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Transmission electron microscopy of critical point dried tissue after observation in the scanning electron microscope.

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Transmission Electron Microscopy of Critical Point
Dried Tissue after Observation in the
Scanning Electron Microscope '
Department of Anatomy, Harvard Medical School and Harvard School of
Dental Medicine, Boston, Massachusetts 021 15 and Department of
Anatomy, University of New Mexico, School of Medicine,
Albuquerque, New Mexico 87131
Embryonic and adult rodent tissues were fixed and prepared for
scanning electron microscopy by dehydration in ethanol followed by critical
point drying with liquid carbon dioxide or Freon 13 (E. I. du Pont de Nemours,
Inc. 1. After coating the dried specimens with evaporated metal, the tissues were
studied by scanning microscopy. The same tissues were subsequently embedded
in Epon-Araldite, thin sectioned and examined by transmission electron microscopy. The cytological details in these specimens were comparable to tissues embedded directly, without drying or metal-coating. With this technique it is
possible to identify with greater certainty the structures responsible for surface
contours revealed by the scanning electron microscope.
Much useful information can be obtained by examining the surface of a biological specimen with the scanning electron microscope (SEM). However, there
is often some uncertainty a s to whether
a particular surface contour is actually
part of the underlying cell or adherent
extraneous material. Moreover, with scanning microscopy the precise identity of
underlying components that are responsible for surface contours is purely conjectural. Thus there are obvious advantages
in being able to examine the same specimen with the transmission electron microscope in thin sections cut perpendicular to
the surface previously studied by scanning
electron microscopy. This allows definitive
identification of the structures responsible
for the surface contours.
There have been several reports of preparations for scanning electron microscopy
that were subsequently viewed in thin section by transmission electron microscopy.
However, there was considerable distortion
and loss of ultrastructural detail due to air
drying (Barber and Boyde, '68) or freezedrying (Wickham and Worthen, '73). Partial infiltration of the tissue with plastic
prior to metal coating helps to preserve
ANAT. REC.,176: 245-252.
cellular details (Cleveland and Schneider,
'69) but this method has its limitations
with regard to observation of the surface
details. The use of critical point drying
with liquid C02 (Anderson, '56) or Freon
(Cohen, Marlow and Garner, '68) is now
widely accepted as the method of choice
for drying biological specimens. This
method avoids distortion by the high surface tension forces of air drying and ice
damage in freeze drying. In a recent paper
Wickham and Worthen ('73) compared
thin sections of material dried by the critical point method using Freon 13 with
sections of specimens prepared by freezedrying. They reported good ultrastructural
preservation of cells only after critical
point drying. They did not, however, illustrate the detailed preservation of membrane structure in their critical point dried
tissue or demonstrate the metal coating
on the specimen in sections. In the present
paper we reaffirm the superiority of the
critical point drying method for SEM preparation and illustrate the retention of ultrastructural details in specimens viewed
Received April 6 '73. Acce ted May 11, '73.
1 This work was bupported [y Ford Foundation grant
710-0036, NIDR grant T01-DE00278,and NIH grant
AM 7578.
over an hour period, infiltrated with EponAraldite 6005 (Mollenhauer, '64) and
propylene oxide ( 1:1) for three hours, and
embedded in Epon-Araldite which was then
cured at 60°C for 36 hours. Thin sections
Golden hamster embryos (LVK strain, were prepared on a Sorvall MT-1 microCharles River Breeding Laboratories, Inc.) tome, stained with uranyl acetate and lead
were removed from the uterus 8.5 days citrate and examined in JEOL 100 B or
post-coitally and placed in 3% glutaralde- AEI 6 B transmission electron microscopes
hyde in 0.1 M phosphate buffer (pH 7.4) (TEM).
Control samples of both hamster periat room temperature for three to four
hours. After a rinse in the same phosphate derm and mouse jejunum were processed
buffer, the tissues were postfixed in 1% through dehydration as described above
osmium tetroxide in 0.1 M phosphate buf- and not prepared €or critical point drying
fer at room temperature for one to two nor metal coating. Subsequently the tishours and dehydrated at room temperature sues were placed in propylene oxide at
through graded ethanol concentrations (35, room temperature for 15 minutes, then
50, 70, 95, 100% ). After complete dehy- propylene oxide : Epon-Araldite ( 1 : 1) for
dration, infiltration with iso-amyl acetate four to six hours, and finally embedded i n
for two hours was followed by critical point Epon-Araldite and cured at 60°C for 36
drying with liquid CO, in a critical point hours. Appropriate thin sections were
bomb (Anderson, '56).
