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

[Jack Ratliff]

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 <dmlaud <@t> 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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