Area and Volume estimation on Stereology

Stereological Techniques for
<strong>Area</strong> <strong>and</strong> <strong>Volume</strong> Estimation
Susan Hendricks, Ph.D.
Staff Scientist,
All material copyright © 2010 MBF Bioscience – MicroBrightField, Inc.

What to expect in this webinar
• <strong>Area</strong> <strong>and</strong> <strong>Volume</strong> Estimation
• Exporting Data <strong>and</strong> Evaluating your data
• Submit questions within the question window
• After the webinar… http://forums.mbfbioscience.com
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Balance effort with accuracy
• Design-based stereology is a set of
unbiased methods for quantitative analysis
of the size, shape <strong>and</strong> number of objects.
• The Cavalieri i method is a stereological
l
probe for quantifying regional volumes.
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Cavalieri Method
Image courtesy of wikipedia
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Calculation of <strong>Volume</strong>
<strong>Volume</strong> of the object
is estimated by
summing the areas
<strong>and</strong> multiplying by
the slice thickness.
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Unbiased results require unbiased data
collection
• Anatomical boundaries
• Systematic, R<strong>and</strong>om
Sections
• Section Interval
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A grid point represents an area
• Each point
represents a given
area.
• Consistent marking
is critical
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Marking Grid Points
Adapted from Lutz Slomianka’s website: www.lab.anhb.uwa/edu/au
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Unbiased Marking Decisions
• Examine the boundaries
• Be consistent!
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Choosing parameters
Striatum <strong>Volume</strong>
<strong>Volume</strong> (mm 3 )
Es stimated
15
14
13
12
11
1000 500 400 250 100
Grid Size (m)
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Choosing parameters
Grid Points
5000
Numbe er of Poin nts
4000
3000
2000
1000
0
1000 500 400 250 100
Grid Size
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Accuracy versus Precision
• High Accuracy, Low Precision
• High Precision, Low Accuracy
• High Precision, High Accuracy
With sampling, a given estimate of a
population will vary from the true
number. The goal of stereology is to
ensure that the individual sampling
error does not overshadow the
difference due to experimental
manipulation.
True
number
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Post your questions now …
A smart experimental design will save
time without sacrificing accuracy.
Questions
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Example: Cerebellum vs Striatum
• Using a contour
Advantage:
If you are using
the contour for
another analysis
Disadvantage:
Tedious if the
region is
convoluted
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Cavalieri Method in Stereo Investigator ®
• Set up serial section manager
to organize sections
• Focus on slide or load in
image
• Set Cavalieri grid dimensions
• Place markers on the grid
points that overlay the ROI
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Marking Grid Points
There are 3 ways to mark grid
points:
• Paint markers with <strong>and</strong> without a contour
• Marquee mode
• Replace mode
Example: Lesion or Tumor studies
• Use 1 marker to paint entire ROI in paint
mode
• Use replace mode to replace the 1st
marker with a 2nd lesion/tumor marker
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Cavalieri Estimation – Painting in Contour
Cavalieri grid spacing: 400 m
<strong>Area</strong> estimate: 2,720,000 m 2
Contour planimetric area: 2,813,630 m 2
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Evaluating Cavalieri Results
• Generate unbiased <strong>estimation</strong>s of area <strong>and</strong>
volume of all regions evaluated
• Export results to Excel ® or copy to clipboard
• Gunderson Coefficient of Error, m=0 is the
appropriate CE for most applications
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Evaluating Cavalieri Results
Data File Marker Estimated <strong>Area</strong>
(µm²)
Estimated
<strong>Volume</strong>
(mm³)
<strong>Volume</strong> Corrected
for OverProjection
(mm³)
CE
(Gundersen),
m=0
CE
(Gundersen),
m=1
Block
Advance
(µm)
Periodicity Grid Size
(µm)
Sections Count
Control 1.ASC Striatum 42187500 12.66 12.20 0.02 0.01 60 5 250 11 675
Control 2.ASC Striatum 42437500 12.73 12.31 0.02 0.01 60 5 250 11 679
Control 3.ASC Striatum 43437500 13.03 12.60 002 0.02 001 0.01 60 5 250 11 695
Control 4.ASC Striatum 39250000 11.78 11.36 0.02 0.01 60 5 250 11 628
Control 5.ASC Striatum 44562500 13.37 12.93 0.02 0.01 60 5 250 12 713
Experimental1.ASC Striatum 29733300 8.92 8.53 0.02 0.01 60 5 250 12 450
Experimental2.ASC Striatum 24366600 7.31 7.12 0.02 0.01 60 5 250 10 423
Experimental3.ASC Striatum 28566600 8.57 8.12 0.03 0.01 60 5 250 9 441
Experimental4.ASC Striatum 19633300 5.89 5.75 0.02 0.01 60 5 250 11 322
Experimental5.ASC Striatum 20333300 6.10 5.99 0.02 0.01 60 5 250 11 387
• Average volume of control ROI = 12.17mm 3
• Average volume of experimental ROI = 7.358mm 3
• Results can be displayed in microns or millimeters
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Evaluating Cavalieri Results
• Results will list the number of markers placed per
section for each animal
Number of Cavalie eri Points
200
150
100
50
0
20 30 40 50
Section
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Combining Methods to Create a Story
• Density: express the number of objects
per unit volume
– Measure the volume <strong>and</strong> number
independently so that you know the reason
underlying the density change
• Avoid the Reference Trap: volume should
represent the region, not the population
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Conclusion
Using Stereo Investigator <strong>and</strong> a smart
experimental design will save you time
without sacrificing accuracy.
Questions
Visit the Forum!
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