DO NOT save each section as a
new file. Use the workflow to add
new sections, as described above,
until all the sections in an animal
are traced and counted.
NOTE:
One animal = one file.
and past the bottom of the tissue until
it is completely out of focus and then
slowly focus back up until something
just comes into focus. This is the bottom
of the tissue.
Note: If more than one person is doing
the counting for one experiment, all the
counters should sit down and come to
a consensus as to what is the top and
bottom of the tissue because if they
choose differently, the difference will be
reflected in the results.
As far as choosing cells to count (place
a marker on), pick a point of the cell
(such as the top of the cell, the widest
perimeter of the cell, the nucleus, the
widest perimeter of the nucleus or the
nucleolus) and when that point of the
cell comes into focus, place a marker on
it, as long as it falls into the rules of the
counting frame discussed in step 6 and
falls within the dissector height (shown
as green in the Z meter). If this point of a
cell comes into focus while in the guard
zone (shown as red in the Z meter) the
software will block counting it.
Once all the sites have been visited, click
on Add New Section in the workflow
below the blue start counting arrow.
The software will then return you to step
2 in the workflow for the next section.
Follow the workflow down to Step 11
again. Continue counting each sec-
tion and adding new sections until all
the sections the particular animal are
completed.
Step 12: View Sampling Results
Once counting is complete, the best way
to view the results is to click on Display
Probe Run List. Once this list pops up,
highlight all the like contours in each
section. For example, if there is only one
contour per section, highlight all of them
(hold down the Ctrl key to select more
than one) and click on View Results. If
the sections have mirrored contours
Left and Right, highlight all the left con-
tours and View Results. Later, highlight
all the right contours and View Results
for that. To see the results for the com-
bined contours, highlight all the right
and left contours and View Results.
Interpretation of Results
The first items to appear in the results
window are the parameters for your
study. Marker will show the estimated
total cell numbers. Do not be alarmed
if some of the results are 0. Here is the
explanation of the different results:
Estimated total by optical fractionator:
If you manually entered the thickness
at the beginning of counting (and did
not measure while counting), then you
use the first output in display probe run
list. This calculates the cell number and
bases the counts on the manual thick-
ness that you entered prior to doing the
cell counts. This is the *LEAST accurate
estimated value* unless you are 100%
sure of the thickness of all of your sec-
tions. If you did not enter the thickness
manually this number will equal zero in
the results, which can be changed by
clicking on Edit Mounted Thickness.
Estimated total mean measured thick-
ness:
This second value calculates cell
number based on the average thick-
ness of the tissue. You would use this
if you measured section thickness at
every sampling site (or periodically dur-
ing counting) and it will calculate the
counts based on the mean thickness
throughout the tissue. This is the *MOST
accurate* of the cell estimates and we
recommend that our customers mea-
sure at every site and use this estimate
for their data analysis.
Estimated total number by weighted
thickness:
This third value calculates
cell number by taking into account very
wavy tissue. So if you noticed that there
was a lot of variation between sampling
sites in terms of section thickness, you
should use this cell count estimate.
You can also see the various CEs by
clicking on them. Clicking on Planim-
etry will display the volume and area for
the structure.
Software Bugs: (Save Often)
• If No Live Image
If no live image appears check camera activ-
ity LED on top of camera body; if it is green
it is on but not talking to SI or NL. If Green
do this: (Amber flicker = Active)
1. Close software
2. Turn off Master switch
3. Restart Computer
• If Software locks up
1. Turn off master switch
2. Restart Computer
3. Turn on Master switch
4. Login to clear logbooks; depending on
the crash, ITG logbooks may not close until
a user has logged in to the computer.
• Before You Leave:
Close the software and restart the computer
instead of log out. This will ensure all back-
ground processes are stopped, improving
stability for users after you.
