3D MORPHOMETRY
Quantification of Microvasculature
by SEM and 3D Morphometry
B. Minnich1, H. Bartel1, H. Leeb2, E.W.N. Bernroider3, W.D. Krautgartner4, A. Lametschwandtner1
1
Department of Vascular - and Performance Biology, Salzburg, Austria
Institute of Statistics, University of Vienna, Vienna, Austria
3
Institute of Information Processing, Vienna, Austria
4
Department of Electron Microscopy and Digital Image Acquisition, Salzburg, Austria
2
Keywords: vascular corrosion castings, stereoscopy, myocardium
S U M M A RY
A new method for accurate dimensional and
angular measurements enables for the first
time quantitative measurements in resin casts
of delicate microvascular networks not accessible by other methods (e.g. intravital light
microscopy,
confocal
laser
scanning
microscopy). Here we briefly describe casting
of the microvascular networks of the canine
myocardium, analysis of vascular casts by scanning electron microscopy (SEM), digital acquisition of SEM stereopairs by slowscan framegrabbing and apply three-dimensional (3D)
morphometry to measure cast vessel diameters, lengths, intervascular and interbranching
distances using a recently introduced method.
Results of 3D and 2D measurements are compared and the method is discussed in respect
to its impact on the study of highly complex
vascular networks of tissues and organs in
health and disease.
INTRODUCTION
The cardiovascular system consists of a muscular pump (heart) and a set of hierarchically
connected tubes (arteries, veins and capillaries) with internal diameters down to ~ 5 µm
(capillaries). With few exceptions only, blood
vessels form three-dimensional networks
which primarily serve to transport oxygen,
nutrients and hormones to and carbon dioxide, metabolites and heat away from the body
cells. To adapt transport capacities to the
needs of different tissues and organs the system is finely tuned by several mechanisms
which narrow (constrict) or widen (dilate)
arteries and/or veins and decrease or increase
blood flow and blood pressure.
Nowadays, low viscosity polymerising resins
enable to fill (cast) the entire circulatory system after blood has been flushed out [1]. After
all organic materials of and around blood vessels have been removed by maceration in
strong alkali only the plastic vascular casts
remain and can be inspected in great detail in
the SEM after appropriate preparation. Resins
replicate finest details of the inner (luminal)
surface of blood vessels and so vascular casts
reveal imprints of endothelial cell nuclei and
endothelial cell borderlines on their surface
which allow a straightforward identification
Figure 1:
Canine heart. Vascular corrosion casts. Arrows mark direction of blood flow. (a) Macroscopic frontal view. Bar = 2 cm. (b) Transverse section (LM) of
the heart wall (from enboxed area in (a). Note supplying and draining coronary vessels (bottom) and the ventricular surface (s) (top). Asterisks mark
"conductive bridges" fixing the cast to the specimen holder. Bar = 2 mm. (c) Myocardial capillary network (SEM). Note the undulating capillaries and
the draining venule (v). Intercapillary distances outline the location of cardiomyocytes. (d) Intramyocardial arteriole with characteristic arterial surface
imprint patterns, circular impressions of smooth muscle cells (small arrows) and "plastic strips" representing plastified smooth muscle cells (asterisks).
SEM. c= capillaries. (e) Intramyocardial venule with circular impressions of smooth muscle cells (small arrows). SEM. (f) Intramyocardial venule with characteristic oval endothelial cell nuclei imprints (small arrows). SEM. (g) Coronary vein with valve (LM). s= valve sinus. Bar = 1 mm. (h) Venous valve (SEM).
Detail view. Note the second valve at the entrance of the small venule (small arrows).
of castings as arteries or veins (Fig 1) [2]. Capillaries can be diagnosed easily by their small
diameter. Moreover, any narrowing of a vessel
lumen by a single or groups of contracted vas-
MICROSCOPY
cular smooth muscle cells (sphincters) [3] or by
atherosclerotic plaques and any widening of
vessels (e.g. aneurysms, outpouchings in
tumour blood vessels) are reliably replicated
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(Fig 1).
