**1. Introduction**

Histopathology is very widely used, not only as a diagnostic tool but also as an examination tool, monitoring the effects of therapy or estimating a disease's prognosis. Microscopes are necessary equipment for carrying out histopathological functions. Histopathological preparations rely on microscopes, as does result examination [1].

A microscope is a tool for viewing cell and tissue structures that cannot be seen by the naked eye because of their micron or smaller size. The cell is the smallest unit of life. Several cells that come together and perform the same function are referred to as tissue. Organs are several tissues that come together and form the same function. The microscope was first introduced by Zacharias Jansen and his father in 1591 [2], and it was then perfected by Leeuwenhoek in 1673. Furthermore, along with the need to see certain tissue structures, microscopes evolved from light to fluorescence microscopes [2].

Histopathology is a technique for making histological preparations for diseased tissues. These techniques include fixation techniques, tissue processing, tissue sectioning, and tissue staining. Fixation techniques are needed for hardening, keeping the cell death process from continuing, and keeping structural molecules from being damaged. Tissue processing aims to remove water from the intracell and replace it with a medium that can harden the cell structure to make it easy to cut. Tissue sectioning refers to cutting the prepared specimen into several slices. Finally, tissue staining aims to color the cell and tissue structures with specific staining according to the examination's purpose [3].

Microscopy and histopathology technique development are fundamental things to know when making examination preparations and reading tissue preparations from appropriate controls to ensure good examination quality [4].

#### **2. Microscope development**

A microscope is a tool used to view tissues and organs' microscopic structures. Microscopes work the same way eyes work. Light, which is an electromagnetic spectrum, is transmitted to the cornea and through the eye's diaphragm (pupil) until it reaches the retina. Whereas in light microscopes, the light that hits the specimen preparation is transmitted by the microscope diaphragm and then captured by the objective and ocular lens for the eye. The resulting image's magnification depends on the objective and ocular lenses' sizes [2]. The following are the microscopes commonly used for a histopathology examination.

#### **2.1 Light microscopy**

Early in the sixteenth century, the first microscope was assembled by Jansen and his father. It used the light from the sun (**Figure 1**). The scientists at that time did not feel comfortable using this microscope because the light source did not fall on one point, thus interfering with the view. Sun or white light consists of several color spectrums. When it falls on a simple lens like Jansen's work, it will make the colors scatter according to their wavelength. This is known as chromatic aberration. This became the basis for Leeuwenhoek in 1673 to make two lenses with different mediums. Colors that degraded due to certain wavelengths could be absorbed by the lens medium. The achromatic is a lens medium that can correct two spectrums of blue and red, while an apochromat medium is a lens medium that corrects two spectrums of green and yellow [2, 3]. Leeuwenhoek's microscope can magnify a maximum of 300 times the resulting image. At the time, he could identify bacteria, muscles, teeth, and blood cells [3]. The microscope's components were then developed to correct chromatic aberration with the discovery of lens mediums that fit examination needs. Leeuwenhoek's principal work was the basic development of the light microscope (**Figure 2**).

Light microscopes' components and models evolve with their needs and uses. Currently, apart from being a diagnostic tool, light microscopes are also used for teaching and learning purposes. Several types of microscopes have been established with their respective advantages for teaching purposes. The double or triple ocular lens and light microscope models that are connected to the computer with or without application are widely used. **Figure 3** shows the resulting image with a light microscope.

*Introduction of Histopathology DOI: http://dx.doi.org/10.5772/intechopen.110225*

**Figure 1.** *Jansen's microscope with the single lens source [2].*

**Figure 2.** *Light microscopy model in general source [2].*

#### **2.2 Electron microscopy**

The electron microscope was first assembled by Ernst Ruska in 1920. This microscope's purpose is to view biological ultrastructures and identify diseases that are difficult to explain. Its working principle is to use light with short wavelengths

**Figure 3.** *Image of the spinal cord by light microscope with 400 times magnification.*

**Figure 4.** *Image of nucleus and RER neuron cell by TEM.*

to produce better resolution and magnification. Resolution is the ratio between the wavelength and the width of the diaphragm opening [3, 5].

