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Osteoporosis is a skeletal disorder characterized by decreased bone strength which affects the increased risk of fracture. Emerging evidence discovered that osteoporosis is associated with reduced bone density and bone quality. Therefore, analysis of bone morphology can afford insight into the characteristics and processes of osteoporosis. Electron microscopy, one of the best methods, can directly provide ultrastructure evidence for bone morphology. Here, we describe an experimental procedure for electron microscopy preparation and analysis of the resulting images, especially scanning and transmission electron microscopes, to analyze bone morphology in animal models of rats. Compared to other bone analyzers such as atomic absorption spectrophotometer, ultraviolet–visible spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, and X-ray diffraction, scanning and transmission electron microscopes are still important to strengthen visual analysis, and a better understanding of this method could be significant to examine bone morphology.
Department of Physics Education, Universitas Indraprasta PGRI, Indonesia
Rum Sapundani
Department of Electromedical Engineering, Universitas Muhammadiyah Purwokerto, Indonesia
*Address all correspondence to: nengnendenmulyaningsih@gmail.com
1. Introduction
Osteoporosis is a metabolic disorder causing bone mineral density to decrease and changing the bone structure [1]. It is a degenerative disease whose initial symptoms are not known with certainty. Someone who suffers from osteoporosis will usually experience complaints if the stage is severe [2, 3]. Bones with osteoporosis will experience a decrease in mechanical strength so they are prone to fracture, and will easily crack or become brittle if exposed to a hard object. It is characterized by low bone mass and structural breakdown of bone tissue. Some parts of the body that are at risk for osteoporosis include the spine, pelvis, femur, tibia, pelvic bones, wrist bones, and other bone parts dominated by the trabecular bone [4, 5, 6].
Osteoporosis can be diagnosed clinically using bone mineral density measurements. At present, bone densitometry is the standard method for diagnosis and treatment monitoring. However, it still possesses significant drawbacks because it cannot give information about the structural manifestations of the disease. Frequently, bone mineral density is analyzed using x-ray or ultrasound imaging methods. In x-ray imaging such as dual-energy x-ray absorptiometry (DEXA) and quantitative computer tomography (QCT), the intensity of the image is correlated to the mineral density of the tissue. In ultrasound, the intensity of the image reflects changes in the frequency and amplitude of sound waves traveling through tissue. X-ray procedures employ ionizing radiation, which can have a damaging impact in sufficient doses. Ultrasound, although harmless, offers only a small field of view, which can restrict measurement accuracy. In addition to bone density, bone quality which includes bone microarchitecture is also a concern. Recent developments in imaging, especially electron microscopy, can now give detailed information about the effects of architecture on disease progression and regression in response to treatment. However, before the diagnosis is made, of course, it is necessary to study and research in a sample or biological material to determine the process of bone remodeling and osteoporosis. The samples analyzed generally use rats as animal models. It takes a long time to make rats osteoporosis naturally. Therefore, rats were given treatment to condition the occurrence of osteoporosis. Some of the common actions taken to condition osteoporosis rats are by giving them a calcium-deficient diet or by performing ovariectomy on these rats [7, 8, 9, 10].
Several characterization tools that can be used to analyze mouse bones include X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Ultraviolet (UV)-visible Spectroscopy, or Atomic Absorption Spectroscopy (AAS). However, these tools provide information in the form of numbers or graphs. A promising imaging modality for morphological analysis of both cortical and trabecular bone is electron microscopy. The types of electron microscopes commonly used to analyze bone morphology are Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM). Figure 1 illustrates the different imaging modalities, between SEM and TEM, which were used to analyze the morphology of the rat femur bone.
Figure 1.
(a) Image of SEM scans, and (b) image of TEM scans on femoral bones of rats from the osteoporosis group.
This review focuses on the emerging methodology of quantitative electron microscopy to assess the bone structure and morphology of osteoporotic rats. For more than 10 years, numerous approaches have been investigated to obtain quantitative image-based information on bone architecture, both trabecular bone, and cortical bone. An indirect method that does not require resolution at individual trabecular scales and can therefore be performed at any skeletal location, a recoverable component of the degree of total transverse relaxation. Therefore, electron microscopy-based structure analysis is technically demanding in terms of the required image acquisition. Other requirements that must be fulfilled involve motion correction and image registration, both of which are important to achieve the reproducibility required in repeated studies. The main targeted clinical application involves the prediction of fracture risk in femoral rats conditioned by osteoporosis due to ovariectomy.
An electron microscope is a type of microscope in which the illumination source is an electron beam. Illumination itself is a process of light coming to an object. There are electron microscopes that have high image resolution, even magnifying objects on the nanometer scale, which are produced by the controlled use of electrons in a vacuum captured on a fluorescent screen. The first electron microscope was introduced by an engineer and professor from German, Ernst Ruska (1906-1988), in 1931, and the same principles behind his prototype still dominate modern Ems [11, 12].
