Opinion TRENDS in Neurosciences Vol.24 No.

1 January 2001 11

Two-dimensional ‘unbiased’ data, and have contended that this method

also rests on a variety of assumptions11. It has even
been asserted that 2-D techniques might yield an

versus three- accurate assessment of numerical density, even when

differences in the size of objects are present12, if this
potential confounding effect is corrected

dimensional cell mathematically using an equation described by

M. Abercrombie in 1946 (Ref. 13). There are other
factors, however, that must also be considered when

counting: a practical assessing the relative strengths and weakness of

various cell-counting methodologies. In the discussion
that follows, these issues are explored from the

perspective vantage point of several theoretical and practical

considerations that might influence the precision and
cost-effectiveness of object-counting in the brain (see,
for example, Fig. 1).
Francine M. Benes and Nicholas Lange Over the last several decades, there has been a
tremendous amount of emphasis within the
stereology community for reducing estimation bias,
In recent years, it has been argued by some neuroanatomists that three- and relatively little attention has been given to
dimensional (3-D) counting approaches must be used in studies of neural reducing an estimate’s variability or to the possible
systems, so that ‘unbiased’ counts of neurons can be obtained. By contrast, bias of an estimate of variability14. More recently,
two-dimensional (2-D) cell-counting methods are said to be ‘assumption- stereologists have devoted greater effort to these
based’ and to yield inaccurate results.Working from the premise that all latter important areas15–18. Overall, the properties of
scientific methodologies are assumption-based and suffer from inherent estimators of cell number are never entirely devoid of
biases, the current review considers the relative strengths and weaknesses of ‘model-based,’ ‘assumption-based’ or ‘design-based’
2-D versus 3-D counting approaches.This comparison is from the standpoint of premises, and the common stereological (and
predictive performance with respect to bias, variance and fidelity to the actual mathematically convenient) assumption of complete
spatial arrangements of cells in the tissue under study.When these spatial randomness for complex systems, such as the
considerations are taken, together with the human resources that are required brain, does not apply in practice (Fig. 2).
in using either methodology, 2-D methods offer more practical alternatives that
might even provide more scientifically accurate estimates compared with their
3-D counterparts.
some investigators have begun to
challenge the premise that optical
‘The cerebral cortex is…composed of several layers
where neuropil and cells show characteristic types
disector counting yields ‘unbiased’
and densities. This will evidently impose certain data...
sampling conditions and require appropriate
adaptations of the general stereological methods.’
H. Haug, in E.R. Weibel, 19791. The structural neuroscientist interested in cell
counting is confronted with the treacherous
Two recent articles, a commentary2 and a review3, intersection of theory and application. It is extremely
have prompted an intense debate as to whether rare that a person can become expert in both worlds.
so-called ‘unbiased’ stereological (three-dimensional, The theorist might find applied data ‘interesting,’ yet
or 3-D) methods of cell counting are superior to so- might not really care about the scientific implications
Francine M. Benes*
Laboratory of Structural called ‘assumption-based’ (two-dimensional, or 2-D) of his or her theory in practice. The empirical scientist
Neuroscience, McLean approaches. In essence, these articles reflect a shift in might seek out the best mathematical abstractions
Hospital, Belmont, MA the field of neuroscience toward the use of optical and apply these ideals to a problem without adequate
and the Program in
Neuroscience and Dept of
disector counting techniques4–9 for determining the understanding of the underlying assumptions.
Psychiatry, Harvard total number and numerical density of neurons, Although all empirical methods employ assumptions
Medical School, Boston, synapses and other neural structures10. Indeed, there and principles, current stereological theory is falsely
MA, USA.
is now a widespread belief that this latter approach believed to be almost assumption-free19. It is far more
*e-mail: benesf@
mclean.harvard.edu must be used for all studies in which objects are being dangerous to think that one employs no assumptions
counted, whether they are neuron somata or axon whatsoever, than it is to make one’s assumptions
Nicholas Lange
Statistical Neuroimaging
varicosities3. A central issue that drives this position explicit and testable. As we attempt to show below, it is
Laboratory, McLean is the fact that differences in object size between two simply incorrect to think ‘unbiased counting methods’
Hospital, Belmont, MA groups under investigation can result in distortions of equate to ‘assumption-free methods’. Most
and the Dept of
their apparent number and density10. More recently, importantly, the central assumption of stereological
Psychiatry, Harvard
Medical School, Boston, however, some investigators have begun to challenge theory – that objects are arranged in a completely
MA, USA. the premise that optical disector counting yields random fashion – does not apply to the brain. A generic

http://tins.trends.com 0166-2236/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0166-2236(00)01660-X
12 Opinion TRENDS in Neurosciences Vol.24 No.1 January 2001

