Romanian Journal of Morphology and Embryology 2007, 48(3):257–261
ORIGINAL PAPER
Scanning electron microscopy of
vascular corrosion casts – standard
method for studying microvessels
ILEANA GIUVĂRĂŞTEANU
Department of Anatomy,
“Carol Davila” University of Medicine and Pharmacy, Bucharest
Abstract
Scanning electron microscopy (SEM) of vascular corrosion casts (microvascular corrosion casting/SEM method) is a standard method,
which allows three-dimensional visualization with good resolution of the normal and abnormal microvessels, including the capillaries of
various organs and tissues. SEM of vascular corrosion casts can obtain qualitative as well as quantitative informations important to
anatomists, pathologists and clinicians. Considering these, the history, the advantages and the main steps of this technique including
general morphological characteristics of vascular casts observed in SEM are reviewed in this paper. Corrosion casts done by the author
representing the microvascular organization of the rat liver and kidney observed in SEM are, also, presented.
Keywords: microvessel, corrosion cast, SEM.
Introduction
An adequate study of the normal function and the
pathological condition of an organ is based upon the
exact understanding of the microvascular architecture of
that organ [1].
The arrangement of microvasculature has been
studied by light microscopy of serial sections or
tissues injected with various dyes, contrast agents and
resins [1–3]. However, light microscopy cannot reveal
the three-dimensional organization with adequate
resolution [1]. Therefore, the currently used method in
the routine examination of the vascular system is
scanning electron microscopy (SEM) of corrosion casts,
also referred to as microvascular corrosion casting/SEM
method [2]. It is a standard method, which allows threedimensional visualization with good resolution of the
microvessels including fine capillaries [2, 4, 5].
In this paper the history, the advantages and the
main steps in producing and preparing vascular
corrosion casts for SEM study including, also, some
morphological characteristics of the vascular casts
observed in SEM are presented.
History of vascular corrosion casting
and SEM methods
The method of corrosion casting has been known
since the 16th century when Leonardo da Vinci made
the first casts by injecting dissolved wax into bovine
cerebral ventricles and heart chambers [2, 6].
During next centuries, the anatomists tried to
improve the casting media, the method of injection, and
the method of removing the surrounding tissues in order
to produce more accurate replica of the biological
structures.
The modern corrosion casting methods are based
upon the idea of Jan Schwammerdam who in the late
17th century dissolved the surrounding tissues after wax
injection into the arteries, veins and ducts [6]. Before
the 19th century, Ruysch and Bidloo used a low melting
point metal alloy to make casts of the bronchial tree
[3, 6]. By the end of 19th century, Paul Schiefferdecker
introduced celloidin dissolved in organic solvents for
casting media, and Storch used celluloid instead
celloidin-making casts more resistant after drying [6].
Hinman (1923) presented the basic principles of the
corrosion casting method and pointed out that lowering
the viscosity of the injection mass one can produce
casts of the very small vessels [6]. In 1930 injectants
such as celluloid solution, polymerizing resins
(Plastoid) and latex gum were used to make casts for
light microscopy [2]. In 1950s, a great advance in the
corrosion casting technique was made when new
synthesized resin products (acrylic resins, polyester
resins and silicone) were used as casting media [3].
Von Ardenne M (1938) and Zworykin VK (1942)
developed the basic principles of scanning electron
microscope [7, 8]. The scanning electron microscope is
using a primary electron beam generated by a cathode,
focused by objective lens toward the sample and
scanned across the surface of the sample by a pair of
electromagnetic deflection coils. SEM images are
produced by detecting and converting into signals
secondary electrons that are released by the sample due
to the excitation by the primary electron beam. The
image is build up on the screen by mapping the detected
signals with primary beam position. Due to its great
power of magnification and large depth of field the
scanning electron microscope is capable of producing
high-resolution images of a sample surface with
characteristic three-dimensional appearance.
258
Ileana Giuvărăşteanu
However, it was just in 1970 when the SEM
was used by Nowell to examine the airway system of
the avian lung replicated with a latex medium
(Cementex N-1971-S) [9].
In 1971, a great advance was made when Takuro
Murakami introduced the semi-polymerized methyl
methacrylate resin for the SEM study of corrosion
casts [10]. This semi-polymerized resin has been
considered the most appropriate casting medium for the
SEM study of microvascular beds due to its low
viscosity, which allows the infusion of the small vessels
including the capillaries and its adequate propriety of
electron reflection [3].
