Histology PDF

Histology: is the science of studying microscopic structures of tissues and organs of the body. -Histology is a Greek word (Histo=tissue, logia = science) also called microscopic anatomy. -In order to study the microscopic structure of tissue, we need to use...

Histology: is the science of studying microscopic structures of tissues and organs of the body. -Histology is a Greek word (Histo=tissue, logia = science) also called microscopic anatomy. -In order to study the microscopic structure of tissue, we need to use a microscope. Microscopy It is an instrument that magnifies an image and allow visualization of details that cannot be seen by unaided human eye. Types of the microscope: 1-Light microscope (LM): Commonly used in teaching labs. 2- Electron microscope (EM): include Transmission EM and scanning EM. I-Light microscope: Parts of LM: It consists of 1- A light source for illumination of a specimen such as day light or lamp. 2-Acondenser to focus the beam of light at the level of specimen. 3-Astage to place the slide on it. 4-Objective lens to gather the light that has passed through the specimen. There are various magnification powers of objective lens such as ×4, ×10, ×40, ×100. 5-Ocular lens (eye piece) may be one in uniocular microscope or 2 lens in binocular microscope. Image showing light microscope. II-Electron microscope (EM): Electron microscopes are based on the interaction of tissue components with beams of electrons. The wavelength in an electron beam is much shorter than that of light, allowing a 1000-fold increase in resolution. The illumination source of the electron microscope (TEM) is a beam of electrons. Lenses are electromagnetic lenses.  Some electrons interact with atoms in the section.so electrons are being absorbed or scattered or passed through the specimen with no interaction. Electrons reaching the objective lens form an image that is then magnified and finally projected on a fluorescent screen or a charge- coupled device (CCD) monitor and camera. The resulting image is white and black in color. The resolving power of the TEM is 0.2-0.5 nm. Types of EM: 1-Transmission electron microscope (TEM) 2-Scanning electron microscope (SEM) Functions of microscope: 1-Magnification 2- Resolution. Magnification: is the increase the linear dimension of an object without optical defect. Final magnification of the microscope = magnification of an objective lens× magnification of ocular lens. LM magnifies an object to 1000 times while EM can magnify it up to 100000 times. Resolution: It is the ability of the microscope to produce separate images of 2 adjacent objects Limit of resolution: It is the shortest distance between 2 objects below which they appear as one or not seen at all. Limit of resolution of human eye is 0.2 mm while it is 0.2 micron for LM and 0.1 nanometer for TEM. Factors affecting resolution power: 1- lenses used. 2-wavelength of light 3-tissue thickness 4-quality of fixation 5-Staining intensity. *To examine tissue by LM, it must be thin enough to allow passage of light through it. Also, we need sufficient level of contrast of the tissue to be examined by LM. This leads to preparation of the tissue for examination. Tissue preparation I-Preparation of fixed tissues and cells (Paraffin technique): Most of slides you will study in the lab are formalin fixed, paraffin embedded, and Hematoxylin and Eosin (Hx&E) stained sections. Steps: 1-Sampling: the sample may be taken from animals such as mice and rats to be used in research purposes or human beings for diagnosis of the diseases. Sample taken by surgical excision of a living human called biopsy. Sample taken postmortem called autopsy. Size of the sample is usually small 0.5 cm *1.0 cm or less. It should be fixed as soon as possible to avoid postmortem changes (putrefaction &autolysis). 2-Fixation: It prevents postmortem changes. It makes the tissue easy to processed and stained. Most used fixative is 10% formalin. Other types of fixatives can be used such as Bouin̕ s solution. 3-Washing to remove excess fixative. 4-Dehydration (removal of water) by using ascending grades of ethyl alcohol (70%-90%-95%-100%) to avoid shrinkage of the cell. 5- Clearing by using organic solvents such as xylol. Xylol removes alcohol and it is miscible for paraffin wax.. 6-Impergnation means infiltration of the specimen with melted paraffin wax. The melting point of paraffin is 58 c. This step is done in a hot air oven. 7-Pouring the melted paraffin and specimen in special blocks and let it to hardens in room temperature. 8-Sectioning means slicing the tissue blocks into 5-10 micron thickness of the section by a microtome. 9-Mounting: The sections are allowed to float in warm water bath and then collected on a clean glass slide to be stained. 10-staining: We stain the tissue to allow visualization of cells and distinguish its various components. Hx&E is the general histological stain (staining most of the cells in the same way). Hx&E is a water-soluble stain consisting of 2 components. 1-Hematoxylin is a basic dye: it stains acidic structure that have an affinity for basic dyes and are termed basophilic structures. These structures appear blue in color. Nucleic acids are acidic so bind to basic dye(basophilic). 2-Eosin is an acidic dye: it stains basic structure that have an affinity for acidic dyes and are termed acidophilic or eosinophilic structures. These structures appear red in color. Cytoplasmic proteins are basic so bind to acidic dye(acidophilic). Special staining methods are used to demonstrate special cell and tissue components such as silver stain for demonstrating Golgi complex, PAS for demonstrating glycogen, iron Hx for demonstrating mitochondria and Sudan III for demonstrating lipid droplets.