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Two-Dimensional Versus Three-Dimensional Cell Counting: A Practical Perspective
Two-Dimensional Versus Three-Dimensional Cell Counting: A Practical Perspective
Two-Dimensional Versus Three-Dimensional Cell Counting: A Practical Perspective
1 January 2001 11
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12 Opinion TRENDS in Neurosciences Vol.24 No.1 January 2001
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Opinion TRENDS in Neurosciences Vol.24 No.1 January 2001 13
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14 Opinion TRENDS in Neurosciences Vol.24 No.1 January 2001
Neuroscientists employing the optical disector often increasing as either a, d or both increase, as one would
use many disectors with small window sizes.This expect. If one defines an ‘emptiness probability’ p as
makes sense only if the objects in question are the probability of an empty window, then
distributed completely at random within the sample
p = e–λa
and if one is interested only in object number and not
in their spatial arrangement. Complete spatial if the objects are distributed completely at random
randomness is mathematically equivalent to a within the sample.The information re-expressed in
Poisson assumption.Technically, this is how one terms of emptiness probability p is found to be
would arrive at a recommendation for small window
I = a2dp/(1–p)
size under such an assumption. If a is the area of a
square window with cell packing density λ and n is the for a large number of disectors. Maximizing
average number of cells observed per window information across disectors by varying window size,
counted with d disector windows, then an estimate of one finds that the optimal mean number of cells
λ defined as: counted per disector is
∧
λ = n/a nmax = λ amax = 1.59
has standard deviation equal to and is associated with an emptiness probability of
about 20%. Information remains high within the range
σ ( λˆ ) = λ /( ad ) of 0.7 to 3.0 cells per window when objects exhibit
complete spatial randomness. However, as shown in
The (Fisher) information for , related inversely to the text, this is not the case for neuronal arrangements
variance, is in the brain, and hence use of small window sizes is ill
advised, regardless of the number of windows and
I = ad/λ their placements throughout the sample.
with a large window size will have the added advantage predictions of spatial arrangements of these cells
of producing data-driven estimates of precision. that are biased because one has not paid adequate
Precision and bias of an estimate are two attention to important neuroanatomic facts. As
fundamental issues in stereology and bear directly on noted, even if only estimating the number of
the comparison of 2-D and 3-D methodologies. Using a pyramidal neurons in these two layers, an estimate’s
‘bull’s eye’ analogy from elementary statistics, correct precision will also be biased if a completely random
use and understanding of these terms have been distribution of cells is assumed. By contrast, spatial
recently addressed3, providing examples of estimators distributions of glia are completely random across all
that are biased and unbiased, precise and imprecise. neocortical layers21. In this case, adopting an overly
One can go further and combine bias, precision and complex estimator of glial patterns that includes
prediction error in a single, simple formula to further layer effects will result in complicated predictions
understand relations between these important that perform poorly because of the attention given to
concepts (Fig. 4). Consider two targets, one of which spatial features that do not matter for glia. Thus,
shows an estimator that is slightly off-target, yet is sampling window size and location in the (x,y) plane
precise, exhibiting low variability around its average. interplay with the degree of exclusion or clustering
The other target shows an estimator whose average and thereby have a marked effect on the amount of
position is right on target, yet is imprecise, because its precision and bias that is inherent in estimates of cell
locations around its average are highly variable number and density.
(Fig. 4). In these cases, bias and variance combine to
yield the prediction error. Some form of bias/variance The z-axis
tradeoff is required in order to avoid too much bias The principal strength of the optical disector
(‘underfitting,’ or not taking important features into method is that it eliminates biases arising from
account) on the one hand, and too much variance differences in object size, shape and orientation by
(‘overfitting,’ or accounting for unimportant features) obtaining ‘unbiased’counts along the z-axis3. Using
on the other30,31. this approach, the tissue section must be thick
A concrete example of a bias/variance tradeoff is enough to obtain samples from a through-focus
shown in the primary motor cortex. As noted above21, series of image planes (Fig. 5a,b). Most histological
neurons are more clustered in layer I, but in layer II sections that are mounted on glass slides collapse
they are further apart than complete randomness along the z-axis when the tissue dries.