Since then many other authors have used the
microvascular corrosion casting/SEM method to study
vascular or non-vascular structures of various organs
and tissues under different conditions and during
development and aging [3, 11–15].
Casting media and steps in the
microvascular corrosion casting/SEM
method
Casting media
The casting or injection media must have physicochemical properties making them adequate for the
SEM study of microvessels, such as low viscosity
(to pass through capillaries); rapid and evenly
polymerization; minimal shrinkage during hardening;
resistance to corrosion, cleaning, dissection and drying
procedures; allow replication of endothelial structures;
electron
conductivity;
resistance
to
electron
bombardment [5, 9].
The semi-polymerized methyl methacrylate resin
introduced by Murakami (1971) for the SEM study of
corrosion casts combines all these proprieties and
therefore, it has been the most widely used casting
medium for replicating microvasculatures [2].
The methyl methacrylate mixtures can be produced
in the laboratory by warming methyl methacrylate
monomer with a catalyst (benzoyl peroxide) or exposing
it to ultraviolet light [4].
There are also commercially produced partially
polymerized methacrylates such as Mercox, Technovit,
Trylon, Batson’s plastic no. 17 [2, 4].
The latex or silicone rubber compounds (Cementex,
Vultex, Geon, Microfil) have some disadvantages
(e.g. lower resistance to corrosion and drying
procedures, inconsistent replication of luminal surface
microstructures) which make them less appropriate to
replicate the fine vascular microstructures [2, 4].
Recent studies [16, 17] show that a new
polyurethane based resin (PU4ii) has superior physical
characteristics (low viscosity, timely polymerization,
minimal shrinkage) which allow high quality replication
of the small vessels including the endothelial cell
imprints. The casts are highly elastic and resistant so
that they can withstand postcasting tissue dissection.
PU4ii allows, also good quantitative analysis and its
inherent fluorescence is sufficient to reproduce casts
with/without tissue using confocal microscopy [16].
Steps in the microvascular
casting/SEM method
corrosion
The microvascular corrosion casting/SEM method
can be used on excised human materials (organs/
tissues) or experimental animals and includes several
steps [2, 9, 10, 18–20].
Precasting treatment
Prior to injecting the casting media into the blood
vessels, the complete removal of the blood is necessary
in order to fill the entire vascular bed and to obtain
endothelial cell imprints on the surface of the cast.
For this reason, the target organs are perfused with
saline or Ringer solution through a cannula placed
closed to the target organ or far from the target organ
(ventricle, descending aorta).
Injection of casting medium
Immediately after the precasting treatment,
the injection medium is prepared by adding to the main
reagent (resin) a catalyst/accelerator to initiate
polymerization. The casting media can be injected
into the blood vessels using a syringe or a syringe
connected to a perfusion apparatus with a flow meter.
It is important to adjust the injection pressure in order to
fill the vascular bed. For instance, in the rat a resin
perfusion raging from 90 to 120 mmHg gives the best
filling and cell replication.
Polymerization (hardening) of casting medium
The injected specimens must be placed in a hot
water bath (600C) for 12–24 hours to accelerate
or to complete the polymerization of the perfused
casting medium. This immersion reduces the
corrosion time and keeps the injected specimens in
their natural form.
Corrosive treatment
The surrounding tissues must be removed
(macerated) to observe the vascular cast with the SEM.
For this purpose, the injected specimen is immersed into
a highly concentrated (15–20%) sodium hydroxide or
potassium hydroxide solution (at 600C, overnight or
longer).
Cleaning of corrosion casts
Washing the cast in running water is important to
remove the white saponified materials resulted from the
maceration of tissues rich in lipid with sodium
hydroxide.
Dissection of corrosion casts
Gross dissection and microdissection must be
carried out to expose the structures of interest.
Cast samples are obtained by cutting the specimen into
blocks.
Drying the casts
Air-drying or freeze-drying (immersion of casts in
distilled water and slowly frozen) can be used. Usually,
the vascular casts are dried in air without any detectable
distortion or dislocation of the fragile parts of the casts.