Fig. 1. Characteristic bias in 2-D techniques10. However, proponents of the

six-layered organization
2-D approach maintain that this confound can be
of the human neocortex.
The diagram depicts a controlled through an Abercrombie correction11,12. By
thick section of human I contrast, issues related to the sampling window size
neocortex in which both and sampling in a distorted (x,y,z) coordinate system,
pyramidal and
non-pyramidal neurons
in addition to the question of cost effectiveness have
II
are present throughout not, as yet, been compared for 2-D and 3-D
the layers.The size and methodologies.
density distribution of
these neurons varies
among the laminae and Size of the sampling window in the (x,y) plane
even within a single layer. The sampling window size is one of the most crucial
The large yellow box is factors in determining the fidelity of a cell-counting
III
approximately 150 µm on
one side, whereas the
method (see Fig. 1). A general rule that can be applied
small red one is only to the choice of a window size is to determine what is
50 µm on one side.The needed to obtain a ‘representative’ sample. As shown
large yellow box contains
in Fig. 3a, this is a particularly important issue in
many more cells and can
sample more effectively some brain regions, such as human cortex, where the
the spatial distribution of packing density of neurons is low (Blinkov and
neurons compared with
IV Gleser22). Thus, a sampling window that is small will
the small red box.
tend to produce a rather distorted view of the
distribution of neurons in tissue space (Fig. 1). In
cerebral cortex, for example, the effect of this
V confound would vary on a layer-by-layer basis even
within individual laminae, because of the complex
arrangements of cells that vary according to both size
and numerical density (Fig. 1).
The ‘unbiased’ method of cell counting typically
employs a sample window that is 50 µm on one side10.
Because the average neuronal diameter could be
VI 15–20 µm (e.g. for human pyramidal neurons), the
number of such cells that can be represented in this
small sampling window is actually relatively low
(Fig. 3). This problem could be overcome by using
multiple windows, but this strategy might repeat the
same sampling error and result in a distorted set of
TRENDS in Neurosciences
counts. In the cortex, columns of narrow windows are
typically employed (Fig. 3); however, three or even
five such columns is still a relatively small number
Poisson model is one in which there is no systematic and might be inadequate to overcome the sampling
variation and neighboring regions have neither higher error, which is inherently present in a narrow
nor lower densities20. As ‘practitioners’, we find column. Results obtained with small windows
complete spatial randomness implausible for neural containing 0, 1, or perhaps 2 neurons might be
systems (Fig. 2) in which the spatial arrangements of reproducible, and the common, yet invalid,
neurons are not Poisson21, that is, these arrangements assumption of complete spatial randomness might
vary systematically and have differing numbers and remain unchallenged. However, these findings will
densities in different neighboring regions. lack requisite fidelity to the actual neuronal structure
Another important issue is the fidelity with which of the cortex. A viable alternative is to use a much
cells and other objects can be detected in a 3-D tissue larger sampling window (e.g. 150–300 µm on one
system. In other words, any sampling protocol must side), such as those employed in 2-D cell-counting
be capable of producing a reliable estimate of object methods, to obtain a more accurate assessment of the
numbers that is ‘representative’ of their actual spatial distribution of cells in the (x,y) plane23–25.
distribution in a tissue section. The principal factors At issue here are tradeoffs between window size,
influencing the scientific utility of a counting protocol precision of cell counts and estimation of spatial
are: (1) object size, shape and orientation; (2) distributions. Assuming a spatial field of cells
numerical density; ( 3) sample window size and object distributed in a completely random fashion, which is
spatial distributions in 3-D space, and (4) cost- the assumption made by the 3-D optical disector
effectiveness. Proponents of the optical disector methods, it would make sense to choose a small x–y
method have addressed extensively the influence of window size and a large number of disectors. Under
cell size, shape and orientation on the determination such an assumption, the expected number of cells and
of numerical density, believing this to be a source of the variance of an estimate of this number are

