Using BoneJ on non vertebrate skeletons

I have been applying BoneJ to shellfish, which includes crustaceans and mollusks. In most of these skeletons CaCO3 is used but many focal hardened structures such as mandibles have been shown to include Carbonate Apatite, i.e. what could be called bone. I have recently described the general use of trabeculae of bone to harden the carapace of the big clawed lobsters including American Lobster, European Lobster and Norway Lobster (i.e. scampi). These three species have relatively thin carapaces that seem to the touch to be very thick because of this use of bone to harden them. My theory about these carapaces is that they use both CaCO3 and bone for different purposes. Irrespective of my theory the densities of the CaCO3 types (calcite, amorphous CaCO3, aragonite (in mollusks) require that I be able to delineate these several distinct densities and visualize the structures that they represent. I would like help in thinking through the approaches to use which will allow me delineating at least 3 density structures. I have analyzed lobster carapace structure using the thickness module in BoneJ
I am having some problem in getting the table of thicknesses that were plotted as colored spheres spanning the structure in the AVI above and could use some handholding or directions as to how to change the thickness module parameters to auto save the thickness numbers or at least see them in an ImageJ table that I can save. These numbers may be critical in determining if the calcite-layer-thickness is a vulnerability of the carapace to lobster shell disease.
The binary slices were created from microCT slices and the binary slices were saved as TIFs. I then select particular series of binary TIFs to load and the thickness module of BoneJ was run producing a series of thickness coded slices. I am expecting I need to set parameters in the thickness module to activate recording the thickness values rather than just plotting them.

@mdoube Any thoughts?

Here is a microCT voxel section of the lobster carapace showing the three densities I need to visualize. A surface layer is calcite which quite discretely in other analytic views (such as polarized light) is different from the amorphous CaCO3 which forms stalactites coursing down through the outer layer (exocuticle). These CaCO3 stalactites are continued by what I have called Bouligand spirals which are CaCO3 densities that follow the chitin/protein layers that are laid down like plywood layers with each layer of chitin/protein rotated producing the so-called Bouligand layers of cuticle. These Bouligand spirals taper down and in this section can be seen to connect with a basal granule of similar density. A lower density between the exocuticle stalactites is the carbonate-apatite (bone) trabeculae which I have been able to visualize using expensive software AnalyzePro which I was able to rent for three months. With that software I was able to record STL files of the various density surfaces. Here is a view of the shell-disease lesion of a lobster carapace with green-trabeculae in which you can see the fenestrations in the trabeculae through which the CaCO3 stalactites flow. The surface red-calcite layer is seen first and then made transparent. The purple-strands are outlines of the dermal gland canals that pour secretion onto the cuticle surface. An Epizootic Shell Disease Lesion on an American Lobster carapace in stereo pair view. I provide this detail to help in understanding the detail I wish to explore with ImageJ and perhaps the BoneJ module.

You should automatically get a table with mean, maximum, and SD thickness values, in columns labelled Tb.Th, which is notation from the bone field that means ‘trabecular thickness’. If you don’t get a Results table, something is very wrong and we’ll have to look into it further.

For more detailed statistics you can run a histogram command (hit [h] on your keyboard), selecting to use the full pixel range and a stack histogram. You can export the bin and count values by hitting the ‘list’ and ‘copy’ buttons on the histogram display.

Would you call it bone, though? Bone tissue should contain osteocytes and a collagenous organic phase produced by osteoblasts. The mineral species alone is not enough to designate the tissue type. Really interesting application - thanks for using BoneJ!

1 Like

I have seen that table with Tb.Th but it has a single line and I was expecting more data. I wanted to see a list of the data including the sample size. I have essentially a biometry masters (after my PhD) with heavy theory and want to see the distribution so I will certainly feel around to find the [h] at the appropriate(?) time.

Wow, I need to look into hitting the [h] key. I have BoneJ installed under ImageJ. Does it present itself differently or in a different version under another environment such as FIJI which I have installed but rarely use as a platform?

Arthropod cuticle and vertebrate bone are both extracellular and so the ‘bone’-trabeculae of lobster cuticle could be being generated in the same way as vertebrate bone. The epidermis underlying the cuticle secretes the Ca, PO4 and CO3, chitin and proteins, some of which I have studied (Telfer & Kunkel), needed to create the environment in which the bone forms analogous to the osteocytes of vertebrate bones.

Telfer WH and JG Kunkel. 1991. THE FUNCTION AND EVOLUTION OF INSECT STORAGE HEXAMERS. Ann Rev Entomol 36: 205-28.

The thickness map is essentially a 3D list of the raw thickness values with a LUT so you can visualise them, so getting stats of those values is all you really need, I think.