Until very recently, SEM studies on vascular
casts were mostly descriptive. Some investigations applied planimetry, point counting
(stereology) or grey level image analysis to
gain quantitative data on vessel diameters,
lengths or vascular densities [4-8]. As these
techniques neglected the spatial extension of
blood vessels they underestimated distances.
Now techniques are available which allow
morphometry of three-dimensional networks
of cast blood vessels [9-12]. These methods are
PC-based and allow quantitative measurements of vessel diameters, vessel lengths,
intervascular and interbranching distances as
well as branching angles in space. They use
digitised stereopaired SEM images with a
defined tilt (e.g. 6˚) [10]. Data can now be subjected to statistics and any significant difference between vascular networks can be
shown.
M AT E R I A L S A N D M E T H O D S
Vascular castings
The heart of a dog terminated at the end of a
clinical experiment was excised, plastic tubings
were inserted into coronary arteries via the
stump of the cut aorta, and tied in place. Then
the coronary circulation was flushed with
warm heparinised saline (1000 IE/1000 ml;
37˚C) to remove the blood. After clear reflux
from the stumps of the cut caval vein, 40 ml of
a polymerising resin, Mercox-Cl-2B (Ladd
Research Inc., Burlington, VT, USA), diluted
4+1 (v+v) with monomeric methylmethacrylate (MMA)(containing 1.25 g of paste MA per
aliquots of 20 ml MMA) were injected with
manual pressure by hand-held syringes. For
further processing of injected specimen and
vascular casts, i.e. tempering, maceration,
cleaning, drying, mounting, sputtering, we
refer to previous work [13-14].
Figure 2:
Point setting in the stereopaired images for calculation of the third dimension (Z) using the parallax. Screenshot of the measurement dialog showing
intervascular distance and vessel diameter measurements on SEM stereopairs of the canine heart.
Scanning Electron Microscopy
Vascular casts were examined with an SEM
(Stereoscan 250, Cambridge Instruments, Cambridge, U.K.) at an accelerating voltage of 10
kV, a working distance of 10 mm and a spot
size of 5 or 6.
Qualitative aspects
The dense three-dimensional vascular network of the canine heart (Fig 1a-c) was excellently casted. Upon inspection by SEM, the surfaces of vascular casts revealed the characteristic endothelial cell nuclei imprint patterns
enabling a clear identification of arteries (Fig
1d) and veins (Figs 1e,f). Circular narrowings
resulting from impressions of contracted vascular smooth muscle cells were found in arteries (Fig 1d) and veins (Fig 1e). Valves were present on coronary and intramyocardial veins
(Figs 1g,h). Courses of individual vessels could
be followed over long distances and branching patterns and connectivities could be studied in great detail.
Digital Imaging
Stereopaired images with a tilt angle of 6˚
around the x-axis were acquired using a slow
scan frame grabber (Orion 5.10; E.L.I. sprl.
Brussels, Belgium) with an initial resolution of
3840 x 3840 pixels and an image depth of 8 bits
(256 grey levels). Generally, images then were
reduced to 480 x 480 pixels. This resolution
was sufficiently high for subsequent 3D morphometry.
3D Morphometry
Stereopaired images were imported into the
newly developed PC-based software (3D Morphometry, Minnich and Muska OEG, Salzburg,
Austria) which allowed exact measurements of
distances (vessel diameters, intervascular and
interbranching distances) and branching
angles in 3D space. The method is based on
the calculation of space co-ordinates using the
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MICROSCOPY
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parallax. It considers also the projection mode
(central or parallel) used during SEM work (Fig
2). Measuring was done by marking corresponding points of interests (e.g. outline of a
vessel diameter) in the stereopaired images
using the PC`s input device (Fig 2). The program stored planar co-ordinates (Xleft / Yleft
and Xright / Yright), calculated the corresponding
Z-co-ordinates using the parallax, and applied
trigonometric functions in order to calculate
marked distances or branching angles. Data
were comfortably transferred into spreadsheet programs (e.g. MS Excel) by dynamic
data exchange (DDE).
