 
material. .
Boyde (1972) mentioned that a small amount of 
shrinkage occurs during the critical point during 
process proper while Waterman (1972) felt that a good 
deal of shrinkage occurred at this stage. Our study has 
confirmed Waterman’s viewpoint.
Wheeler et al (1975) were unable to distinguish be­
tween the SEM appearances of specimens dehy­
drated by freeze drying and the critical point tech­
niques. A similar finding in our study with the freeze­
drying, critical point and camphene techniques was 
not unexpected. What was unusual however was that 
the air-dried specimens could not be identified among 
those prepared with the other techniques.
From this study it is concluded that the freeze-drying 
technique produces less tissue shrinkage than the air­
drying, critical point and camphene techniques. The 
amount of shrinkage could not be correlated with the 
appearance of the tissues in the scanning electron 
microscope.
ACKNOWLEDGEMENTS
We are very grateful to Dr. M. Witcomb and his Staff 
of the Electron Microscope Unit for their assistance 
and to Miss B. Slack for typing the manuscript.
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scopy/1972. ed. Johari, 0 .  pp. 257-264. Chicago: IITRI.
Boyde, A. & Wood, C. (1969) Preparation of animal tissues for sur­
face scanning electron microscopy. Journal of Microscopy, 90 
221-249.
Brain, E.B. (1966) Preparation of Decalcified Sections, p. 148. 
Charles C. Thomas: Springfield.
Cohen, A.L. (1974) Critical point drying. In Principles and 
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Culling, C .F .A . (1974) Handbook of Histopathological and 
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REFRENCES
Journal of the D.A.S.A. -  March 1977 145


Soft-tissue specimen shrinkage during the preparation for scanning electron microscopy

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Soft-tissue specimen shrinkage during the preparation for scanning electron microscopy

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