Keep using BoneJ under ‘vanilla’ ImageJ for the time being, if it suits your purposes. We are working on BoneJ so that it will integrate seamlessly with up-to-date Fiji, but that’s some distance from being user-ready. It is possible to use BoneJ with Fiji, but you have to hold your Fiji back to Java6 (and not let it update to Java8), which is a bit messy. Best for now to continue as you are.[quote=“JoeK, post:5, topic:4917”]
Arthropod cuticle and vertebrate bone are both extracellular

I disagree with you here. Bone tissue comprises cells embedded within matrix, so it cannot be extracellular. Bone has a mineralised extracellular matrix, but is inherently a highly (surprisingly) cellular tissue. It’s also of mesodermal origin, unlike epidermis which is ectodermal. Bone-like apatite mineral in arthropod cuticle is very interesting, I’d simply resist calling it ‘bone’.

The same argument that arthropod shell/cuticle is a ‘live tissue’ with microvilli extending from the epidermal cells into the extracellular matrix of the cuticle is made by my line of arthropod biologists. The cuticle protein and chitin fibers are laid down by the epidermis and then mineralized in an extracellular compartment as I see the traditional bone of vertebrates is also … the carbonate apatite being solidified outside of the cell membranes of the cells that condition the space for bone development. Arthropods are inherently planar-epithelial animals. They have very few solid 3D tissues. In the epithelial plane of the epidermis special organules develop from one cell dividing twice to produce 4 cells. One cell becomes either a gland cell or a sensory neuron. A second cell becomes socket epidermal cell to bound a third cell which becomes a canal for gland secretion or a sensory neuron canal to the surface bristle or dome. The canal forming cell in several lobsters, including slipper lobsters and my big clawed lobsters, produces an carbonate-apatite lining to the canal. That carbonate apatite can vary in carbonate composition including the canal closest to the exterior which can be pure apatite with no carbonate. The surface of that ‘bone’ is fluoridated as time goes on seemingly from the seawater given it extreme surface layering. I have proposed that the more traditional apatite in the canals prevents microbial dissolution of the canal than if it were made with the traditional CaCO3 of the major shell/cuticle. Being hardened with fluoride the organule canals are more like a tooth/fish scale which we are also studying using zebrafish scales which have their bone forming cells similar to skeletal bone formation.
It very well may be that the organule canal forming cell is our best single cell invertebrate model for bone formation. Some of my electron microprobe scans of neonatal mouse bone show that it is the very surface bone adjacent to capillaries that is fluoridated, which I imagine is known and published somewhere, but in my arthropod oriented research was reassuring of the fact that bone is fluoridated after first being established by the bone forming process.
The argument about bone not being able to be produced by an ectodermal tissue has I think been disproved in jaw development where some jaw bones are actually ectodermal in origin. I will have to confer with my colleague Drew Noden (who mentioned it in a seminar) on that point.
Fig 1. Diagram of lobster cuticle structure.
Figure 1. Model of American lobster cuticle and surrounding environment. The epidermal layer illustrates generalized epidermal cells and specialized cells of a simple organule composed of a Canal Cell, Socket Cell and Gland Cell with their overlying cuticles. The generalized epidermal cells produce a pavement type cuticle which spans between the organules. Calcite (green layer) and and amorphous calcium carbonate (ACC), are forms of calcium carbonate. Phosphatic mineralization is indicated in colored fields of purple. Trabeculae of CAp (purple lozenge shapes) are interconnected to provide rigidity. In the canal wall Ca:P ratios are indicated going from 2 to 7 out to in the CAp (CAp) trabeculae marked with Ca:P ratio of 7:1. A dashed purple line beneath the calcite layer indicates phosphate deposits. Horizontal wavy lines indicate the closer layering of the chiitin protein in the exocuticle than the endocuticle. Solubilization of the calcite through the epicuticle produces Ca2+, HCO3- and OH- plus a high pH unstirred layer providing an antimicrobial effect at the cuticle surface. Fluoride F ¯ diffuses in from the environment to create fluoridated CAp.

That’s super-interesting, thank you for educating us.

You are absolutely correct - craniofacial bones derive from neural crest ectoderm - although these cells do an epithelial to mesenchymal transition. Your mineralised cuticle looks to me more like dental enamel - which is a really ectodermal/epitheilal derived mineralised tissue - than bone. Maybe the fluoride story fits in here, too?

Ahaa, I was unaware of that distinction about teeth being epithelial derived mineralization. But fish scales are the precursors of teeth, no? We have been also exploring the ion flux associated with scale development and I thought they had something like the traditional osteoblasts and octeoclast type of bone deposition. In any event both tissue type somehow create an environment where carbonate apatite crystalizes(?) out into a rigid structure. My Dusseldorf and Linz decapod colleagues want to call it amorphous CaPO4 (which I think they mean that it is micro-crystalline rather than soluble) but the whole issue of crystallinity in carbonate apatite is somewhat muddy. I am looking forward to applying the suggestions you have provided for how I can mine my microCT data with BoneJ.