Adult Swiss-Webster mouse (Charles
River Breeding Laboratories, Inc.) jejunum
Scanning electron microscopy of liquid
was placed in a 0.1 M cacodylate buffered
(pH 7.4) 2% paraformaldehyde 3% glu- CO, critical point dried embryos revealed
taraldehyde fixative containing 0.01% tri- numerous microvilli which project from
nitrocresol (It0 and Karnovsky, '68) a t the surface of the periderm cells. Along
room temperature for two hours, and post- the borders of adjacent cells, many microfixed with cacodylate buffered 1% osmium villi were arranged in rows that sharply
tetroxide for two hours. The tissue was demarcated rhe junctional areas between
washed in 0.05 M maleate buffer (pH 5.4) cells (figs. 1, 2 ) . Thin sections of such
and treated with 1% uranyl acetate in areas studied by TEM clearly revealed
0.05 M maleate buffer (pH 6.0) for one these microvilli. However, in thin sections
hour and dehydrated with ethanol. After their profiles cannot easily be distinguished
dehydration the tissue was infiltrated with from surface folds or plications. Fixed
increasing concentrations of Freon TF adult mouse intestine, dried by the critical
(E. I. duPont de Nemours, Inc. also known point method using Freon 13, was exas Freon 60) in ethanol and finally in amined in the SEM. On the luminal sur100% Freon TF for one half hour.
face of the absorptive cells numerous
The specimens were then dried by the hemispherical protrusions which correcritical point method using Freon 13 in sponded to the tips of the microvilli forma Bomar critical point apparatus (SPC ing the striated border were seen (fig. 5).
900) and were affixed to aluminum slugs Confirmation of these structures as the
with silver conductive paint and placed on apices of microvilli was made by thin secan Omnirotating stage in a Denton tioning the same material. General cytoVacuum DV 504 evaporator. When a mini- logical preservation was retained and at
torr was at- high magnification the trilaminar appearmum vacuum of 5 X
tained, a coating of gold or gold-palladium ance of membranes is apparent (figs. 6,
(60:40) was evaporated on the rotating 7). Nuclear and cytoplasmic ultrastructure
specimens. Coated specimens were exam- (figs. 3, 7 ) did not seem to be significantly
ined in a JEOL JSM U-3 SEM at 25 KV u p altered when compared to the same tisto two hours. After observations were made sue prepared by conventional methods
in the SEM, the specimens were placed for transmission electron microscopy
into propylene oxide, changed 3 or 4 times (figs. 8, 9).
i n the SEM that are comparable to tissues
processed in the usual manner and not
dried prior to embedding.
The gold or gold-palladium coating
(figs. 4, 6 ) is a 250-500 A thick layer of
heterogeneously sized particles. In the intestine, only the rounded tips of the microvilli and the glycocalyx between microvilli are coated with gold-palladium. This
explains; the hemispherical appearance of
these microvilli in the SEM. The metal
coating on the embryo was not found beneath densily packed surface projections
(fig. 2 ) nor on the under surface of long
horizontally oriented microvilli. Despite
this uneven coating, no tissue “charging,”
indicative of tissue damage due to beam
absorption, was noted in this specimen.
Positive and direct correlation of SEM and
TEM observations on the same specimen
allows study of the surface in three dimensions with subsequent confirmation of ultrastructural cytology. If the underlying
structure in a particular SEM field required further identification, it is possible
to mark an area with a micromanipulator
so that this precise area can be sectioned
for study by transmission electron microscopy.
The general preservation in critical
point dried tissue examined by scanning
electron microscopy and subsequently embedded and sectioned for transmission
electron microscopy is indistinguishable
from similar tissues prepared for conventional transmission electron microscopy.
Both the carbon dioxide and Freon critical
point drying are practical, routine procedures. ’Tissue may be stored in iso-amyl
acetate for several weeks without apparent
loss of cytological integrity, however, prolonged storage in Freon TF seems to result in a loss of the trilaminar appearance
of membranes. Although the reason for
this altered appearance is not clear, i t may
be due to extraction of membrane components by Freon TF. If prolonged Freon
TF immersion is avoided, CO, and Freon
critical point drying results in comparable
retention of cytoplasmic ultrastructure.