R E S U LT S
Quantitative aspects
Using 3D morphometry we were able to measure structural parameters of myocardial
blood vessels (means ± SEM), namely diameters (luminal): capillaries 5.79 ± 0.24 µm,
venules 14.41 ± 2.79µm, arterioles: 10.69 ±
1.07µm; interbranching distances: venules
22.93 ± 2.29µm, arterioles: 28.52 ± 1.52µm,
and intervascular distances: venules 12.14 ±
0.72µm. Branching angles were not measured
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in these specimens.
Measuring distances (e.g. lengths of vessels)
on single micrographs, here referred to as 2D
morphometry, resulted - due to the high depth
of focus of SEM images - in up to 44.47%
underestimation of true lengths when compared with multiple lengths measurements
available in the 3D morphometry system (Fig
4). The multiple lengths measurement tool in
our system proved particularly useful when
measuring lengths of the undulating intramyocardial capillaries (Fig 1c).
CONCLUSIONS
SEM of microvascular corrosion casts was introduced as a purely qualitative method to
describe the fine distribution of blood vessels
within tissues and organs [1]. In combination
with modern three-dimensional morphometry
techniques [9-12] it has become a quantitative
method to measure vessel parameters. Extensive Monte Carlo experiments and reproducibility tests revealed a very low measurement error (1 ± 0.5%; Fig 3) and found that the
dimension of measured structures relative to
the width of the measurement field (fv) is a
critical factor [10]. This strongly contrasts with
measurements in 2D where errors of up to
58% may occur [15]. This accuracy of 3D measurements together with advances in specimen preparation and improvements in injection technology now make SEM of microvascular corrosion castings applicable to the study
of microvascular beds of a wide range of tissues and organs during development, juvenile,
adult and aged states, both under physiological or pathological conditions. Presently, few
studies only use this method to demonstrate
the vascular beds of tumours [16-17]. Better
knowledge of the 3D arrangement of tumour
blood vascular networks can greatly contribute to a better understanding of tumour
blood circulation and therapies based upon it.
3D MORPHOMETRY
The technique finally allows us to prove if postulated optimality principles for the construction of vascular networks [18] match the real
situation in the circulatory system and other
fluid transporting systems [19-20].
REFERENCES
1. Murakami T. Application of the scanning electron
microscope to the study of the fine distribution of the
blood vessels. Archivum Histologicum Japonicum 32,
445-454, 1971.
2. Miodonski A. et al. Rasterelektronenmikroskopie von
Plastik-Korrosions-Präparaten: morphologische
Unterschiede zwischen Arterien und Venen. Beiträge
Elektronenmikroskopischer Direktabbildungen von
Oberflächen (BEDO) 9, 435-442, 1976.
3. Aharinejad S. et al. Scanning and transmission
electron microscopy of venous sphincters in the rat
lung. Anatomical Record 233, 555-568, 1992.
4. Kratky R.G. et al. Quantitative measurement from
vascular casts. Scanning Microscopy 3, 937-943, 1989.
5. Nelson A.C. Study of rat lung alveoli using corrosion
casting of the lung. A state-of-the-art review.
Scanning Microscopy 1, 1733-1747, 1987.
6. Schraufnagel D.E. Microvascular corrosion casting of
the lung. A state-of-the-art review. Scanning Microscopy
1, 1733-1747, 1987.
7. Zeindler C.M. et al. Quantitative measurements of early
atherosclerotic lesions on rabbit aortae from vascular
casts. Atherosclerosis 76, 245-255, 1989.
8. Zeith R. et al. Eine Methode zur quantitativen
rasterelektronenmikroskopischen Analyse des
myokardialen Kapillarnetzes anhand von
Korrosionspräparaten. Beiträge Elektronenmikroskopischer
Direktabbildungen von Oberflächen (BEDO) 17,
203-208, 1984.
