SPECIAL

COMMUNICATIONS

A principle
for counting
tissue
structures
on random
sections1
EWALD
R. WEIBEL2
AND DOMINGO
M. GOMEZ3
Department of Medicine, Columbia University, College of Physicians and
Surgeons; and Cardio-Pulmonary
Laboratory, First Medical
and Chest
Services (Columbia University Division), Bellevue Hospital, New York City

or b) estimating
directly the number of these structures
in the volume
of a section of finite thickness (1-4).
This paper deals with a principle
on which a new
method
of counting
structures
contained
in a given
volume
can be based. Essentially,
the number
N of
such structures in the unit volume is derived from the
number
n of transections
through
these structures
counted in the unit area of random cut surfaces or histological sections, by applying
the general equation

counting

iv

k.n312

( I >

where the coefficient k will depend on the shape of these

structures
and on their volumetric
density, as will be
shown later. This method is applicable
if the following
four conditions
are satisfied :
I) The investigated
bodies must be randomly
distributed
within
the containing
volume.
This means
that any section through this volume, in whatever direction and at whatever
level, should pass through
the
same number
of bodies, so that a constant number
of
transections
is found per unit area of section. If the unit
area is infinitely
large, as compared
with the largest
possible cross section of the bodies, this should be true
in the strict sense. In practice,
however,
the standard
deviation
of the counts per field must be small.
2) The structures
to be investigated
must be comparable to finite bodies, each one well defined in every
direction.
Furthermore,
their shape must be such that
any section through
one structure may only yield one
single transection.
It is, however, not required
that all
bodies be of the same shape, provided
that typical
shapes,
e.g., spheres, cylinders,
etc., are randomly
distributed.
3) Typical
size groups must be randomly
distributed
and the largest possible size of the body must be small,
as compared
with the total containing
volume, although
the sizes of the bodies need not be homogeneous,
as demonstrated below.
4) The section on which transections are to be counted

URING
RECENT
YEARS studies have been in progress
in this laboratory
which should establish the means for a
correlation
of structure
and function
of the human
lung. An accurate quantitative
study of the architecture
of the human lung, in addition
to its morphological
interest, will serve as an indispensable
base for a physicomathematical
analysis of various aspects of lung physiology, thus leading
directly
toward
a correlation
of
structure and function of this organ.
In the course of these quantitative
morphological
studies the problem
arose how best to determine
the
number
of certain structures,
for example,
alveoli or
alveolar ducts. Most earlier investigators
have inferred
such numbers by a) comparing
the dimensions
of single
structures
with the over-all
dimensions
of the organ,
Received
for publication
3 I May I 961.
l This investigation
was supported
by a research
grant
from
the New York Heart Association,
and by Grant H-2001
from the
National
Heart Institute.
2 Investigator,
Health
Research
Council
of the City of New
York.
3 Associate
Director,
National
Institutes
of Health
Training
Grant HTS-5443.
343

Downloaded from http://jap.physiology.org/ by 10.220.32.247 on December 13, 2016

EWALD
R., AND DOMINGO
M. GOMEZ.
A principle
tissue structures on random sections. J. Appl.
Physiol.
1962.-A
new principle
is derived
from
17~2) 1 343-348.
mathematical
considerations
which
will allow
the calculation
of the number
of bodies contained
in the unit volume
by counting the number
of transections
on the unit area of a random
section.
The simple
equation
involves
two coefficients
related
to the volumetric
density
of the bodies in the volume,
and to
their configuration.
Means
for the accurate
determination
of
these coefficients
are given. The verification
of this principle
in
model
experiments
showed
a high degree
of reliability
of the
method
with average
errors of estimate
of 2-4y0.
The method
has been applied
to the counting
of alveoli
in five normal
human lungs, which
were found
to number
an average
of 300
million,
with a striking
constancy
from lung to lung.
WEIBEL,

for

WEIBEL

344

AND GOMEZ

If we now mix m different

sets of granules,
whereby
shape and size are constant within
each set, but vary
from set to set, then the total number N of granules in
the unit volume,
their total density e, and the total
number
n of transections
on the unit area of cut surface are
N=