would predict (i.e. they exhibit exclusionary Consequently, a section that is 20 µm thick might
spacings; see Fig. 2). Therefore, if a simplistic have a final thickness of only 5–6 µm once it has
Poisson assumption is employed, this will produce completely dried on the slide. For the optical
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Opinion TRENDS in Neurosciences Vol.24 No.1 January 2001 15
As an example of the scientific importance of analyzing scale r. If K(r) is less than πr2, then there are fewer cells
spatial distributions of neurons, consider the careful than would be expected under a Poisson assumption,
identification and positional recording of pyramidal as a result of the presence of exclusionary ‘sub-
neurons in layer II of human cingulate cortexa,b. One Poisson’ distances between neurons at that scale. If
quantitative descriptor of spatial pattern for such a K(r) is greater than πr2, then there are more cells than
study is a function, K, that depends on point–pair would be expected under a Poisson assumption,
distances, r. This function is defined as followsc,d: because of ‘supra-Poisson’ clustering at that scale.
Therefore, a new function defined by
K(r) = λ–1E (number of other cells at a distance ≤r
from an arbitarily chosen cell) (1) K ∗ (r ) = Kˆ ( r ) / π − r
In equation (1), λ is the intensity of a null hypothesis will accentuate non-Poisson departures in spatial
Poisson process and E is the expectation operator arrangements.The square root is applied to K(r)/π as
(which, in this case, takes the form of a weighted an appropriate ‘variance stabilizing’ transformation.
average). In stereological fashion, under the null Estimation of the K* function from the (x,y)
hypothesis, the value of K(r) for every distance r is coordinates of pyramidal neurons in layer II of human
equal to πr 2, the area of a circle of radius r centered cingulate cortex revealed exclusionary spacings of up
on each observed cell. An approximately unbiased to roughly 60 µm between cellsb.The small window
estimate of K(r) isc: sizes and ‘skips’ of systematic random sampling with
optical disectors preclude observation of such
Kˆ ( r ) = ( a / n )∑ wij−11( dij ≤ r ) / n (2) arrangements at distances greater than the spatial
i≠ j
extent of the disector window (50 µm × 50 µm) minus
In equation (2), n is the observed number of cells in a average neuronal diameter (15–20 µm). Such a
sampling window of area a, a/n is an estimate of λ−1, finding would not have been possible using the
and the weighted average common optical disector sampling strategy with a
small window size in the (x,y) plane.
∑ wij−11(dij ≤ r ) / n
i≠ j References
a Benes, F. and Bird, E. (1987) An analysis of the arrangement
replaces E(·). In this average, 1(dij ≤ r) is equal to 1
of neurons in the cingulate cortex of schizophrenic patients.
when dij, the point–pair distance between cells i and j, Arch. Gen. Psych. 44, 608–616
is less than or equal to r, and otherwise is equal to 0. b Diggle, P.J. et al. (1991) Analysis of variance for replicated
Finally, there is a correction for edge effects, wij, equal spatial point patterns in clinical neuroanatomy. J. Amer. Stat.
