Microscopy of Bone
and Step-by-Step Sample Preparation

Ulf Griesmann, Gaithersburg, Maryland, USA


Bone is an educational and rewarding subject for the amateur microscopist. In this article I describe the preparation of bone samples for optical transmission microscopy and illustrate the remarkable observations that can be made with rather modest means. The preparation methods described here may also be of interest to students and their teachers who want to take a closer look at one of the most astonishing, and astonishingly complex, natural materials. The first part of this article describes the preparation and mounting of bone thin sections. In the  second part, several micrographs are discussed that were made using a simple LOMO Multiscope microscope (known as Biolam outside the US).

Bone is opaque and must be prepared in the form of thin sections for the brightfield transmission microscopes that are most readily available to amateur microscopists or in educational settings. One advantage for the amateur is that the preparation of bone specimens does not require the use of dangerous chemicals for fixing and staining, because most features can be observed without elaborate specimen preparation. Without a saw microtome, making thin sections is tedious work, but the procedure described in the following paragraphs yields sections that allow for surprisingly good images. Fig. 1 shows a piece of bovine (cow) long bone, about one inch in length, that was cut from a fresh leg bone with a saw. The bone section was cleaned with warm water.

Bovine long bone
Figure 1: About one inch (25 mm) long section of bovine long bone.

A complete imaging of the complex three-dimensional structure of bone requires multiple transverse, longitudinal, and radial sections. Here I will concentrate on the preparation of a transverse bone section. The finished bone section will be bonded to a microscope slide and so the first step is to grind flat and polish the part of the bone that will be glued to the slide. This grinding step is illustrated in Fig. 2. The grinding is done using emerald paper from a DIY market where paper with grit sizes down to 600 is commonly available. Further polishing was done using a set of Micro-Mesh polishing pads (shown in Fig. 3) for wood polishing with up to 12000 grit, which achieve a mirror-like finish. The samples can be prepared either wet or dry, but it is much easier to bond a wet sample to the microscope slide because suitable water compatible glues, e.g. surgical glues, tend to be very expensive. The bottom side of the bone does not have to be finished all the way to a mirror finish because it will not be viewed with the microscope, but it should be fine ground without large, visible scratches.

Grinding the bottom of the bone
                sample
Figure 2: Grinding the "bottom" of the bone sample.

Micro-Mesh polishing pads
Figure 3: Micro-Mesh polishing pads used for bone polishing.

Bone
                    slice
Figure 4: Bone slice with one fine-ground side.

Once the bone piece has been ground to a fine finish, the piece is clamped in a vise and, using a small hacksaw, a slice as narrow as possible is cut from the bone as is shown in Fig. 4. Next, the bone slice, like the one in Fig. 4, is cut up into about 5 mm x 5 mm chips which are bonded to microscope slides using a clear epoxy glue. Fig. 5 shows two chips, one a transverse section, the other a longitudinal section, being clamped to microscope slides while the epoxy glue cures. The clamping is important to make sure the glue layer between slide and bone chip becomes as thin as possible. Fig. 6 shows the result - a bone chip that is about 1 mm thick bonded to a microscope slide. Wet samples can be glued with acrylic superglue, which is somewhat water compatible because the curing reaction is catalyzed by water, but the result is a bond that is much more fragile than the epoxy bond and much less likely to survive the subsequent grinding and polishing. Most of the ones I tried peeled off during grinding. Bone chips that are kept for later glueing can be stored in isopropanol if they are destined to become dry samples or de-ionized water in case they will remain wet. Purists can use physiological saline solution or Ringer's solution to temporarily store wet samples.

Bone chips being glued
                      to a microscope slide
Figure 5: Bone chips being glued to microscope slides.

Bone
                      chip bonded to slide
Figure 6: A 5 mm x 5 mm x 1 mm bone chip bonded to a microscope slide.

The bone sample must now be ground and polished to a thickness between 25 m and 30 m. If the bone section becomes too thin there will be nothing left to look at; if it is too thick, the structures near the surface will be confounded by features that are buried inside the section. The thin grinding is the most difficult part of the sample preparation and it will require some practice. Holding the slide between thumb and middle finger and supporting the back with the index finger, the thickness of the sample can quickly be reduced by grinding with emerald paper as is shown in Fig. 7. People with allergies or concerns about, heaven forbid, mad cow disease may want to wear a face mask when preparing dry sections to avoid inhaling bone dust. At this stage it is easy to get bored and grind too fast; it is better to go slow than to start over. The thickness of the section must be monitored with a micrometer as is shown in Fig. 8. Once the thickness drops below about 200 m a finer grit must be used for a while and then the next finer grit and so on. The purpose of the polishing is to remove the scratches and the damage that is introduced in the sample by the larger grit abrasives. As the section thins, the grit size should be reduced until a 25 m thick section is obtained that is polishded to a mirror finish with the finest available grit. A successfully polished section looks like the one shown in Fig. 9. Finally, the polished section is wiped clean with isopropanol or water and the section is mounted under a coverglass. Dry sections can be mounted with Mount-Quick (Daido Sangyo Co. Japan) or a similar medium, wet sections may be mounted in glycerin gelatin. (For wet mounted sections, the edges of the coverglass must be sealed with varnish.) Fig. 10 shows a finished slide. A 25 m thick section still appears noticeable opaque. Wet sections mounted in glycerin gelatin will become more transparent over time because glycerin is a clearing agent for bone.