Nl

N2

Nm

i
(6

EN

>

i=l
m
P

Pl

P2

Pm

C
i=l

pi

69

n;

(6 c >

n=

nl +

n2 +

nm =

x
i=l

A cylinder
parallel

which is inclined
to the (JJ, z) plane

at an angle t9 to the x axis

into thin slices of thickness

Continuous
sectioning
of cylinders
angles of inclination
8.

2.

varying

and

ellipsoids

at
whereby

must
sion
with
over,

be very thin, as compared

with the smallest dimenof the investigated
structure,
and as compared
the shortes t distance between the structures. Moreit must be randomlv
oriented.

DEVELOPMENT
ON

CUT

OF

PRINCIPLE

FOR

COUNTING

SURFACES

This principle
1s based on a theorem
developed
bY
Delesse (5) in 1847 which states that the fraction
pv
of a unit volume Y occupied by a given granular
component, which is randomlv , distributed,
is equal to the
fraction ps of the unit area S of a cut surface through
this volume covered by transections of these components.
This theorem is true, regardless of size or shape of the
granules.
If we now consider N granules of equal size and shape
with an individual
volume
u suspended
randomly
in
the total volume V, then it follows from Delesses theorem that

n-S

= p*s

(3)

where n is the number

of transections
of the granules
found on the area S of a cut surface through the volume;
S is the mean cross-sectional
area of one granule of a
given shape obtained
by random
sectioning
in any
direction;
it is related to the volume as
V =

p.j312

(4)

where p depends on the shape of the granules. This will

be discussed later.
From equations 2, 3, and 4 we derive the relationship
between
the number
N of granules
of equal size and
shape in the unit volume and the number n of transections on the unit area of a cut surface:

iVi

(7)

312
N1

dx.
FIG.

between

n,

PlJ6l

N2

Nm

will be different
for each set
on the configuration
of the corresponding

PI

depending
granules :
Vl =

P2

P1*Sl

-312

Pm

-312
= pys,

02

Vm =

Pm

.y

(8)

Since, within
each set, the individual
volume
ui
to the
is constant, the density pi is directly proportional
number
of granules
N; ; and, since the mean crosssectional area 1; , as defined in equation 4, is also constant
for each set, the number
n; of transections
is also proportional
to pi . Expressing
N; , pi and n; as fractions
4 of N, e and n, respectively, we obtain this set of premises :
nl

Nl

frN;

Pl

he;

N2

t2N;

P2

t2e;

n2

Nm

ErnN;

Pm

Em@;

?Zm =

Eln

(9)

t2n

f mll

Introducing
the corresponding
relations
of equation
g into the set of equations in no. 7, and taking the sum
of all Ni from Nl to N, , as in equation 6a, we arrive at
the relation
between
the total number
N of granules
in the unit area of a cut surface for a mixture of granules
of varying size and shape:

( >
m

N=

n3l2

ti

=i=l

F i

(4

l -*IA?

The shape factor ,8 introduced

in equations 4 and 6
is solely dependent
on the configuration
of the granules
and is independent
of their over-all size. If the bodies in
our m sets have the same configuration,
so that
p1 =

p2

but if they still are allowed

set, equation IO becomes

pm

&

to vary in size from

set to

n3l2
N

0.4;

(5)

which

is identical

with

equation 5, since by definition

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FIG. I.
is sectioned

Accord ing to equation 5, the relationship

and n; for ea ch set is obviously

PRINCIPLE

FOR

345

COUNTING
m

MEAN
(4

i=

cz:
i=l

We arrive at the conclusion

that the relation
(eq. 5)
between the number of granules in the unit volume and
the number
of transections
in the unit area of a cut
surface is independent
of a variation
in size of the granules, provided
their
configuration
is geometrically
similar.
This principle
can, therefore,
be applied
to
the counting
of a mixture
of granules of varying
size
as long as the /3; of all the granules is the same. If the
shape is varying,
the relative frequency
& of a given
IO,
component
enters the relation,
as shown in equation
so as to provide a composite coefficient for ,8.
EXTENSION