to the proportion of the circumference of a circle of Assoc. 86, 618–625
c Ripley, B.D. (1976) The second-order analysis of stationary
radius dij centered at cell i contained within a square
point processes. J. Appl. Prob. 13, 255–266
or rectangular window. If K(r) is approximatety πr2, d Stoyan, D. et al. (1987) Stochastic Geometry and Its
then the arrangement of cells is Poisson at spatial Applications, John Wiley & Sons
disector method, this problem is overcome using two this case, although shrinkage prior to sectioning can
different strategies. The first is to imbed the tissues be less than with celloidin, the collapse along the
in celloidin, a resin that allows the blocks to be cut z-axis can be as great as 67%. Thus, a tissue section
at a thickness of 40 µm; but, unfortunately, this can that is 70 µm thick could collapse to a thickness of
result in up to 40–70% shrinkage prior to sectioning 23 µm (Fig. 5b). Importantly, this collapse probably
(Fig. 3). The second approach is to cut sections that occurs in a non-linear fashion, with the greatest
are not imbedded in resin at a much greater degree of distortion occurring at, or near, one or both
thickness of 70–80 µm if imbedding interferes with of the two surfaces. An epoxy resin, such as Epon,
specialized staining techniques, such as in could theoretically eliminate the problem, but these
immunohistochemistry. For the celloidin-imbedded materials are generally brittle and, under ordinary
sections, in spite of their rigidity, there is still circumstances, not suitable for sectioning at a
significant collapse (~35%) along the z-axis. Using a thickness of 5 µm or greater. Modifications of this
z-axis encoder and through-focus measurements, form of processing could potentially overcome these
the final thickness of slide-mounted celloidin problems; however, such methods require
sections is approximately 26 µm. Together with the specialized expertise and equipment and it would
shrinkage prior to sectioning, the distortions not be practical for most laboratories to develop this
inherent in this approach are considerable. Using capability. In any case, the failure to take these
the second approach, sections that are not imbedded various factors into account in ‘unbiased’ cell
in resin exhibit a more-marked contraction along counting results in the possibility that such counts
the z-axis when they are mounted on glass slides. In are confounded by indeterminate degrees of
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Opinion TRENDS in Neurosciences Vol.24 No.1 January 2001 17
physical and mental fatigue impact significantly on optical disector has been proscribed as the sole
the speed with which the project can proceed. method of choice for cell counting. Some young
For optical disector counting, the level of difficulty investigators are opting to avoid any form of
is much greater compared with 2-D counting. First, microscopic quantification whatsoever, lest they be
the work must be conducted with a 100 × objective criticized for using a 2-D approach. If this trend
lens so that the depth of field is narrow enough to persists, it could have a negative impact on the
permit an accurate disection in the z-axis. Second, quality of the science that is produced by the field.
optical disection through the z-axis requires that the
investigator move the sampling field meticulously Conclusions
through a continuous depth of field in the tissue. Based on the discussion above, it seems obvious that
Going deeper into the tissue, there is a significant loss both 2-D and 3-D cell counting are capable of providing
of spatial resolution as a result of the light-scattering estimates of the total number and numerical density of
properties of the tissue and/or of the imbedding resin. objects in the brain and each approach has its own
Not surprisingly, mental and physical fatigue are relative strengths, weaknesses and biases. A reasonable
much greater using this method compared with 2-D conclusion that might be drawn from the above
counting. Even more significant is the fact that the discussion is that it is prudent to employ a common sense
overall length of time required for the data collection approach in selecting a methodology for quantifying cell
is greater. Whereas, a 2-D counting study might numbers, features and distributions in neural tissue.
require six months to collect the necessary data, the Although it is certainly appropriate in some cases to plan
optical disector counterpart might require as much as to ‘do more less well’32, as suggested by some, the
one and a half years. Overall, the difference in structural neuroscientist might want to exercise an
personnel cost to undertake a 3-D project could be alternative strategy of aiming to ‘do less much better.’
three times greater than that of a 2-D analysis. Do the Weighing the relative costs and benefits of each, in
Acknowledgements
This work was supported
data obtained with the optical disector technique relation to the scientific question being asked, offers a
by grants from the justify the additional expenditure of resources? rational way of choosing an appropriate methodology.
National Institutes of Practical concerns such as these are particularly
Health (MH00423,
pressing for young investigators establishing new ‘The truth as near as we can get at it…’ Miller and
MH42261, MH31154,
MH31862, NS37483) and research programs with relatively small amounts of Carlton, 1895, the earliest published use of the
the Stanley Foundation. funding. A disturbing new trend has arisen since the disector principle33.
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