2.1 Principle
Electron microscopy uses signals generated by the interaction of the electron beam with the sample to gain information about its structure, morphology, and composition. The process and major parts of an electron microscope are:
Electrons are produced by the electron gun
The electron beam is concentrated on the sample by condenser lenses.
About 100 kV – 1000 kV accelerating voltage is employed between the tungsten filament and anode to move electrons down the column.
The sample to be observed should be fabricated very thin, or minimal 200 times thinner than that observed in optical microscopes. A very thin sample with a size of 20-100 nm was sliced and put in the sample holder.
The electronic beam traverses the sample and electrons are scattered relying on the thickness or refractive index of different areas of the sample.
The denser sample areas will scatter more electrons so that the image displayed in these areas will be darker because fewer electrons hit this area of the screen. Contrarily, the transparent areas will look brighter.
The electron beam leaving the sample is transferred to the objective lens which will make a magnified image.
The eyepiece then renders the final image for further magnification.
2.2 Types of electron microscope
Electron microscopes are categorized into three types based on operating styles:
2.2.1 Scanning electron microscope (SEM)
Nowadays, scanning electron microscopy (SEM) is a robust and effective imaging instrument. It is employed for scanning surfaces with a magnification from 1 m to 1 nm which depends on the hardware used to create the electron beam with various lenses and vacuum systems. Further, it is integrated with an energy dispersion spectrometer to combine the elemental analysis potential on the sample surface. SEM imaging has new characteristics those are backscattering electrons and secondary electrons which increase the scanning potential. The electron gun includes the main parts of the SEM components. With the existence of different magnetic lenses and vacuum systems, SEM has become a unique imaging tool [13].
The characterization method with SEM can deliver visual information on the morphology of the bone surface. SEM images can also be analyzed with an image processing program such as ImageJ, with the output in the form of a histogram of pixels that can provide information about the cavities in the bone and their distribution. From the histogram, bone quality can be known quantitatively by looking at the average pixel value and the percentage of cavity intensity. Schematically, the scan with SEM is shown in Figure 2.
Figure 2.
Schematic flow diagram of a scanning electron microscope [14].
From Figure 2, Electron Microscopes utilize electrons beam to illuminate a sample and construct an image with high magnification. The electrons from the electron source passing through the condenser lenses, aperture, scanning coil, objective lens, detectors and hit the gold-coated sample positioned on its holder. The condenser lenses center the electron beam in a specific area corresponding to the sample and thus generate the image. Electrons hit the sample surface thereby producing the secondary electrons which are detected by the secondary electron detector and transformed into a signal delivered to a monitor scanner.
Conventional SEM relies on the emanation of auxiliary electrons from the sample surface. As its large focus depth, the SEM is the EM analog of the stereo light microscope. It gives nitty-gritty pictures of the cell surface and the whole life form. It can moreover be worked for molecule checking and measuring, and for handle control. A SEM, it is so called, because it forms the image by scanning a focused electron beam onto the sample surface in a raster design. The primary electron beam interacting with atoms nearby the surface induces particle emission at any location in the raster. The emissions, for instance, include low energy secondary electrons, high energy scattering electrons, X-rays, and photons that then can be gathered by distinct detectors, and their relative quantities are converted to brightness at every equivalence point on the cathode ray tube (CRT). Due to the considerably smaller raster size than the CRT screen display, the resulting image is the image magnification of the sample. SEMs are equipped with proper equipment such as secondary detectors, backscattering, and X-rays, which can be functioned to analyze the topography and atomic composition of the sample and the surface distribution of immune labels [15, 16].
2.2.2 Transmission electron microscope (TEM)
Transmission electron microscopes are exploited to examine thin samples (parts of tissue, molecules, etc.) that electrons can traverse to produce a projected image. TEM is analogous to a conventional light microscope. Schematically, the scan with TEM is presented in Figure 3.
Figure 3.
Schematic flow diagram of a transmission electron microscope [14].
In Figure 3, the TEM applies high-energy electrons for imaging. It has been developed since the 1938’s. Its operation requires a very high voltage of about 500 − 1000 kV with a resolution reaching 0.1 nm. During TEM operation, the electrons beam is generated and transmitted through an ultra-thin sample. Then, the unscattered electrons are transmitted through the sample and hit the fluorescent screen at the bottom of the microscope, thus producing an image. By changing the gun voltage, the electron velocity can be modified which in turn changes the image. Commonly, TEM generates a grayscale image that exhibits lighter and darker regions. The lighter regions demonstrate regions with a large number of transmitted electrons while the darker ones represent a lower number and denser regions in the sample. The sample used in TEM should be prepared thin enough for electrons to be transmitted [17].