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(a) Neurons
Exclusion Clustering
−0.10 −0.05 0.00 0.05 0.10
(b) Neurons
Exclusion Clustering
−0.10 −0.05 0.00 0.05 0.10
Fig. 2. Non-Poisson
neuronal arrangements in
primary motor and
anterior cingulate cortex.
Non-Poisson neuronal
arrangements in all layers
of primary motor cortex
(Brodmann area 4) are
shown in (a) and of the
anterior cingulate cortex
(Brodmann area 24) are
shown in (b) for 12 control
subjects. A total number
of 2098 neurons and 3443
glia were examined
according to a descriptive
index of ‘exclusion’
(shown in red) or
‘clustering’ (shown in
green); see Ref. 21 for
computational details. For
neurons in both regions,
note clustering at layer I
and exclusion in all other
layers. Such non-Poisson
neuronal distributions
contrast sharply with
those of glia, which are
neither strongly
exclusionary nor
clustered at all layers and
in both regions. Instead
glia exhibit spatial
patterns that are close to
being completely
random.
Opinion TRENDS in Neurosciences Vol.24 No.1 January 2001
Glia
Layer Exclusion Clustering
−0.10 −0.05 0.00 0.05 0.10
I I
II
III
IV
V
VI
Glia
Layer Exclusion Clustering
−0.10 −0.05 0.00 0.05 0.10
I
II
III
IV
V
VI
TRENDS in Neurosciences
identical (being a feature of every Poisson probability
distribution). Thus, a small window size could indeed
result in a small standard error and a high degree of
reproducibility. However, these features alone do not
necessarily reflect the degree of natural variability
and the complexity of the system under study. Hence,
results from such a simplistic approach would not
necessarily be generalizable. As shown in Fig. 2, given
that the distribution of neurons in cortical sections is
not completely random21, recommendations that rest
on this faulty assumption are unreliable. Early
analyses of optimal window sizes for in vitro culture
and plant specimens26,27 employed the
mathematically convenient Poisson assumption of
complete spatial randomness and indicated that
expected counts of 0.7 to 3.0 cells per window are
optimal (see Box 1 for a derivation of this result).
Indeed, in an informal survey of recent applications of
the disector principle, a majority reported mean cell
counts per disector within this range. More recently,
Howard and Reed28 have advised an x–y window size
that records between 2–5 cells on average, pointing
out that counting 0–1 cells per window might be
prohibitively tedious.
There is a major problem with the preceding
analytical argument (Box 1) and small window size
recommendation: in the brain, neurons are not
distributed in a Poisson fashion (Fig. 2), but instead
show inter-neuronal spacings that are much larger or
smaller than would be predicted by such an
assumption21. Only glial cells exhibit Poisson
randomness throughout the cortical matrix (Fig. 2).
Mathematical and statistical details supporting these
findings are given in Box 2. Therefore, it makes sense to
design a sampling plan that employs a larger window
size, so that an accurate assessment of the spatial
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13
(a) (b)
I
II
II
III
III
V
V
VI
VI
WM
WM
TRENDS in Neurosciences
Fig. 3. Low-power photomicrographs showing layers I–VI of the
cortical mantle in human anterior cingulate cortex. (a)The section was
prepared from a formalin-fixed tissue block that was cryoprotected,
frozen and cut on a cryostat at a thickness of 20 µm.The packing density
of cells is low and there are large areas of neuropil surrounding the
neuronal cell bodies. On the left side of the section, a contiguous series
of red boxes (each 150 µm × 150 µm), depict how a column of the cortex
would be sampled using a 25 × objective, with a depth of field that
would include all of the cells in the section. By contrast, on the right side
of this section, a similar series of boxes is depicted of ~50 µm × 50 µm.
Within the larger boxes on the left, there is a relatively large number of
cells included that reflect the density distribution of neurons within
each of the laminae. In the smaller boxes on the right, however, each
box contains a very small number of cells and, in some cases, there are
no cells present at all. (b)This section was taken from approximately the
same portion of the block as that shown in (a), except that the tissue was
dehydrated, imbedded in celloidin and cut at a thickness of 40 µm. Note
the shrinkage of the cortical mantle across layers I–VI with (a). In
addition the packing density in (b) is higher as a result of both the
shrinkage of the tissue and the greater thickness of the section.The
optical disector counting method would employ a column of small
boxes similar to those shown in the right of (a). A 100 × objective would
be used to define two focal planes through the z-axis in this section.
Abbreviation: WM, white matter.
distributions of cells can be obtained. Another
important reason for a large window size is an
additional requirement of several recent strategies for
assessing the precision of estimated counts16,17. These
methods employ probability distributions that differ by
a multiplicative constant from those of the more
commonly used point–pair distances described here29
(Box 2). Thus, even if one is interested only in cell
counting, recording spatial locations of cells in disectors
14 Opinion TRENDS in Neurosciences Vol.24 No.1 January 2001
Box 1.When does a small sampling window size make sense?
