**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**.

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

#### **Figure 2.**

*Schematic flow diagram of a scanning electron microscope [14].*

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**.

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

**Figure 3.** *Schematic flow diagram of a transmission electron microscope [14].*

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].
