[Histonet] Re: Cortical bone image analysis fluorescent calcein measurement (Damien Laudier)

[Damien]

Hi Jamie,

Goal 1:

For fluorescent label measurements, you’re definitely better off measuring
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 bone
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 parameters
and this will require more derivative calculations on your part after
principle data collection; a great option if you’re comfortable doing 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 the 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