Neuroscientists employing the optical disector often
use many disectors with small window sizes.This
makes sense only if the objects in question are
distributed completely at random within the sample
and if one is interested only in object number and not
in their spatial arrangement. Complete spatial
randomness is mathematically equivalent to a
Poisson assumption.Technically, this is how one
would arrive at a recommendation for small window
size under such an assumption. If a is the area of a
square window with cell packing density λ and n is the
average number of cells observed per window
counted with d disector windows, then an estimate of
λ defined as:
∧
λ = n/a
has standard deviation equal to
σ ( λˆ ) = λ /( ad )
The (Fisher) information for ␭, related inversely to
variance, is
I = ad/λ
with a large window size will have the added advantage
of producing data-driven estimates of precision.
Precision and bias of an estimate are two
fundamental issues in stereology and bear directly on
the comparison of 2-D and 3-D methodologies. Using a
‘bull’s eye’ analogy from elementary statistics, correct
use and understanding of these terms have been
recently addressed3, providing examples of estimators
that are biased and unbiased, precise and imprecise.
One can go further and combine bias, precision and
prediction error in a single, simple formula to further
understand relations between these important
concepts (Fig. 4). Consider two targets, one of which
shows an estimator that is slightly off-target, yet is
precise, exhibiting low variability around its average.
The other target shows an estimator whose average
position is right on target, yet is imprecise, because its
locations around its average are highly variable
(Fig. 4). In these cases, bias and variance combine to
yield the prediction error. Some form of bias/variance
tradeoff is required in order to avoid too much bias
(‘underfitting,’ or not taking important features into
account) on the one hand, and too much variance
(‘overfitting,’ or accounting for unimportant features)
on the other30,31.
A concrete example of a bias/variance tradeoff is
shown in the primary motor cortex. As noted above21,
neurons are more clustered in layer I, but in layer II
they are further apart than complete randomness
would predict (i.e. they exhibit exclusionary
spacings; see Fig. 2). Therefore, if a simplistic
Poisson assumption is employed, this will produce
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increasing as either a, d or both increase, as one would
expect. If one defines an ‘emptiness probability’ p as
the probability of an empty window, then
p = e–λa
if the objects are distributed completely at random
within the sample.The information re-expressed in
terms of emptiness probability p is found to be
I = a2dp/(1–p)
for a large number of disectors. Maximizing
information across disectors by varying window size,
one finds that the optimal mean number of cells
counted per disector is
nmax = λ amax = 1.59
and is associated with an emptiness probability of
about 20%. Information remains high within the range
of 0.7 to 3.0 cells per window when objects exhibit
complete spatial randomness. However, as shown in
the text, this is not the case for neuronal arrangements
in the brain, and hence use of small window sizes is ill
advised, regardless of the number of windows and
their placements throughout the sample.
predictions of spatial arrangements of these cells
that are biased because one has not paid adequate
attention to important neuroanatomic facts. As
noted, even if only estimating the number of
pyramidal neurons in these two layers, an estimate’s
precision will also be biased if a completely random
distribution of cells is assumed. By contrast, spatial
distributions of glia are completely random across all
neocortical layers21. In this case, adopting an overly
complex estimator of glial patterns that includes
layer effects will result in complicated predictions
that perform poorly because of the attention given to
spatial features that do not matter for glia. Thus,
sampling window size and location in the (x,y) plane
interplay with the degree of exclusion or clustering
and thereby have a marked effect on the amount of
precision and bias that is inherent in estimates of cell
number and density.
The z-axis
The principal strength of the optical disector
method is that it eliminates biases arising from
differences in object size, shape and orientation by
obtaining ‘unbiased’counts along the z-axis3. Using
this approach, the tissue section must be thick
enough to obtain samples from a through-focus
series of image planes (Fig. 5a,b). Most histological
sections that are mounted on glass slides collapse
along the z-axis when the tissue dries.
Consequently, a section that is 20 µm thick might
have a final thickness of only 5–6 µm once it has
completely dried on the slide. For the optical
Opinion TRENDS in Neurosciences Vol.24 No.1 January 2001
Box 2. Quantitative description of spatial pattern: an example
As an example of the scientific importance of analyzing
spatial distributions of neurons, consider the careful
identification and positional recording of pyramidal
neurons in layer II of human cingulate cortexa,b. One
quantitative descriptor of spatial pattern for such a
study is a function, K, that depends on point–pair
distances, r. This function is defined as followsc,d:
K(r) = λ–1E (number of other cells at a distance ≤r
from an arbitarily chosen cell)
In equation (1), λ is the intensity of a null hypothesis
Poisson process and E is the expectation operator
(which, in this case, takes the form of a weighted
average). In stereological fashion, under the null
hypothesis, the value of K(r) for every distance r is
equal to πr 2, the area of a circle of radius r centered
on each observed cell. An approximately unbiased
estimate of K(r) isc:
Kˆ ( r ) = ( a / n )∑ wij−11( dij ≤ r ) / n
i≠ j
In equation (2), n is the observed number of cells in a
sampling window of area a, a/n is an estimate of λ−1,
and the weighted average
∑ wij−11(dij ≤ r ) / n
i≠ j
replaces E(·). In this average, 1(dij ≤ r) is equal to 1
when dij, the point–pair distance between cells i and j,
is less than or equal to r, and otherwise is equal to 0.
Finally, there is a correction for edge effects, wij, equal
to the proportion of the circumference of a circle of
radius dij centered at cell i contained within a square
or rectangular window. If K(r) is approximatety πr2,
then the arrangement of cells is Poisson at spatial
disector method, this problem is overcome using two
different strategies. The first is to imbed the tissues
in celloidin, a resin that allows the blocks to be cut
at a thickness of 40 µm; but, unfortunately, this can
result in up to 40–70% shrinkage prior to sectioning
(Fig. 3). The second approach is to cut sections that
are not imbedded in resin at a much greater
thickness of 70–80 µm if imbedding interferes with
specialized staining techniques, such as in
immunohistochemistry. For the celloidin-imbedded
sections, in spite of their rigidity, there is still
significant collapse (~35%) along the z-axis. Using a
z-axis encoder and through-focus measurements,
the final thickness of slide-mounted celloidin
sections is approximately 26 µm. Together with the
shrinkage prior to sectioning, the distortions
inherent in this approach are considerable. Using
the second approach, sections that are not imbedded
in resin exhibit a more-marked contraction along
the z-axis when they are mounted on glass slides. In
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15
scale r. If K(r) is less than πr2, then there are fewer cells
than would be expected under a Poisson assumption,
as a result of the presence of exclusionary ‘sub-
Poisson’ distances between neurons at that scale. If
K(r) is greater than πr2, then there are more cells than
would be expected under a Poisson assumption,
because of ‘supra-Poisson’ clustering at that scale.
Therefore, a new function defined by
(1) K ∗ (r ) = Kˆ ( r ) / π − r
will accentuate non-Poisson departures in spatial
arrangements.The square root is applied to K(r)/π as
an appropriate ‘variance stabilizing’ transformation.
Estimation of the K* function from the (x,y)
coordinates of pyramidal neurons in layer II of human
cingulate cortex revealed exclusionary spacings of up
to roughly 60 µm between cellsb.The small window
sizes and ‘skips’ of systematic random sampling with
optical disectors preclude observation of such
(2) arrangements at distances greater than the spatial
extent of the disector window (50 µm × 50 µm) minus
average neuronal diameter (15–20 µm). Such a
finding would not have been possible using the
common optical disector sampling strategy with a
small window size in the (x,y) plane.
References
a Benes, F. and Bird, E. (1987) An analysis of the arrangement
of neurons in the cingulate cortex of schizophrenic patients.
Arch. Gen. Psych. 44, 608–616
b Diggle, P.J. et al. (1991) Analysis of variance for replicated
spatial point patterns in clinical neuroanatomy. J. Amer. Stat.
Assoc. 86, 618–625
c Ripley, B.D. (1976) The second-order analysis of stationary
point processes. J. Appl. Prob. 13, 255–266
d Stoyan, D. et al. (1987) Stochastic Geometry and Its
Applications, John Wiley & Sons
this case, although shrinkage prior to sectioning can
be less than with celloidin, the collapse along the
z-axis can be as great as 67%. Thus, a tissue section
that is 70 µm thick could collapse to a thickness of
23 µm (Fig. 5b). Importantly, this collapse probably
occurs in a non-linear fashion, with the greatest
degree of distortion occurring at, or near, one or both
of the two surfaces. An epoxy resin, such as Epon,
could theoretically eliminate the problem, but these
materials are generally brittle and, under ordinary
circumstances, not suitable for sectioning at a
thickness of 5 µm or greater. Modifications of this
form of processing could potentially overcome these
problems; however, such methods require
specialized expertise and equipment and it would
not be practical for most laboratories to develop this
capability. In any case, the failure to take these
various factors into account in ‘unbiased’ cell
counting results in the possibility that such counts
are confounded by indeterminate degrees of
16 Opinion TRENDS in Neurosciences Vol.24 No.1 January 2001
(a) Low bias (b) No bias
Low variance High variance
TRENDS in Neurosciences
Fig. 4. Bias/variance tradeoff. ‘Bias’ refers to how far off-center the
estimates are, on average.The problem is that one does not know what the
true center actually is, that is, the true number of cells. Low bias means
high accuracy. ‘Variance’ refers to the spread of estimates about their
average. Low variance means high precision. ‘Prediction error’ is taken
here as the ‘mean squared error’ of the estimates, the two being
