**4. Image processing techniques**

After obtaining the image from the electron microscope, the next step that needs to be done is to analyze the resulting image. Several applications that can be used to process the output image of an electron microscope, including ImageJ, Matlab, Python, OpenCV, Dragonfly, HyperSpy, and others. Each has its own advantages and disadvantages. However, on this occasion, we will review the analysis of images from

**Figure 5.** *SEM image of the rat femur bone, (a) sham, (b) osteoporosis due to ovariectomy.*

electron microscopy using the ImageJ application. **Figure 5** shows the results of SEM imaging of sham rat femur (a) and osteoporosis due to ovariectomy (b).

**Figure 5** shows an SEM image of a rat bone taken from the femur at 1000x magnification. **Figure 5(a)** SEM image of the femur bone of a 13-week-old sham rat, visually it can be seen that the surface is denser, there are no large cavities found. This is different from the SEM image shown in **Figure 5(b)**, the image was taken from the femur bones of rats with osteoporosis due to ovariectomy treatment. The surface is clearly visible in the presence of wider cavities. The picture was taken when the rats were 21 weeks old or 9 weeks after being given ovariectomy. In accordance with the results of previous studies, the rats began to show the characteristics of osteoporosis at the ninth week since ovariectomy [36].

#### *Analysis of Osteoporosis by Electron Microscopy DOI: http://dx.doi.org/10.5772/intechopen.104582*

The characteristics of osteoporosis are clearer from the SEM image that has been analyzed with the help of the ImageJ application as shown in **Figure 6**. **Figure 6(a)** results of the analysis of the sham femur, the black color is thicker and fused together, indicating that the bone is still solid. This is supported by the results of the [37] study which showed that the bones of sham rats contained minerals such as calcium, magnesium, and phosphorus which were still normal. Meanwhile in **Figure 6(b)** the results of the analysis of the femur bones of rats treated with ovariectomy, it appears that the color is lighter, with the black parts that have started to break off and are thinner. This is because ovariectomy treatment can cause a decrease in the hormone estrogen in the body. With a decrease in the hormone estrogen, bone resorption by osteoclasts increases, and conversely osteoblast activity becomes inhibited [38–40]. As a result, bone density will also decrease, and osteoporosis occurs [41]. In addition, a decrease in the hormone estrogen can also increase the resorption of calcium (Ca) in bone, so that bone mass will decrease [42, 43]. Even the absorption of Ca in the intestine also decreases and the excretion of Ca through the kidneys increases [44–46]. All these conditions cause parathyroid hormone activity to increase and bone density to decrease which in turn triggers osteoporosis [47, 48].

Quantitatively several parameters that can be known from SEM image analysis with Image J application include particle diameter, percentage of voids, or porosity analysis. Particle diameter analysis for the same sample as previously mentioned is shown in **Figure 7**. **Figure 7(a)** shows the particle diameter size of the sham rat femur bone ranging from 1.5 to 34.4 μm. The particle diameter experienced a significant increase in the ovariectomized femur bone, the highest size reaching 150.2 μm as shown in **Figure 7(b)**. Larger particle sizes tend to be more porous, as a result, are more brittle [49].

Likewise, TEM images can be analyzed and obtained the same information as for images from SEM. The output of the porosity analysis can also be carried out, some quantitative data can be obtained from the results of the porosity analysis, namely the pore volume and the percentage of pores. Some of these parameters can be used as a reference for osteoporosis analysis in bone, especially in experimental animal models.
