Optical Fractionator Workflow Continued...
100x-oil). Objective choice will depend
on tissue type and staining. Note: objec-
tives with magnification lower than 40x
will not give you enough depth of view
to count accurately especially when cells
are close to each other in Z.
Step 5: Measure Section Thickness
The calculations for Optical Fractionator
require the measured thickness of the
tissue post processing. Ideally, measure
the tissue thickness at every sampling
site for the most accurate cell estimates.
Measured thickness from all counting
sites will then be averaged and used in
the calculations.
1. Check the Measure the section while
counting option and leave the other op-
tions unchecked.
2. Set your Evaluation Interval for
measuring to 1. Every site will require a
thickness measurement.
Step 6: Define the Counting Frame
Size
Set counting frame size to have 1-5
countable objects within.
Counting Frame tip: Choose a unique
identifying point of your cell or object
of interest, such as cell top, nucleus
top, nucleolus, widest cell perimeter, or
widest nucleus perimeter. Consistently
count objects only when the identifying
point is in focus.
Remember, also that the counting frame
can be placed during this step and the
software will always display it where you
placed it. Click and drag in the center,
or anywhere on the screen and that will
be the location (click) and size (drag)
that will be used while using the optical
fractionator.
Step 7: Define SRS Grid Layout
Set the grid size so that there are ap-
proximately 10 counting frames in the
region of interest (ROI). Remember, this
is just an approximation. The number
of counting frames per ROI will never
be constant, because contours change
size in each section and because the SRS
Grid Layout is randomly thrown down
before every run to help reduce Bias.
If the sections have a history of hav-
ing very uniform thickness, choose to
set the Evaluation Interval to be larger
than one. Possible options could be
at every 3rd sampling site. This can
be done to save time.
As for Guard Zones, it is recommend-
ed that the guard zone be a minimum
of half the thickness of the object
being counted.
NOTE:
Section Thickness measurements & Guard Zones
Step 8: Define Dissector Options
Set Guard zones so that an over- or
under-estimation of cell totals does not
occur. Do not count cells that fall into
the guard zone. Guard zones are used
to ensure objects counted are not from
an area where potential knife damage
can occur either by being cut in half, or
plucked from the tissue during micro-
tomy. In practice, we recommend that
the guard zone be a minimum of half the
thickness of the object you re count-
ing. The dissector height will define
the depth in which you will count cells.
This will also depend on how thick the
tissue is. For example, a section that is
20 microns thick after processing, could
have a top guard zone of 4 microns, with
a dissector height of 12 microns, leav-
ing by default a bottom guard zone of 4
microns. If tissue varies a lot in thickness,
choose a dissector and guard zones that
will fit into the thinnest piece of tissue.
Step 9: Save Sampling Parameters
Save the sampling parameters that were
determined in the pilot study. Then for
subsequent animals, choose the saved
sampling parameters from Step 1: Set up
the Subject and skip steps 5-9.
Step 10: Select Markers for Counting
Choose a marker for counting and use
this same marker for subsequent sec-
tions. Change the name of the marker
to match the type of cells counted by
going to the Display menu, Display Set-
tings, and the Marker Tab. Click on the
marker and rename it. This name will
then appear in the marker tool bar and
will be easily visible for you to select it
for counting. On reload of a Data set the
software will ask to use marker names if
they are not present.
Step 11: Count Objects
In this step choose from a list of the
sections and contours that were drawn
for each section, by highlighting the con-
tour of a specific section to count and
click on the blue arrow. The stage will
move to the first site and the software
will prompt to measure the top and the
bottom of the tissue. To find the top of
the tissue focus up past the top of the
tissue until it is completely out of focus.
Slowly focus back down onto the tissue
and as soon as something on the tissue,
whether it is a cell or connective tissue
comes into focus. This is the top of the
section. To measure the bottom of the
section, focus all the way down through