9. Malkusch W. et al. A simple and accurate method for
3-D measurements in microcorrosion casts illustrated
with tumor vascularization. Analytical and Cellular
Pathology 9, 69-81, 1995.
10. Minnich B. et al. 3-dimensional morphometry in
scanning electron microscopy: A technique for
accurate dimensional and angular measurements
of microstructures using stereopaired digitized images
and digital image analysis. Journal of Microscopy 195,
Figure 3:
Accuracy of the 3D method. Results from Monte Carlo experiments. (a) Simulation of lengths measurements (uniform distribution; central perspective
projection; n = 5,000) (b) Simulation of angular measurements with 15°, 30°, 60°, 90°, and 120° angles (uniform distribution; central perspective projection; n = 25,000). Note that errors in measurements increase exponentially if the length of a measured structure (respectively the side lengths of a
triangle enclosing the measured angle as well as the angle itself) decreases in a linear manner relative to the field width of the SEM view (% fv). This
figure was published in [10] and is reproduced with the permission of the copyright holder.
23-33, 1999.
11. Komatsu F. et al. Measurement of geometrical
dimensions using scanning electron microscopy.
Journal of Electron Microscopy 48, 407-415, 1999.
12. MeX, Alicona GdBr, Berchtesgaden, Germany, 1999.
13. Lametschwandtner A. and Weiger T. Scanning
electronmicroscopy of vascular corrosion caststechnique and applications: updated review.
Scanning Microscopy 4, 889-941, 1990.
14. Aharinejad S. and Lametschwandtner A. Microvascular
corrosion casting in scanning electron microscopy.
Springer Verlag, Berlin-Wien, pp 380, 1992.
15. Minnich B. and Lametschwandtner A. Length
measurements in micro-vascular corrosion castings- 2D
versus 3D-morphometry. Scanning 22, 173 – 177, 2000.
16. Konerding M.A et al. Evidence of characteristic vascular
patterns in solid tumours: quantitative studies using
corrosion casts. British Journal of Cancer 80, 724-732, 1999.
17. Konerding M.A. et al. Impact of fibroblast growth factor-2
on tumor microvascular architecture. A tridimensional
morphometric study. American Journal of Pathology
152, 1607-1616, 1998.
18. Murray C.D. The physiological principle of minimum
work applied to the angle of branching of arteries. Journal
of General Physiology 9, 835-841, 1926.
19. LaBabera M. Optimality in biological fluid transport
systems. Contemporary Mathematics 141, 565-585, 1993.
20. LaBarbera M. Principles of design of fluid transport systems
in zoology. Science 249, 992-1000, 1990.
ACKNOWLEDGEMENTS
The authors are grateful to Ms. Synöve Tholo
for technical and Mr. Andreas Zankl for photographic assistance and to the Royal Microscopical Society for copyright permission (Fig
3).
Author’s details: Mag.Dr. Bernd Minnich, Department of Vascular- and Performance Biology, Institute of Zoology, University of Salzburg, Hellbrunnerstr. 34, A-5020 Salzburg, Austria. Tel: +43 (0)662
8044 5607, Fax: +43 (0)662 6389 5697.
Email: [email protected]
Figure 4:
Comparison of lengths measurements using (a) 3D-morphometry – anaglyphic (red/green) image (ComServ, Minnich & Muska OEG, Salzburg, Austria) and (b) 2D-morphometry (Optimas 6.5 Image analysis software, Media Cybernetics L.P., Carlsbad, CA, USA). Three examples of multiple lengths measurements showed the following results: Length 1 (7 segments): 49.72 µm (3D) vs. 48.60 µm (2D); Length
2 (6 segments): 72.48 µm (3D) vs. 40.20 µm (2D); and Length 3 (9 segments): 61.96 µm (3D) vs. 55.42 µm (2D). The amount of measurement error in 2D varies from 2.25 % (Length 1), 10.55 % (Length
3) to 44.47 % (Length 2) according to the spatial extension of the vessels measured.
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