Area and Volume estimation on Stereology
Area and Volume estimation on Stereology
Area and Volume estimation on Stereology
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Stereological Techniques for<br />
<str<strong>on</strong>g>Area</str<strong>on</strong>g> <str<strong>on</strong>g>and</str<strong>on</strong>g> <str<strong>on</strong>g>Volume</str<strong>on</strong>g> Estimati<strong>on</strong><br />
Susan Hendricks, Ph.D.<br />
Staff Scientist,<br />
All material copyright © 2010 MBF Bioscience – MicroBrightField, Inc.
What to expect in this webinar<br />
• <str<strong>on</strong>g>Area</str<strong>on</strong>g> <str<strong>on</strong>g>and</str<strong>on</strong>g> <str<strong>on</strong>g>Volume</str<strong>on</strong>g> Estimati<strong>on</strong><br />
• Exporting Data <str<strong>on</strong>g>and</str<strong>on</strong>g> Evaluating your data<br />
• Submit questi<strong>on</strong>s within the questi<strong>on</strong> window<br />
• After the webinar… http://forums.mbfbioscience.com<br />
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Balance effort with accuracy<br />
• Design-based stereology is a set of<br />
unbiased methods for quantitative analysis<br />
of the size, shape <str<strong>on</strong>g>and</str<strong>on</strong>g> number of objects.<br />
• The Cavalieri i method is a stereological<br />
l<br />
probe for quantifying regi<strong>on</strong>al volumes.<br />
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Cavalieri Method<br />
Image courtesy of wikipedia<br />
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Calculati<strong>on</strong> of <str<strong>on</strong>g>Volume</str<strong>on</strong>g><br />
<str<strong>on</strong>g>Volume</str<strong>on</strong>g> of the object<br />
is estimated by<br />
summing the areas<br />
<str<strong>on</strong>g>and</str<strong>on</strong>g> multiplying by<br />
the slice thickness.<br />
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Unbiased results require unbiased data<br />
collecti<strong>on</strong><br />
• Anatomical boundaries<br />
• Systematic, R<str<strong>on</strong>g>and</str<strong>on</strong>g>om<br />
Secti<strong>on</strong>s<br />
• Secti<strong>on</strong> Interval<br />
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A grid point represents an area<br />
• Each point<br />
represents a given<br />
area.<br />
• C<strong>on</strong>sistent marking<br />
is critical<br />
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Marking Grid Points<br />
Adapted from Lutz Slomianka’s website: www.lab.anhb.uwa/edu/au<br />
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Unbiased Marking Decisi<strong>on</strong>s<br />
• Examine the boundaries<br />
• Be c<strong>on</strong>sistent!<br />
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Choosing parameters<br />
Striatum <str<strong>on</strong>g>Volume</str<strong>on</strong>g><br />
<str<strong>on</strong>g>Volume</str<strong>on</strong>g> (mm 3 )<br />
Es stimated<br />
15<br />
14<br />
13<br />
12<br />
11<br />
1000 500 400 250 100<br />
Grid Size (m)<br />
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Choosing parameters<br />
Grid Points<br />
5000<br />
<br />
Numbe er of Poin nts<br />
4000<br />
3000<br />
2000<br />
1000<br />
<br />
<br />
0<br />
1000 500 400 250 100<br />
Grid Size<br />
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Accuracy versus Precisi<strong>on</strong><br />
• High Accuracy, Low Precisi<strong>on</strong><br />
• High Precisi<strong>on</strong>, Low Accuracy<br />
• High Precisi<strong>on</strong>, High Accuracy<br />
With sampling, a given estimate of a<br />
populati<strong>on</strong> will vary from the true<br />
number. The goal of stereology is to<br />
ensure that the individual sampling<br />
error does not overshadow the<br />
difference due to experimental<br />
manipulati<strong>on</strong>.<br />
True<br />
number<br />
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Post your questi<strong>on</strong>s now …<br />
A smart experimental design will save<br />
time without sacrificing accuracy.<br />
Questi<strong>on</strong>s<br />
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Example: Cerebellum vs Striatum<br />
• Using a c<strong>on</strong>tour<br />
Advantage:<br />
If you are using<br />
the c<strong>on</strong>tour for<br />
another analysis<br />
Disadvantage:<br />
Tedious if the<br />
regi<strong>on</strong> is<br />
c<strong>on</strong>voluted<br />