2.2.3 Reflection electron microscope (REM)
Another type of development of the electron microscope is the reflection electron microscope (REM). The REM is an electron microscope that has almost the same way of working as TEM, the difference is that REM uses the detection of electron reflections on the object’s surface. The sample is semi-infinite and the surface to be observed is almost parallel to the electron beam. The transmitted spot may or may not be observable, depending on the sample size as shown in Figure 4. This technique is specifically used in combination with the Reflection High Energy Electron Diffraction (RHEED) technique and the reflection high-energy loss spectrum (RHELS) technique.
Figure 4.
These ray diagrams illustrate (a) TEM and (b) REM [18].
REM could be a combination of imaging, diffraction, and spectroscopy procedures for the characterization of topography, crystal structure, and composition of surfaces of single crystals. High-energy electrons are occurring at looking points to the surface and reflected electrons are utilized to create a REM picture. Utilization of REM in analyzing osteoporosis in bone is still rarely done, because REM has several drawbacks including REM images are shortened in the direction of electron events and high resolution is only achieved in the normal direction, so that in analyzing surface topographic details more than one azimuth is needed. Meanwhile, bones that are not homogeneous can produce different images in each image. These techniques are applicable to metal [19], semiconductor [20], crystal surfaces [21], surface reconstructions and phase transformations [22], correlation between topographical features and reconstructions, directions, distribution, and motion of surface steps, dislocations on surfaces, nucleation and growth of films, and surface reactions [23].
Sample preparation for REM is the same as for other types of electron microscopy, i.e., it must be ensured that the surface is sufficiently flat and clean. The size of the sample should fit the microscope sample holder by about 3 mm. Then inserted into the electron microscope with a surface normal perpendicular to the optical axis. The nominal size of a REM sample is no more than 1 mm3 for a sample holder which gets 3 mm grids. Hence, the perceptible surface is about 1 mm or less. The lower restrain of the surface area is approximately 10 μm in diameter.
Electron microscopy (EM) is a method to obtain biological and non-biological samples’ images with a high-resolution. This method is frequently employed in biomedical research to examine the detailed structure of tissues, cells, organelles, and macromolecular complexes. High-resolution EM images are produced from the use of electrons having very short wavelengths as the illumination source. EM is used in conjunction with numerous additional methods (e.g., thin cutting, immune labeling, and negative staining) to answer specific questions. EM images can deliver crucial information about the structural and morphological basis of bone. Several results of prior studies that investigated bone with EM are presented in Table 1.
Scanning electron microscopy (SEM)
Transmission electron microscopy (TEM)
The SEM image exhibited that the longer the time after ovariectomy, the greater the degree of damage seen in the tibial cavity [24]
Acicular crystals of apatite with approximate dimensions of ~20–30 nm by 5 nm. Gap zones and overlap zones in collagen fibrils [25]
The group of ovariectomized rats had histograms that increasingly shifted more black areas. Areas that were black or dark relate to cavities in the bone [26]
Apatite crystals that resemble tablet form, in the control group have a longer size, and for groups of ovariectomized rats there was a decrease in size both length and width [26]
Network organization in trabecular bone showing topographical details [27]
Different calcium phosphate minerals morphologies in the bone extracellular matrix: dense granules, globular aggregates of needle-like apatite, and mature fibrous minerals [28, 29]
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 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.
SEM image of the rat femur bone, (a) sham, (b) osteoporosis due to ovariectomy.
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].
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, 39, 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, 45, 46]. All these conditions cause parathyroid hormone activity to increase and bone density to decrease which in turn triggers osteoporosis [47, 48].
Figure 6.
SEM image of rat femur after analysis with ImageJ, (a) sham, (b) osteoporosis due to ovariectomy.
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].
Figure 7.
Particle diameter size of the rat femur bone, (a) sham, (b) osteoporosis due to ovariectomy.
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.
Imaging at the nanoscale is very important to analyze the quality and structure of bone morphology. This review examines the images produced by electron microscopy of the femur bones of rats under sham conditions and osteoporosis due to ovariectomy. The scanned electron microscopy image with the help of the ImageJ application provides information that the femur bones of ovariectomized rats show signs of osteoporosis. Some of the parameters that characterize the cavities in the ovariectomized femur appear wider, with the edges of the cavity appearing to be cracked. In addition, the particle diameter also increased by an average of 77.16%. Therefore, electron microscopy is one of the best approaches, which can directly provide ultrastructural evidence for bone morphology, and furthermore, the results of this bone morphology analysis can provide insight into the characteristics and processes of osteoporosis.
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Written By
Neng Nenden Mulyaningsih and Rum Sapundani
Submitted: 16 February 2022Reviewed: 21 March 2022Published: 07 May 2022
Open access peer-reviewed chapter
