Stereo Investigator and Neurolucida 2000: An Overview

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Stereo Investigator and Neurolucida 2000 An
Overview

• Imaging Technology Group Forum
• 9 November 2000

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What is Stereology?What are Stereo Investigator
and Neurolucida, and what are the capabilities of
each program? Who might use the Stereology
Workstation?
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Stereology is a statistical methodology for
estimating the physical properties of
3-dimensional structures from 2-dimensional
images or from thin sections or sequences of
thin sections. Stereology allows you to estimate
quantities of interest it's important for when
you can't count or measure because such
procedures would be too labor intensive or
otherwise difficult.
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MicroBrightField, in Burlington VT, is a small
company that produces what is clearly one of
best software packages available for
the implementation of stereological methodology.
MicroBrightField characterizes their
stereology software as design-based, compared
to older, model-based methodologies. This is
said to have many distinct advantages, rendering
model-based stereology obsolete.
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Two kinds of stereology 1) Model-based "the
old stuff" you don't want to be doing, for some
of the reasons listed below 2)
Design-based a) "unbiased" b) gives known
levels of accuracy using coefficient of error
(CE) c) reasonably efficient protocols d)
considered to be "assumption-free
("assumption-free" is jargon particular to
stereology)
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In design-based stereology, the protocols,
or probes, are intended to be independent of
the size, shape, orientation, and distribution of
the objects being examined. Particles of
interest, such as cells, cellular components, or
other specific features, can be big, small, thin,
round, clustered, or distributed
evenly. Abercrombie Correction is model-based
it works for round cells only and is based on
assumptions it is now considered historical.

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Stereo Investigator vs Neurolucida Neurolucida
was developed before Stereo Investigator. Stereo
Investigator was designed as a tool for
implementing a large variety of design-based
stereological protocols. Neurolucida has a more
restricted purpose it is a tool for the
implementation of neuroanatomical mapping, neuron
tracing, and 3D serial section reconstruction.
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Stereo Investigator
Estimations 1) Area, tracing a closed
contour 2) Volume, after measuring section
thickness used with the Cavalieri estimator to
determine estimated volumes 3) Population Counts
(i.e., of specific cell types). For thick
sections the appropriate probe is the Optical
Disector that for thin sections is called the
Physical Disector
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Stereo Investigator
Permits population estimates within an area,
depending upon how much information is available.
For example, if the area of a region of interest
is unknown, the Optical Fractionator protocol may
be used to obtain a reliable (valid) population
estimate. Several modifications of this may also
be used, e.g., a volume density population
estimate may be made using what is called the
NvVref technique, which has been incorporated
into a modification of the Optical Fractionator.
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Stereo Investigator
Regions of interest may be mapped, provided that
an area is defined by the user, who must
determine the boundaries of a region. This
software is also designed to permit and
facilitate systematic random sampling, including
the performance of such procedures in and across
serial sections.
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Stereo Investigator
Permits the utilization of counting frames, in
2 or 3 dimensions. Consideration of focal depth
(the Z dimension) differentiates 3D from 2D
counting frames. Counting frames, in turn,
highlight the importance of what are called
guard zones in this type of work. Guard zones
are thin layers, at the top and bottom of each
section, that are purposefully ignored in
counting. Various aberrations or distortions of
the tissue are possible, and more likely, in
these areas.
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Stereo Investigator
Guard zones are typically 3 to 5 microns thick at
the top surface of a section and the diameter of
a cell at the bottom of a section (for
vibratome- or cryotome-cut sections). Distortions
or aberrations in these zones include cell
plucking, from the action of the knife blade,
tearing, and section shrinkage, which can be much
more pronounced in the Z dimension compared to X
and Y. Sections prepared in plastic (i.e., Epon
or Spurrs resin) and sectioned using a diamond
knife may have less distortion and permit one to
establish thinner guard zones or perhaps even
ignore the concept.
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Stereo Investigator
Stereological Probes, which are protocols with
specific purposes, or estimators, may be divided
into two categories 1) Global estimators
involve large areas, i.e., larger than the field
of view. Typically they would involve counting
cells or determining areas such as the volume of
a region of the brain. 2) Local estimators relate
more typically to objects that are smaller and
within the field of view, such as cells. For
example, a local estimator might be used to
derive a cell volume.
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Stereo Investigator
examples of Global estimators Optical
Fractionator Physical Disector Cavalieri, Merz,
Weibel, and cycloid probes Local
estimators Nucleator Surfactor Planar Rotator
Optical Rotator
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Stereo Investigator
The Disector (di sector) is a 3D probe whose most
important quality is its sampling of objects with
a probability that is proportional to their
number, as opposed to their size. In this way no
assumptions are made about size or shape of the
objects. The Disector derives its name from the
concept that in its simplest form only two
sections are sampled in order to put it to use.
As a concept, however, the Disector does not
stand alone it is included as a part of other
probes, e.g., the Physical Disector or the Linear
Disector.
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Stereo Investigator
Optical Disector a version of the Disector that
works on the same principles except that it may
be used to gather information from within one
thick section instead of from at least two
thinner sections. The Optical Disector utilizes a
3D counting frame and guard zones like the
Disector, it is an important component of other
probes but does not stand alone.
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Stereo Investigator
The Fractionator is a method that permits
systematic random sampling of populations of
objects, such as cells within tissue sections. It
may be combined with the Disector to form the
probe called the Physical Disector combined with
the Optical Disector, it becomes the Optical
Fractionator. Stereo Investigator includes these
and many other protocols that are designed to
allow the implementation of systematic random
sampling in a way that is rigorous, replicable,
and statistically valid.
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Stereo Investigator
Stereo Investigator also provides editing
functions that permit the user to modify traced
data, either immediately after collecting it or
at any time later. In addition, there is a
module called Virtual Slice that we currently do
not have activated on our stereology workstation.
It duplicates the tiling/montage functions we
have on the MCID system, and for best results
(autofocus and finer depth control) it requires
using a LUDL rather than a Prior stage
controller. MBF claims that Virtual Slice will
outperform the analogous functions of MCID.
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Neurolucida
As noted, Neurolucida is the precursor to Stereo
Investigator. It is primarily intended for 1)
2D mapping, contours, open or closed (open
contour has no fixed interior) 2) 3D
reconstruction 3) 3D neuron reconstruction
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Neurolucida
Neurolucida, as its name implies, may be used
with a drawing tube, or camera lucida. This
tracing/drawing function may be duplicated using
video capture or performed on-screen, as with
Stereo Investigator.
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Neurolucida
One important function of Neurolucida is its
capacity for Serial Section Reconstruction. This
requires, of course, multiple serial sections of
tissue and the ability to find and chart
recognizable features from section to section.
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Neurolucida
Enables and facilitates the tracing of branching
objects (tree tracing), and the tracing of trees
through serial sections. Differentiation is
made, in the software, of splicing endings to
ending, endings to beginnings, and beginnings to
beginnings, etc. All of these objects can be
color-coded, traced in differing line
thicknesses, and differentiated by type of
structure (i.e., axon vs dendrite).
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Neurolucida
Contains provisions for tracing (and
incorporating within the rest of the sections)
sections that have been mounted upside down.
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Neurolucida
Data gathered during the reconstruction of a
branched nerve through many serial sections may
be put into a sophisticated Solid Modeling
program that we presently do not have activated
in our system. This allows one to enter the nerve
or other complex object in virtual space,
permitting what may be an enlightening tour
through the 3-dimensional structure.
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The Last Slide
Almost everyone performing cell counting will be
working in the brain. There are other versions
of Neurolucida software available for processing
data. Thank you.