The types of electron microscopes are the scanning electron microscope (SEM), the transmission electron microscope (TEM), and the cryo-electron microscope (Cryo-EM) [5–7]. SEM is a type of electron microscope that can only take ultrasound images on surfaces, such as the skin's epidermal layer. TEM is a type of electron microscope that can take ultra-cell images of cytoplasmic organelles such as mitochondria and rough endoplasmic reticulum (RER). Cryo-EM is a type of electron microscope that can produce ultra-cell images up to atoms in 3D for single particle analysis, such as on certain proteins, with better resolution [5–7].

The difference in preparation between a light microscope and SEM or TEM is that the specimen's maximum size before processing is cut at 1 mm3 as well as its fixative medium, which will be discussed in detail in topic 3 [3]. The differences in preparation between the light microscope and Cryo-EM are specimen screening and preparation, data acquisition, image processing, and structure validation [6]. **Figure 4** shows an image result from EM.

#### **2.3 Phase contrast microscopy/interference microscopy**

The working principle of a phase-contrast microscope or interference microscope is that the light is blocked by the specimen, thus producing a **"**halo**"** in the resulting image. A halo pattern will form on the bright and dark bands when light passes through the specimen. Besides the halo, the resulting image is also a color gradation and a quantitative index of refraction. This microscope shows biological structures in larger pieces than other types of microscopes [3].

#### **2.4 Fluorescence microscopy**

Fluorescence is light that is reflected by objects or specimens that are lit at a certain wavelength. Objects or specimens that can emit fluorescence alone are called fluorophores, also called primary fluorescence or autofluorescence. Some examples are porphyria, elastic fiber, collagen, vitamin A, and lipofuscin [3].

Specimen fluorescence can also be induced by adding a substance, such as a certain antibody painted with a fluorescence agent like blue, green, or ultraviolet fluorochrome [2]. This was first introduced by Coons, Creech, and Jones in 1941.

Fluorescence microscopy's development from two dimensions (2D) to three dimensions (3D) makes pathological models in biological tissues clearer. In 3D fluorescence microscopy, axial scanning is required for several specimen pieces that make up the volume. Data is transferred to a computer with certain applications. The required variables can be calculated quantitatively or only qualitatively [8]. This microscope's weakness is that the entire field of view will show fluorochrome, including areas that are not the focus of research [3]. This weakness became the basis for confocal microscopy development.

#### **2.5 Confocal microscopy**

Confocal microscopy enhances the image results from fluorescence microscopy. Confocal microscopes have a pinhole component to only focus the image on the part being examined. Microscopic imaging results can be seen at one time [3].

Histopathology has an important role in the diagnosis. However, ordinary histological techniques after a biopsy are not able to describe details such as the boundaries of cancer cell development that must be taken during the operation at the same time (during surgery). To overcome this, the confocal microscope technique was developed into confocal laser endomicroscopy (CLE). The CLE technique greatly improves the surgical outcome and prognosis of most cancers [8].

The CLE technique requires intravenous injection of fluorosphere contrast enhancer before surgery begins. Sodium fluorescence (SF) is a type of fluorosphere that has been studied. It has a composition of 5 mL in 10% saline solution and must be injected just before surgery. The second type is 5-aminolevulinic acid (5-ALA) with a composition of 20 mg/kg, which is injected 3 hours before surgery. The time of contrast enhancer administration from seven studies gave different results in terms of cell contour [8].

Pathological feature observation results using the CLE technique were better than those with ordinary histological techniques. The contour cells could be differentiated in terms of vascular proliferation, cellular density, and irregular cell phenotype

**Figure 5.**

*A. Confocal imaging of CLE during operation. B. Convivo ex vivo CLE image that show low cellular density (red circle). Source [8].*

between high-grade gliomas, low-grade gliomas, and normal glia at the same time during surgery. Based on research, the imaging results were better when using SF to show the cells' contours (**Figure 5**) [8].