OF

PRINCIPLE

TO

COUNTING

ALONG

y is similar to ,8 and relates the mean

l of a given two-dimensional
figure

COEFFICIENT
BODIES

( I 7)

where Scz,e) is the cross-sectional

any value of x and 8. The total
is, then,
u =

( I 3)

AND

VARIOUS

I
JO

area of the body for

volume u of the body

H(e)
&z,e)-

dx

( r 8)

H(s)= S(e)

where S(d) is the average

cross-sectional
area
obtained by sectioning
the body in one given direction
8,
and H(e) is the projection
length
of the body on the
x-axis at this inclination.
If this body was generated
by revolution
around its
axis, it is sufficient to vary the angle of inclination
0
from o to go0 (Fig. 2) to obtain the mean cross-sectional
area, S, resulting
from sectioning
the body in any
direction :
T I2

SC-7r

7F/2 de

2u

Sce,*dO = -

7r
0

We)
0

4, this mean cross-sectional

area,
As stated in equation
S, is related to the volume u of the body through
a
coefficient ,8 which is independent
of the size of the body,
but depends solely on its configuration.
It will be expressed as a function of the ratio E relating the diameter
of the body perpendicular
to its axis to the axial length
(eqs.

20,

25).

The values of s and ,B have been calculated

S

(I 9)

for various

( I 4)

= 1%

For circles of anv size:

y=-

1.273

7r

If this
volume
granules
counting
granules,

line is thrown on a random section through

a
containing
granules,
the number
N of these
in the unit volume can also be obtained
from
the number n of passages of the line through
by using the equation

where
k;

(I 6)
FIG.

which

for spheres has the value 6 =

.767.

to ratio
sphere.

3. Values
of
E of diameter

coefficient
over

of
length

configuration,
for cylinders,

p, with
ellipsoids,

respect
and

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The coefficient
linear intercept
to its areas:

n 2
YP

AREA
,6 FOR

Consider
a body of given shape (see below) with its
axis lying in the plane, X, y, of a three-dimensional
coorat
dinate system, X, y, z (Fig. I), and being inclined
an angle 0 to the x-axis. A slice of infinitely
small thickness, dx, cut parallel to the y, < plane will have a volume

LINES

Rosiwal
(6) has generalized
the theorem
of Delesse
pL of a line passing
(5) in showing
that the fraction
through a granular
component
of the containing
volume
is equal to the planimetric
fraction ps of a cut surface
covered by transections of the component
and, therefore,
equal to the volumetric
density pv of the granules in the
total volume. We can, therefore,
extend our principle
to the counting
along a line of known length L thrown
at random
on a surface containing
two-dimensional
figures.
If we count the number n of times this counting < line
the two-dimensional
of unit length passes through
figures, th .e number
n of figures in the unit area fol lows
from
?Z=

CROSS-SECTIONAL

OF CONFIGURATION

WEIBEL

346
bodies, in particular
for cylinders
and revolution
ellipsoids. Most of the shapes which are frequently
encountered in biology can be assimilated
to one of these tYPical bod
Cylinders. The mean cross-sectional
area, S, for cylinders of various values of
E =-

(4

was calculated
according
to equation rg. The projection
length of cylinders at any inclination
0 is given by

SC=

0
D
-

21 into

equation

rg,

one

obtains

2
l

%E)

