Dear Furqan, how exactly do you prepare bone samples for electron microscopy? You can use the characteristic D-spacing of collagen as a criterion of structure preservation. Normally it is 67 nm (in thin sections of native mineralized bone imaged by TEM). In demineralized/ air-dried bone it is 57-63nm, i.e collagen fibrils are shrunk. Though, in demineralized/high pressure frozen/freeze-substituted/embedded samples the D-spacing is 67nm that means no warping occurred.

Dear Furquan Shah,

Good question....but....

the problem lies in the methods used itself... so you have to rely on what you get out from “live” measurement, eventually marking “live” tissue in a way that you can find the markers also in your LM or EM-specimens. Once you have established “live” measurement compared to measurement in fixed state (the comparison with collagen D-Banding offered by the previous poster Natalie REZNIKOV is certainly ONE possibility… but then, what to do if there is no collagen in the specimen?), and consequently you will be pursued by other obvious extrinsic possibilities of adulteration of measures, depending on the method(s) you use:

e. g. measures shrinking/swelling artifacts during and after complete dehydration, infiltration into resin, polymerization and last but not least distortions by cutting your resin embedded specimens (LM as well as T-EM). The latest introduction of artificial measurement will be set by viewing and documenting analogically or digitally your sections (or parts of these) under the e-beam (distortion depending on type of grid, used kV, thermal conductance/heat dissipation and resin quality = stability in the e-beam).

So your “quantitative data” at the end of the viewing process are only semiquantitative if not only relative data/measurements which (I believe) are only valid for the specimen under examination since measures could vary in other specimens – even if they are from the same source or have been processed in one and the same preparation schedule (e.g. heterogeneity of structural density in even small portions of tissue, esp. in bone: more/less calcified regions like trabeculae, fibrillar connective tissue network components, vessels, or even cartilaginous components) and if your (T)EM for instance has been calibrated accordingly. So in answering your last statement above “Considering osteoblasts are about 25 µm in size, differences of even 2-3 µm are not acceptable "error" limits for the nano- scale” I think you are right but for me it means that you have to do measurements as precise as possible in “live” state, set measurement markers anyhow and find the measures during and after your processing methods (e.g. combined block surface SEM with following sectioning for TEM 

Serial Block-Face Scanning Electron Microscopy to Reconstruct Three-Dimensional Tissue Nanostructure, e.g.

Denk W, Horstmann H (2004) Serial Block-Face Scanning Electron Microscopy to Reconstruct Three-Dimensional Tissue Nanostructure. PLoS Biol 2(11): e329. doi:10.1371/journal.pbio.0020329, Abstract

@ http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.0020329

Further and more recent publications cf. Google research:

< Serial Block-Face Scanning Electron Microscopy AND Transmission EM> or < SFB-SEM AND TEM OR Transmission Electron microscopy>.

Best wishes, good luck and regards,

Wolfgang MUSS, Salzburg-AUSTRIA

Hi Natalie,

Thank you for the ideas. I've been thinking on those lines myself too. I've also been thinking about comparing cellular organelles sizes. I prepare my samples both un-decalcified resin-embedded and post-embedding decalcified; with focused ion beam milling as the "sectioning" technique.

What do you think about evaluating the "bulk shrinkage" of bone, due to dehydration (and the substitution of water for ethanol/acetone); as well as a function of polymerization shrinkage of the embedding resin?

Best regards.

Hi Dr. Muss,

Could not have described that dilemma any better, myself! Thank you for the ideas.

I have actually read about serial sectioning inside the SEM, but the problem really lies in the sample preparation parameters and all the countless steps involved. And that is exactly what my area of research is. It is rather difficult to interpret the biological processes taking place at biomaterial-tissue interfaces, and if we can't evaluate undistorted interfaces, it may well be impossible to tailor and optimize biomaterial physico-chemistry!

The problem I see in SBFSEM is that it's more or less impossible to section my samples using a diamond knife.

However, I totally agree with you that the eventual data is really semi-quantitative or even relative. I can think of µCT as a method of "live" measurement, and we are in the process of acquiring one very soon, but I wonder what kind of resolution limitations there could be. Do you think there could be any other techniques for "live" measurement of the D-spacing?

Thank you once again.

Best regards.

I would not recommend collagen spacing as an indicator of specimen shrinkage. Collagen is very different from other soft tissue and its shrinkage differs significantly. According to [Changes in collagen structure: drying, dehydrothermal treatment and relation to long term deterioration. T. J. Wess, J.P. Orgel, Thermochimica Acta 365 (2000) 119-128] periodicity of collagen changed due air drying from 67.2 to 64.7 nm. Pretty small difference. Even historic parchment (very long air drying) contained collagen with spacing 63.0 nm. Shrinkage of organelles could also differ from shrinkage of an entire cell. You have really tough task. Of course, mineralized bone will shrink much less than demineralized one, but what about sells in lacunas? In addition, lacunas sizes even in the same region of the same bone are very different (I have not measured size of cells themselves, but common sense – if applicable – these sizes should be somewhat proportional), so if you need statistical comparison of various treatments, then you need to observe a lot of cells. By the way, we often measure lacunae size with SEM on mineralized bone, embedded in resin and polished, as described for example in http://www.ncbi.nlm.nih.gov/pubmed/22308018

Hi Vladimir,

Incidentally, I have been considering measuring the sizes of cells within the lacunae, and the "gap area" between the lacuna wall and the cell, and as you mentioned, ofcourse they should be proportional. But that brought me to another question: what kind of space exists between the cell wall and the lacuna wall (in the living state), say, on a sub-nano/ångström scale even?

I'm not really looking for an "absolute" answer to this problem. I suppose being able to make relative comparisons would also be good enough in a way, the problem is really about "what" do we have that we can reliably compare, from one study to another, from group of samples to another. I have a feeling the solution is really simple and lies right there out in the open, possibly as you very aptly suggested - observing a lot of cells and lacunae..

Thank you =)