If appropriately fixed soft tissues are
dried by the critical point method for scanning electron microscopic study and then
embedded for transmission electron microscopic study, some of the uncertainties of
misinterpreting surface contours seen in
the SEM could be avoided. Furthermore,
for rare specimens, meaningful information may be obtained from the same specimen by both methods without sacrificing
loss of fine detail. An alternative method
for using both types of microscopical observations on the same specimen was recently reported by Erlandsen, Thomas and
Wendelschafer (’73) who remove the
plastic with sodium methoxylate from tissue samples embedded for thin section
transmission electron microscopy, and coat
them with metal after critical point drying. The same sample may at a later time
be reembedded for thin sectioning. It is
clear that if tissues are adequately fixed
and appropriately prepared, the same sample may be used for both SEM and TEM
study. The possibility of being able to modulate between these different types of microscopy of the same specimen with little
loss of ultrastructure adds greater credibility to the interpretation of observations
by either method alone.
Anderson, T. F. 1956 Electron Microscopy of
Microorganisms. In: Physical Techniques in
Biological Research. Vol. 111. G. Oster and
A. Pollister, eds. Academic Press, New York,
pp. 177-240.
Barber, V. C., and A. Boyde
electron microscopic studies of cilia. Z. Zellforsch. Mikrosk. Anat., 84: 269-284.
Cleveland, P. H., and C. W. Schneider 1969 A
simple method of preserving ocular tissue for
scanning electron microscopy. Vision Res., 9:
Cohen, A. L., D. P. Marlow and G. E. Garner
1968 A rapid critical point method using
fluorocarbons (“Freons”) as intermediate and
transitional fluids. J. Microscopie, 7: 331-342.
Erlandsen, S. L., A. Thomas and G. Wendelschafer
1973 A simple technique for correlating SEM
with TEM on biological tissue originally embedded i n epoxy resin for TEM. Scanning
Electron Microscopy/l973 (Part 111) Scanning
Electron Microscopy in Pathology, IIT Research
Institute, Chicago, pp. 349-356.
Ito, S., and M. J. Karnovsky
1968 Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. J. Cell Biol., 39: 168a-169a.
Mollenhauer, H. H. 1964 Plastic embedding
mixtures for use i n electron microscopy. Stain
Techn., 39: 111-114.
Wickham, M. G., and D. M. Worthen 1973 Correlation of scanning and transmission electron
microscopy on the same tissue sample. Stain
Teehn., 48: 63-68.
Hamster embryonic periderm critical point dried with liquid COZ.
A representative area of the head periderm of a n 8.5 day hamster
embryo reveals rows of microvilli prominent along cell borders.
x 3,200.Inset. Microvilli at higher magnification x 13,000.
Taansmission electron micrograph of a portion of a periderm cell
with a junctional area showing microvilli coated with gold (arrow)
x 17,000.
Transmission electron micrograph of nucleus with nucleolus, nuclear
envelope and pore exhibiting representative ultrastructural integrity.
x 32,000.
Transmission electron micrograph of surface of periderm cell with
particulate gold coating measuring 250-500 A in thickness and subjacent Golgi cisternae. x 108,000
Meller, Coppe, Ito and Waterman
Adult mouse jejunal absorptive cells critical point dried with Freon 13.
Scanning electron micrograph of the brush border of two adjacent
intestinal epithelial cells. x 22,000.
Transmission electron micrograph of microvilli with gold-palladium
coating 250-500 A thick on their termha1 contours, A thin coating
of gold-palladium between microvilli (arrow) is deposited on the
glycocalyx. Unit membrane preservation is seen. x 70,000.
Transmission electron micrograph of absorptive cell exhibiting goldpalladium coating and good cytological preservation. x 12,000.
Meller, Coppe, Ito and Waterman
25 1
Meller, Coppe, Ito and Waterman
Control tissues.
8 Hamster embryonic periderm, fixed and dehydrated like the hamster tissue for the SEM,
not prepared for critical point drying or metal coating, and embedded in Epon-Araldite.
An area of junctional microvilli is illustrated. x 21,000.
9 Mouse jejunum processed for the TEM directly illustrating ultrastructural preservation
comparable to that seen in figure 7. Swollen Golgi cisternae are characteristic of mouse
jejunal absorptive cells with the fixation described.
x 15,000.
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point, transmission, observations, microscopy, critical, scanning, electro, tissue, dried
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