(4
-

z+

(&q-2

_-loge

(1/q-

J&h-T-s

+ 4

I&/g-y

The coefficient
P for cylinders
of various
now defined by introducing
equation 22 into
so that

lengths is
equation 4,

where Q) is defined by equation 22. P is a function

of
E alone; it was plotted in Fig. 3. A cylinder with E < I
is elongated;
one with E > I is a flat disc.
Ellipsoids (Fig. 26). The mean cross-sectional
area of
ellipsoids
generated
by rotation
about one of the axes
can also be obtained by applying equation rg. The integration of the inverse of the projection
length, H(e) , for
ellipses,
however,
yields
a very involved
equation.
since the resu .lting very laboriWe omit its presentation,
ous computation
can be avoided
the
bY determining
function 9cE) of the more general equation:
?E = b2+&e)

E =-

where a is the
tion, and b is
soids p is again
equation 24 into

( 2 4)

semigraphical

b
( 2 5)

semiaxis coinciding
with the axis of rotathe semiaxis rotated about a. For ellipa function of E alone, since by introducing
equation 4,

(2 6)
The

values

of ,0 obtained

by equation

26

ss = y&v2

are again

( 2 7)

Therefore,
for a sphere ,& = I .382 . . . .
Polyhedra and prisms. Polyhedra
which can be circumscribed to a sphere will have a value of p slightly higher
than that of a sphere, since the corners will tend to
reduce the value of S. The higher the order of the polyhedron, the closer to the sphere it will be. For elongated
or flat polyhedra,
or for prisms, similar considerations
will have to be made in relating
them to ellipsoids
or
cylinders of corresponding
E. In general, the P for polyhedra will be located
somewhere
between
the p of
cylinders
and that of ellipsoids.
For prisms p may be
somewhat
higher than that of corresponding
cylinders.
DETERMINATION
IN

( 2 3)

corresponding
to equation 22, by a direct
procedure.
The ratio E is, in this case,

plotted on Fig. 3. This curve follows the trend of the

curve for cylinders.
It is located
somewhat
lower,
indicating
that the mean cross-sectional
area of an
ellipsoid
is larger than that of a cylinder
of the same
E. Values of E < I refer to prolate,
those of E > I to
oblate ellipsoids.
Sphere. The sphere is, evidently,
the special case of
the ellipsoid
for which E = I. Its mean cross section is
well defined as

CALCULATING

OF
N

PA RAMETERS
BY

INVOLVED

EQUATION

Number of transections. A field of known area is superimposed on a random section through the volume containing the unknown
number of bodies. All the transections which are entirely
within
the field are counted.
All those transections
intercepted
by the left and upper
field border are also counted, whereas those intercepted
by the right and lower border are neglected.
Coeficient ,& If the bodies which are to be counted can
be assimilated
to one of the typical
bodies described
in the preceding
paragraph,
the ratio E has to be deterfl
mined, as in equations 20 and 25. The corresponding
can then be read from the graph in Fig. 3.
Volumetric density p. The fraction p of the volume occupied by the bodies which are to be counted can be determined
by various methods, of which mainly two are
in use at present. Both these methods operate on random
sections through
the volume containing
the bodies, on
which the counting is also carried out.
One method is based on the principle
developed
by
Delesse (5) and Rosiwal
(6) for the volumetric
analysis
of the composition
of rocks. It has been applied
to
the microscopic
level by Schuchardt
(7), using the socalled integrating
eyepiece
of Leitz. The application
of this method will be extensively
described elsewhere
(in preparation).
The volume proportion,
p, can also
be determined
by the point
counting
method
of
Hennig (8). This method can be applied as well to the
macroscopic
as to the microscopic
range of dimensions.
EXPERIMENTAL

VERIFICATION

OF

PRINCIPLE

The validity of this principle

was checked in a series
of simple model experiments.
A known number of peas

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Introducing
equation
upon integration

AND GOMEZ

PRINCIPLE

FOR

COUNTING

347

7
APPLICATION
OF

ALVEOLI

OF
IN

PRINCIPLE
NORMAL

TO
HUMAN

500

89

61.2

.343

LUNGS

In a study on the dimensions

of the human
lung,
which
will be reported
in detail
(in preparation),

B. Beans
2
3
4

100
295
5

630
90
II52

* Counted number
TABLE

2.