proportional to each other under a Gaussian (normal, bell-shaped curve)
assumption. Mean squared error is equal to the average squared distance
between the estimates and their average. Low prediction error means
high predictive performance. Bias and variance combine to yield
prediction error according to a simple formula: prediction
error=variance+bias2.The figure depicts hypothetical values obtained by
two different estimators (a) and (b), i.e. ‘recipes’ applied to repeated
samples of numerical data to yield summary statistics, or estimates.The
red dots depict the actual observed estimates, positioned identically in
both (a) and (b).These two estimators possess different bias and variance,
and hence different prediction errors. Note the location of the dark cross
hairs near or on the ‘bull’s eye’ in each case. In (a) the estimator might be
slightly off-target on average (‘biased’) yet it might still be a superior
predictor because it exhibits lower variability around its average.
Although the estimator in (b) is theoretically on-target on average
(‘unbiased’), it might yield poorer predictions because it is more variable
than the estimator in (a). Some modern estimation procedures resolve the
bias/variance tradeoff by trading a small amount of bias to achieve great
reductions in variance and thus improve overall predictive performance.
distortion, depending upon where along the z-axis
the optical disector is placed11.
Another important issue is that optical disector
sampling is conducted using 100 × oil immersion
lenses that typically have a narrow depth of field. As
shown in Fig. 5, contraction of the tissue along the z-
axis will have differential effects on the estimation of
the numerical density of objects, depending upon
their size. For example, pyramidal cell density might
be as much as 150% greater in a thin section after
collapse along the z-axis. For non-pyramidal cells, the
density might increase by only 30%. Therefore, for the
optical disector to generate accurate counts, it is
necessary to determine the precise amount of
contraction that has taken place along the z-axis and
the influence this change might have had on the
numerical densities of different cell populations. A
further issue that has not been considered is whether
the compression of the section along the z-axis might
influence the size of objects. As the tissue section
collapses, it is conceivable that cell bodies located
within the section might flatten in such a way that
their apparent cross-sectional size is altered. Because
the collapse along the z-axis is non-linear, its relative
effect on cell size could vary with section depth.
Without controlling for these two confounding effects
of compression on cell density and cell size, the
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advantages accrued from using the ‘unbiased’ optical
disector method will not be appreciated.
An alternative method that can overcome these
limitations of working with thick sections, whether
they are imbedded or frozen, is to work with ‘thin’
sections with a thickness of 20 µm. For such sections,
counting of objects is conducted at a relatively low
magnification (e.g. 25 ×) where the depth of field is quite
large. Under these conditions, all of the cells present
along the entire z-axis of the section are included in the
analysis and the confounding effect of tissue collapse in
this plane is essentially eliminated. These conditions
are routinely employed in 2-D cell-counting paradigms
and represent an important strength of this approach.
Cost-effectiveness
A significant amount of resources must be expended
in order to complete a morphometric analysis. Of
greatest significance are the human resources needed
for stereology. All forms of morphometric analysis are
tedious and time-consuming, regardless of whether a
2-D or a 3-D methodology is employed. Therefore,
when an investigator makes the decision to
undertake a cell counting study, a significant
commitment of human resources must be made to the
project. One must find the ‘right’ individual, who is
willing and able to sit for long periods of time
performing microscopic sampling by focusing
intensely on single cells. In carrying out such work,
x
(a) z
y
(b)
70 µm a z-axis
23 µm a′
Pyramidal Non-pyramidal
cells cells
a 2 3
z-axis
a′ 5 4
TRENDS in Neurosciences
Fig. 5. Effects of z-axis collapse in tissue sections. (a) Diagram showing the
distribution of neurons in the (x,y,z) space of a tissue section. (b) Diagram
depicting the effect of z-axis collapse on the distribution of cells within a
tissue section. When a ‘wet’ tissue section is mounted on a glass slide and
permitted to dry, there is approximately 67.5% shrinkage along the z-axis.
Using a z-axis encoder and a ‘through-focus’ measurement of the distance
between the upper and lower surfaces, a 70 µm thick section, a, was found
to be compressed down to 23 µm in thickness, a′. Based on a computer
simulation, the table below shows estimates of the number of pyramidal
and non-pyramidal cells that would be present within each optical section.
For pyramidal cells a disproportionately high degree of compression of the
larger pyramidal neurons results in a density 150% higher in the collapsed
section. For non-pyramidal cells, the density is increased by only 32.5% in
the collapsed section.
 Opinion TRENDS in Neurosciences Vol.24 No.1 January 2001 17