Thin
                          section grinding
Figure 7: Grinding of a thin bone section.

Measuring section thickness
Figure 8: Measuring the bone section thickness with a micrometer.

Finished thin section
Figure 9: A finished thin section.

Finished slide
Figure 10: A finished slide with cover glass and labels.





With slides finished it is time to look at them under the microscope! Figs. 11 - 15 are made with my modest LOMO Multiscope microscope that is fitted with a Motic 3 megapixel camera. Fig. 11 is a beautiful picture of the fundamental structural feature of compact bone - an osteon. Osteons are bone columns made from concentric layers, or lamellas (lamellae). The green arrow  in Fig. 11 points to one of the lamellas. At the center of each osteon is a conduit, called a Haversian canal, that carries vessels for the blood and lymph supply. The outer boundary of an osteon is the cement line (actually a sheet) pointed to by a yellow arrow in Fig. 11. The Haversian canals at the centers of osteons are connected in a ladder rung-like fashion by canals that are called Volkmann's canals. In Fig. 11, a Volkmann canal can be seen branching off to the right from the central Haversian canal.


bovine osteon
Figure 11: Osteon in a transverse section of bovine long bone (20x, NA=0.65).

Everywhere along boundaries between lamellae in Fig. 11 are small voids that are more clearly seen at larger magnification in Fig. 12. The voids are called lacunas, or lacunae in high falutin (related to the words "lake" and "lagoon"). They are about 10 m long and 4 m wide. Some of the lacunae can be seen as dark shadows in Fig. 12 because they are buried beneath the surface of the bone slice. Each of the lacunae is home to a bone building cell called osteoblast. These cells have laid down a bone lamella until they became imprisoned in the bone that they have created. Their connection to the outside is a vast network of small channels, or canaliculi, which can be seen in Fig. 12 connecting the lacunae within an osteon. Canaliculi do not cross the cement line.


Lacunae and canaliculi
Figure 12: Lacuae and canaliculi in bovine osteon (oil immersion, 60x, NA=1).

When the light that enters the condenser is polarized by placing a polarizer in the filter holder and a second, crossed polarizer at the image plane of the microscope objective, the sub-structure of the bone lamellas becomes visible. Fig. 13 is the osteon of Fig.11 but imaged with polarized light. The green arrows in Figs. 11 and 13 point to the same location. The lamellas appear to have a sub-structure made of layers that respond differently to polarized light, which implies that the structure and organization of the bone in these layers must be different.

Bovine osteon in polarized light
Figure 13: Bovine bone osteon in polarized illumination (20x, NA=0.65).


A particularly beautiful find is Fig. 14, which shows an example of bone remodeling and the creation of a new osteon. In a living animal, bone is constantly modified by carefully orchestrated processes of disassembly and rebuilding. Bone is dismantled by specialized cells, called osteoclasts, which dig tunnels into the bone and release the molecular building materials into the blood stream. The bone tunnels are then populated by osteoblasts that build new osteons from the outside in. The balance of these two processes determines if the bone in a specific bone area is strengthened or weakened. In Fig. 14 the beginning of a new osteon in the space left behind by the activity of an osteoclast is captured and the first lamellas that have been laid down as part of the new osteon can be discerned.

Bone remodeling
Figure 14: Bovine bone remodeling (20x, NA=0.65).

Finally, Fig. 15 is a lower resolution image of a longitudinal section of bonvine bone. The column structure of the osteons in this part of long bone are clearly visible - as are some amateurish bubbles in the mounting medium.


Longitudinal section
Figure 15: Bovine bone, longitudinal section (4x, NA=0.12).

Figs. 11 to 15 are merely a glimpse of the complexity of bone. A real slide is like a strange country that awaits discovery and I hope that some of the readers are inspired to make their own bone thin sections and study them with their microscopes. Higher resolution versions of the bone images are available for download. Readers with an academic bent can go to their library to learn more about bone and how it functions. The famous book by Bruce Alberts et al. "Molecular Biology of the Cell", Garland Science, would be a good start.

All comments to the author are welcomed.

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