I will add to Damien's comments and say that another thing to consider
is the labeling schedule. Typically the smaller the animal the greater
interlabel time period. For example, a smaller window in a mouse tibia/
femur might make it difficult to determine the difference between
single and double labels. On the other hand, the remodeling rate can
also affect this where in say a larger animal like a dog or sheep with
a greater interlabel time period could make you miss your double
labels because your first label could be resorbed.
Lastly, you are better served with your mouse and rat sections if you
can grind your sections thinner at say 20-30 microns. This essentially
will sharpen the labels so that a single label can not be mistaken for
a double label due to a halo effect from label over-expression. You
can accomplish this goal either automatically with say an Exakt
grinder or manually with a suction cup and a little patience.
Regardless, they are all very teachable methods.
I will again encourage you attend the BIOQUANT Image Analysis Meeting
April 21-23 in Nashville as all of these topics will be addressed in
detail from a histology, histopatholgy, and image analysis
perspective. If you can attend, I also encourage you to bring a
representative polymerized specimen and/or prepared slides.
Jack
On Mar 28, 2009, at 10:23 PM, Damien <dml...@gmail.com> wrote:
Hi Jamie,
Goal 1:
For fluorescent label measurements, you’re definitely better off mea
suring
multiple fields using a higher power objective (at least10X).
Accuracy is
paramount when measuring inter-label width and working at a higher
magnification will help to ensure this. Your other option is to
create a
photo-stitched composite image of each sample (you would take multiple
images until you get the entire circumference of the sample) and make
measurements from a static image. Osteometrics *Osteomeasure*
software is
not “overkill” and is considered the simpler of the two popular bo
ne
histomorphometry packages on the market. The beauty of using such
programs
specifically designed for bone histomorphometry is that the
calibration
standards/code are already established/programmed, as well as the
back-end
calculations required to derive MAR. You can certainly make these
measurements using Zeiss axiovision software or any imaging software
(NIH
Image J, Matlab IPT). However, you’ll need to establish your own par
ameters
and this will require more derivative calculations on your part after
principle data collection; a great option if you’re comfortable doin
g this.
Goal 2:
If you’re using your (expensive) precision saw correctly, you should
not be
getting uneven sections. Try reducing your cutting speed and/or
adjusting
the angle of your sample. Also, make sure you’re using a blade of th
e proper
size- relative to the size of the sample. Use a micrometer to gauge
your
cutting adjustments. Once you fix this problem, yes it is possible
on a 1mm
section, but since all confocal microscopes are not created equally,
success
will ultimately depend on the resolving power of your system.
Good luck!
-Damien Laudier
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