physical and mental fatigue impact significantly on
the speed with which the project can proceed.
For optical disector counting, the level of difficulty
is much greater compared with 2-D counting. First,
the work must be conducted with a 100 × objective
lens so that the depth of field is narrow enough to
permit an accurate disection in the z-axis. Second,
optical disection through the z-axis requires that the
investigator move the sampling field meticulously
through a continuous depth of field in the tissue.
Going deeper into the tissue, there is a significant loss
of spatial resolution as a result of the light-scattering
properties of the tissue and/or of the imbedding resin.
Not surprisingly, mental and physical fatigue are
much greater using this method compared with 2-D
counting. Even more significant is the fact that the
overall length of time required for the data collection
is greater. Whereas, a 2-D counting study might
require six months to collect the necessary data, the
optical disector counterpart might require as much as
one and a half years. Overall, the difference in
personnel cost to undertake a 3-D project could be
three times greater than that of a 2-D analysis. Do the
Acknowledgements
This work was supported
data obtained with the optical disector technique
by grants from the justify the additional expenditure of resources?
National Institutes of Practical concerns such as these are particularly
Health (MH00423,
pressing for young investigators establishing new
MH42261, MH31154,
MH31862, NS37483) and research programs with relatively small amounts of
the Stanley Foundation. funding. A disturbing new trend has arisen since the
optical disector has been proscribed as the sole
method of choice for cell counting. Some young
investigators are opting to avoid any form of
microscopic quantification whatsoever, lest they be
criticized for using a 2-D approach. If this trend
persists, it could have a negative impact on the
quality of the science that is produced by the field.
Conclusions
Based on the discussion above, it seems obvious that
both 2-D and 3-D cell counting are capable of providing
estimates of the total number and numerical density of
objects in the brain and each approach has its own
relative strengths, weaknesses and biases. A reasonable
conclusion that might be drawn from the above
discussion is that it is prudent to employ a common sense
approach in selecting a methodology for quantifying cell
numbers, features and distributions in neural tissue.
Although it is certainly appropriate in some cases to plan
to ‘do more less well’32, as suggested by some, the
structural neuroscientist might want to exercise an
alternative strategy of aiming to ‘do less much better.’
Weighing the relative costs and benefits of each, in
relation to the scientific question being asked, offers a
rational way of choosing an appropriate methodology.
‘The truth as near as we can get at it…’ Miller and
Carlton, 1895, the earliest published use of the
disector principle33.

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