.*3
.7
.20

I,

8, M

16,
3, 34,
4, 48,
5, 74,

F
M
M
F

28.8
37.5
24.0

&I

.6

93.5 -6.5
$2.0
30
116

+I.O

Mean

f3.2

per IOO cmz.

Number of alveoli by c0untin.c on sections

A IO2

945
980
2100

2600
2180

.58
.60
.58
.53
.5*

NA Id

52.4f6.45
4g.8f7.10

322f60
2g2f7z

31.1zt7.24
24.8f4.97
27.gf4.56

148f54
11of31

131f31

2g6.Io6fII.I

NAT

304.10

288.106
310.106

286.106
286.106
(f3.9%'0)

V,, = total volume of fixed lungs after histological prepara; PA = volumetric

density Of dVeOli ; nA = number Of alveolar transections per I cm2 histological section; NA = number
of alveoli per I cm3 lung parenchyma; NAT = total number
of alveoli; NAT = group average.
tion

transec-

I.70
2.62

of transections

NAT

FIG. 5. Section of pulmonary parenchyma containing

tions of alveoli. Counting fields superimposed.

2.175

.39
.84
.*5

PA

2,

53 +2.6
tiean

COUNTING

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was embedded
in a known
volume of gelatin (Fig. 4A).
The total volume
of the
peas was measured by water
displacement,
thus allowing
a direct
determination
of
p. After solidification,
the
block was sliced and the
number
of transections
was
counted on the cut surfaces
of each slice. The peas were
assimilated
to
ellipsoids
having a ratio E = .85, yielding a coefficient /3 = 1.42.
The results of these experiments are listed in Table IA.
The calculated
number
of
peas differed, on the average,
by only I .6 % from the actually embedded
number,
a
FIG.
4. Counting model experiments. Photographs of cut surfaces of blocks containing, A,
result which is highly satispeas and, B, peas and beans.
factory.
TABLE
I. Counting of (A) peas and (B) beans
In part of these model experiments,
segments of string
beans of known length and diameter
were embedded
(cylinders) in model exberiments
along with the peas, so as to simulate
cylinders
with
Tot.
varying E (see Fig. 4B). The results of those experiments
VOIU01. 01
n/m*
Exp. No.
netric
B
klatil
in which fair random mixing
(SD of counts
per section
ensity
Hock
had to be smaller than IO %) could be achieved are
listed in Table I B. Although
the accuracy of the estimate
A. Peas
is not as good as with peas, it is still satisfactory, conI
.62
IOI .6 530
520
+I.9
580
2
60.9
354
+I.1
sidering that beans are only roughly
comparable
to
350
630
.365
-1.0
.286
396
400
47.7
3
90
cylinders.
Moreover,
they did not distribute
as ran1152
.26g
40.6
406
f1.5
400
4
domly as peas (Fig. 4B); they often had a tendency
to
.615
III.0
506
+I.*
5
500
483
stick together or to adhere to the wall of the container.
6
408
f2.0
.278
400
49.0
891

WEIBEL

348

GOMEZ

vestigators
varies between
66 million
(3) and 1800
million
( I 0) ; several
figures,
however,
are around
300-400
million
(I I-I 3). These numbers were mostly
obtained by comparing
the estimated volume of a single
alveolus with the estimated total volume of the alveoli;
it is obvious that many errors are inherent
in this appreach.
CONCLUSIONS

A principle
has been developed
by mathematical
considerations
which will allow the calculation
of the
number
N of bodies contained
in the unit volume bv
counting
n transections
through
these bodies on the
unit area of a random
section through
that volume, if
the configuration
of the bodies (coefficient ,8) and their
volumetric
density in the volume (p) are known; means
are given for the determination
of these two coefficients.
The general
equation,
applying
to uniformly
shaped
bodies, which may vary in size, is:

Generalizations
of this equation are given for mixtures
of bodies of different shape, requiring
a composite coefficient ,8 (eq. lo), and for counting
of these bodies along
lines of unit length
(eqs. 13, 15). Under well-defined
conditions,
this principle,
which
has been tested in
model experiments,
may be applied
to many problems
of counting, as well in the macroscopical
as in the microscopical range of dimensions.
It has been applied to the
counting
of alveoli in the normal human lung.
We acknowledge
Dr. Dickinson
W.
suggestions.

our indebtedness
to Dr. Andre Cournand
and
Richards
for their
interest
and stimulating

REFERENCES
I. WILLSON,
2. MACKLIN,

3. ELZE, C.,
457> 1956.
4. HIERONYMI,

H.

G. Am.

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30: 267,
1922.
Inst. Med.,
Chicago
18 : 78, 19-p.
AND A. HENNIG. 2. Anat. Entwicklungsgeschichte
C.

C.

G.

Proc.

Ergebn.

allg.

Path.

path.

M. A. Compt. rend. 25 : 544,

6. ROSIWAL, A. Verhandl.
geol. Reichsamts,
7. SCHUCHARDT,
E. 2. wiss. Mikroskop.
62
8. HENNIG, A. Verhandl.
Anat. Ges. 54: 254,
5. DELESSE,

41 : 1960.

Anat.

I I.

AEBY,

C.

Der

Bronchialbaum

der

p.

1954.

I 43, 1898.

: Springer,
1880.
I 2. MARCUS,
H. Morph.
Jb. 5g : 562,
Lung.
13. VON HAYEK, H. The Human

Saeugetiere

und

Diseases

5 (Part.

des Menschen.

Leipzig

1847.
Wien
: g,
1957.

I 19 :

Am. Rev. Respirat.

g. WEIBEL, E. R., AND R. A. VIDONE.
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Cited by W. BARGMANN in Handb.
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3) : 800, 1936.

1928.

New

York:

Hafner,

1960.

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the number
of alveoli has been counted in five normal
human lungs of different
age, sex, and size (Table 2)
by applying
this principle.
The lungs had been prepared by a standardized
procedure
(g), providing
uniformly
inflated
specimens.
Being rather
densely arranged in the parenchyma
(Fig. 5), the alveoli can be
regarded
for this purpose
as randomly
distributed,
so that the principle
is applicable.
They are small
polyhedral
bodies, for which
the coefficient
of configuration
,8 was estimated to be I .55, between a sphere
and a cylinder of E = I.
The microscopic
integration
method
of Schuchardt
(7) was used to determine
the volumetric
fraction,
occupied by alveoli. As seen in
PA > of the parenchyma
Table 2, alveoli constituted
about 60 % of the parenand about 52 % in the
chyma in the three younger,
two older individuals.
In each lung the number of alveolar transections
was
counted
microscopically
on 50 fields measuring
g. I .
outlined
by an eyepiece reticle.
The fields
I o-3cm2,
were randomly
chosen from five different regions of the
lung. The mean number
of transections
per I cm2
of section was calculated
(Table 2). This figure was then
introduced
into equation 5, with
the corresponding
PA and p, to yield the average number
of alveoli per
The total number of
I cm3 of pulmon arY Pa renchyma.
alveoli in both lungs was then obtained
from mulper I cm3 with go % of the total
tiplying
the number
was estimated
to represent
the
lung volume,
which
parenchymal
volume.
As can be seen from Table 2, the average number of
varies inversely wi th
alveoli per I cm3 of parenchyma
the lung volume. As a result of this the total number
of alveoli is calculated
to be very constant from lung
to lung with an average of 296 million and a coefficient
of variation
of only 4 %.
The total number
of alveoli estimated
by other in-

AND