[Histonet] Cortical bone Image analysis fluorescent calcine measurement

[Jamie E Erickson]

Hi All,
            Question for anyone working with image analysis on Bone..

My 1st goal: Evaluate and purchase image analysis software to measure bone 
formation rate (BFR) and mineral apposition rate (MAR).

        I am trying to measure the area between two fluorescent (calcine) 
labels of cortical bone by image analysis.
This is standard for bone histomorphometrics in all the literature I see 
but as always there is a few details that are missing. 
        Papers don't say what object they use, but for my mouse and rat 
tibia samples, 10x won't get the whole sample in one frame? I have notes 
from someone who has done this and they trace the perimeters to get the 
data (with osteometric software). If I do tracing  at 4x I wonder about 
the accuracy.   Osteometrics is the most common bone morphometric system 
out there but for my cortical bone it seems a little overkill. I would 
like to use Zeiss axiovision software anyone using this? I think people 
are using 10-20x and trace the perimeter of the bone but I am wondering if 
with certain software you can move along the perimeter of the bone without 
stopping the data collection or do you have to collect multiple data sets 
and sum the total to get the perimeter. 

My 2nd goal: Evaluate confocal microscopy imaging of bone for reducing in 
time spent grind samples.

        This method was based on a short communication in Journal of 
Histochemical "rapid method for the assessment of bone architecture by 
confocal microscopy."
Right now I am trying to determine what imaging modality I am going to use 
for these measurements.  My pathologists wants me to do large rough cuts 
(625-1000um) from my isomet saw and see if I can measure these labels 
under confocal  (ie eliminating grinding) , has anyone tried this?
         I can tell you that I can image these samples without a coverslip 
but the samples is uneven due no grinding or placement of sample on the 
slide. The theory is that I can image these samples at 10 or 20x and get 
an image some microns into the sample thus eliminating the uneven (rough) 
upper surface. 
In practice I can get some areas to look great but I can't  focus all the 
way around the bone I lose the image focus possible due to a thicker side 
of bone,, so I think this idea is not practical. Has anyone tried this?

I  think going forward I will grinding samples to about 20um and mount the 
samples in water based media and coverslip. 

If you do this type of work please contact me I'd love to talk about 
this...  As always the powers to be are demanding validation studies of 
this method be done yesterday and I haven't even decided on software.  Not 
to mention that I am new to bone work and I  haven't even ground my first 
bone sample.

Anyway I'll get there, thanks for any help "Happy Friday".

Jamie

_______________________________
Jamie Erickson
Sr. Research Associate II M.S. HTL (ASCP)
Department: DSMP
Abbott Bioresearch Center
100 Research Drive
Worcester, MA 01605-4341
508-688-3134
FAX: 508-793-4895
e-mail: jamie.erickson <@t> abbott.com