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Cavalieri Method in Stereo Investigator ®<br />
• Set up serial secti<strong>on</strong> manager<br />
to organize secti<strong>on</strong>s<br />
• Focus <strong>on</strong> slide or load in<br />
image<br />
• Set Cavalieri grid dimensi<strong>on</strong>s<br />
• Place markers <strong>on</strong> the grid<br />
points that overlay the ROI<br />
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Marking Grid Points<br />
There are 3 ways to mark grid<br />
points:<br />
• Paint markers with <str<strong>on</strong>g>and</str<strong>on</strong>g> without a c<strong>on</strong>tour<br />
• Marquee mode<br />
• Replace mode<br />
Example: Lesi<strong>on</strong> or Tumor studies<br />
• Use 1 marker to paint entire ROI in paint<br />
mode<br />
• Use replace mode to replace the 1st<br />
marker with a 2nd lesi<strong>on</strong>/tumor marker<br />
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Cavalieri Estimati<strong>on</strong> – Painting in C<strong>on</strong>tour<br />
Cavalieri grid spacing: 400 m<br />
<str<strong>on</strong>g>Area</str<strong>on</strong>g> estimate: 2,720,000 m 2<br />
C<strong>on</strong>tour planimetric area: 2,813,630 m 2<br />
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Evaluating Cavalieri Results<br />
• Generate unbiased <str<strong>on</strong>g>estimati<strong>on</strong></str<strong>on</strong>g>s of area <str<strong>on</strong>g>and</str<strong>on</strong>g><br />
volume of all regi<strong>on</strong>s evaluated<br />
• Export results to Excel ® or copy to clipboard<br />
• Gunders<strong>on</strong> Coefficient of Error, m=0 is the<br />
appropriate CE for most applicati<strong>on</strong>s<br />
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Evaluating Cavalieri Results<br />
Data File Marker Estimated <str<strong>on</strong>g>Area</str<strong>on</strong>g><br />
(µm²)<br />
Estimated<br />
<str<strong>on</strong>g>Volume</str<strong>on</strong>g><br />
(mm³)<br />
<str<strong>on</strong>g>Volume</str<strong>on</strong>g> Corrected<br />
for OverProjecti<strong>on</strong><br />
(mm³)<br />
CE<br />
(Gundersen),<br />
m=0<br />
CE<br />
(Gundersen),<br />
m=1<br />
Block<br />
Advance<br />
(µm)<br />
Periodicity Grid Size<br />
(µm)<br />
Secti<strong>on</strong>s Count<br />
C<strong>on</strong>trol 1.ASC Striatum 42187500 12.66 12.20 0.02 0.01 60 5 250 11 675<br />
C<strong>on</strong>trol 2.ASC Striatum 42437500 12.73 12.31 0.02 0.01 60 5 250 11 679<br />
C<strong>on</strong>trol 3.ASC Striatum 43437500 13.03 12.60 002 0.02 001 0.01 60 5 250 11 695<br />
C<strong>on</strong>trol 4.ASC Striatum 39250000 11.78 11.36 0.02 0.01 60 5 250 11 628<br />
C<strong>on</strong>trol 5.ASC Striatum 44562500 13.37 12.93 0.02 0.01 60 5 250 12 713<br />
Experimental1.ASC Striatum 29733300 8.92 8.53 0.02 0.01 60 5 250 12 450<br />
Experimental2.ASC Striatum 24366600 7.31 7.12 0.02 0.01 60 5 250 10 423<br />
Experimental3.ASC Striatum 28566600 8.57 8.12 0.03 0.01 60 5 250 9 441<br />
Experimental4.ASC Striatum 19633300 5.89 5.75 0.02 0.01 60 5 250 11 322<br />
Experimental5.ASC Striatum 20333300 6.10 5.99 0.02 0.01 60 5 250 11 387<br />
• Average volume of c<strong>on</strong>trol ROI = 12.17mm 3<br />
• Average volume of experimental ROI = 7.358mm 3<br />
• Results can be displayed in micr<strong>on</strong>s or millimeters<br />
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Evaluating Cavalieri Results<br />
• Results will list the number of markers placed per<br />
secti<strong>on</strong> for each animal<br />
Number of Cavalie eri Points<br />
200<br />
150<br />
100<br />
50<br />
0<br />
20 30 40 50<br />
Secti<strong>on</strong><br />
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Combining Methods to Create a Story<br />
• Density: express the number of objects<br />
per unit volume<br />
– Measure the volume <str<strong>on</strong>g>and</str<strong>on</strong>g> number<br />
independently so that you know the reas<strong>on</strong><br />
underlying the density change<br />
• Avoid the Reference Trap: volume should<br />
represent the regi<strong>on</strong>, not the populati<strong>on</strong><br />
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C<strong>on</strong>clusi<strong>on</strong><br />
Using Stereo Investigator <str<strong>on</strong>g>and</str<strong>on</strong>g> a smart<br />
experimental design will save you time<br />
without sacrificing accuracy.<br />
Questi<strong>on</strong>s<br />
Visit the Forum!<br />
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