Scanning electron microscopy of vascular corrosion casts – standard method for studying microvessels
259
Figure 1 – Blood vascular bed of the liver. Sinusoids,
central vein (c) and interlobular veins (i)
Figure 2 – Cortex of the kidney with
renal glomeruli (G)
Figure 3 – Corrosion cast of the renal glomeruli (G).
Afferent arteriole (a) and efferent arteriole (e)
having different diameters
Figure 4 – Hepatic sinusoids and
central vein (C)
Figure 5 – Connection of a portal vein branch (V) to the
hepatic sinusoids (arrowhead)
Figure 6 – Portal vein branch (V) accompanied by hepatic
artery branch (A). The arrowhead indicates the
connection of the vein to the sinusoids
260
Ileana Giuvărăşteanu
Fragile casts of fine capillary networks are
recommended to be freeze dried to minimize surface
tension, which can dislocate these fine structures.
Conductive treatment of casts
Since the SEM uses electrons to produce an image,
the cast sample must be electron conductive. Therefore,
the specimen is coated with a heavy metal (gold),
and then observed in SEM with an accelerating voltage
of 5–10 kV.
SEM observation of casts
Valuable and detailed information about the
organization of blood vascular beds of various organs
and tissues of humans or experimental animals
(Figures 1 and 2) can be obtained by SEM observation
of corrosion casts. The vascular organization varies
depending on the structure and function of the organ, in
normal, experimental or pathological conditions and in
development and aging [9]. The complicated
distribution of the capillaries (Figure 3) including the
sinusoids (Figures 4–6) as well as their connections to
the arteries or veins (Figures 5 and 6) can be clearly
visualized by microvascular corrosion casting/SEM
method [2, 4, 5, 10]. The Figures 1–6 are scanning
micrographs of blood vascular casts of the rat liver and
kidney. The author of this paper did the casts during a
training course at the Department of Anatomy,
Okayama University Medical School, Japan.
The casting medium was Mercox, and the protocole
used to obtain and prepare the casts for SEM
observation included steps presented in the above
paragraphs with some specifications: the thoracic aorta
was cannulated for saline perfusion and injection of
Mercox, a syringe was used for the injection of the
casting medium, the liver and the kidney were excised
after hardening, and after the corrosion procedure
(20% sodium hydroxide) and washing in water, the liver
and the kidney casts were dissected and air dried.
Characteristic endothelial cell imprints on the
surface of the casts allowing the identification of the
vessel type (artery/vein), venous valves, intraarterial
cushions, sphincter structures and arterio-venous
anastomoses can, also, be demonstrated by SEM of
corrosion casts [2, 9, 21].
Precise morphometric measurements of vascular
parameters such as vessel lengths and diameters,
interbranching distances (branching frequencies and
number of branching sites), intervascular distances, and
branching angles (between stem and daughter vessels)
can be performed by analyzing the stereo pair images of
vascular corrosion casts [22–24]. These variables define
the overall three-dimensional structure of the vascular
network as well as the basic transport capacity of the
system [24].
Conclusions
SEM of vascular corrosion casts is one of the most
widely used methods nowadays to demonstrate the
three-dimensional architecture of the microvasculature
of various organs and tissues, both in humans and
experimental animals, in different stages of
development and age.
The improved casting methods and the new casting
media used together with advanced imaging
technologies enable to achieve fundamental qualitative
and quantitative information regarding complex
vascular networks both in healthy and diseased organs
which are of great help not only to anatomists but, also,
to pathologists and clinicians.
Acknowledgements
The author sincerely thanks Prof. Takuro Murakami,
Prof. Aiji Ohtsuka and Prof. Takehito Taguchi at
Department of Anatomy, Okayama University Medical
School, Japan for offering practical and theoretical
knowledge about corrosion casting method and
SEM method. The author also acknowledges
Prof. Nicolae Constantinescu at Department of
Anatomy, “Carol Davila” University of Medicine and
Pharmacy, Bucharest, for encouragement and critical
reading of the manuscript.
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Corresponding author
Ileana Giuvărăşteanu, Assistant Professor, MD, PhD, Department of Anatomy, “Carol Davila” University of
Medicine and Pharmacy, 8 Eroilor Sanitari Avenue, 050 474 Bucharest, Romania; Phone +40720–915 471,
E-mail: [email protected]
Received: June 5th, 2007
Accepted: August 20th, 2007