Introduction to Microscopic Techniques PDF

Introduction to cytology and genetics By Prof Dr. Ahmed Said Alazzouni 10/9/2023 DR. Ahmed S. Alazzouni  Microscopes are optical devices which allow observation of objects of microscopic size (less than 70μm) and which are invisible f...

Introduction to cytology and genetics By Prof Dr. Ahmed Said Alazzouni 10/9/2023 DR. Ahmed S. Alazzouni  Microscopes are optical devices which allow observation of objects of microscopic size (less than 70μm) and which are invisible for human eye. The main goal of this chapter is to provide basic information about types and design of microscopes, as well as about principles of work and the use of microscopic techniques in the biomedical disciplines. 10/9/2023 DR. Ahmed S. Alazzouni  Types of microscopes According to type of used radiation the light and electron microscopes are distinguished. According to type of radiation Light microscope (LM) Electron microscope (EM) 10/9/2023 DR. Ahmed S. Alazzouni  Light microscopes Light microscopes use white or ultraviolet light. Sunlight, bulb or vapour lamp is used as source of light. Optical parts are made from cut glass. The resolving power (resolution) of this type of microscopes is 0.2 μm and maximal theoretical magnification is 2 000 times. In practice, the objects are usually observed at a magnification of up to 1 000 times. In the routine conditions, the slides (native or fixed) are observed in the passing light. The method of lighting from above slides is used mainly in fluorescence and inverted microscope. 10/9/2023 DR. Ahmed S. Alazzouni  Light microscope 10/9/2023 DR. Ahmed S. Alazzouni  Electron microscopes Electron microscopes – radiation is a stream of electrons emitted by cathode. The function of optical parts occupy special electromagnetic lens. The resolving power is 0.2 nm and maximal useful magnification can be up to 1 000 000 times. Slides have to be prepared by special techniques (fixation, staining, contrasting, etc.). According to way of sample visualization the transmission electron microscope (TEM; electron beam passes through the preparation impregnated by electron- dense particles) and scanning electron microscope (SEM; electron beam proceeds on the surface of sample and bring information about surface details, shape as well as size of observed object) are distinguished. 10/9/2023 DR. Ahmed S. Alazzouni 10/9/2023 DR. Ahmed S. Alazzouni Types of Electron microoscope 10/9/2023 DR. Ahmed S. Alazzouni  Types of light microscopes Stereomicroscope Stereomicroscopes, also called dissecting microscopes belong to two light microscopes which focus on the same point from slightly different angles. This allows the specimen to be observed in three dimensions. Stereomicroscopes are relatively low power compared with light microscopes – useful magnification is usually below 100 times. They can have a single fixed magnification, several discrete magnifications, or a zoom magnification system. Working distance is much longer than with a typical microscope as well. It allows work to be done on the specimen while it is being observed through the microscope. 10/9/2023 DR. Ahmed S. Alazzouni  Inverted microscope Inverted microscope It is special type of light microscope with changed order of optical part and source of light. Optics is under the slide and light source is above. It is mainly used for monitoring cell cultures, when is necessary to visualize the growing of cells on the bottom of culture vessels (Petri dishes or cultivation flasks). 10/9/2023 DR. Ahmed S. Alazzouni Fluorescence microscope Fluorescence microscope Fluorescence microscope belongs to light microscopes which use vapour lamp as a source of UV radiation. Specially prepared biological samples are illuminated with light of a specific wavelength which induces emission of light with longer wavelength – visible light. There are only few objects with natural ability of fluorescence. For that reason, the special fluorescent dyes (fluorochromes) have to be used. It allows not only observation of cell structures, but also it is very useful method for molecular cytogenetics (e.g. FISH). 10/9/2023 DR. Ahmed S. Alazzouni Polarized microscope Polarized microscope It uses polarized light. The optical part contains special Nicol prisms to generate a beam of polarized light. They are used for observation of structures such as chitin, cellular fibres and crystalline cell inclusions 10/9/2023 DR. Ahmed S. Alazzouni Recommended procedure for microscopic observation Orientation in the microscopic view When observing the specimen in the microscope, position of an object can be Specified by three methods: quadrants – the optical field is divided clockwise into four quadrants I. – IV. concentric circles – central, pericentric and peripheral circle according to clock face 10/9/2023 DR. Ahmed S. Alazzouni Types of slide preparations According to mode of preparation different types of microscopic slides are recognised: impression preparations – a new clean slide is slightly pressed on the surface of the examined tissue and attached cells are observed (e.g. cells of liver or brain); smears – e.g. a small drop of suspension containing cells is placed near an end of a slide and is spread across the slide by the edge of another slide (e.g. blood smear); covered slides containing cell suspension or processed histology tissue covered by cover slip. 10/9/2023 DR. Ahmed S. Alazzouni  Native slides are used to observe physiological manifestations of cell (e.g. movement, cell division, particles ingestion etc.) or its typical shape. Because refractive index of organelles is very similar this technique does not allow observing intracellular structures. For this purpose phase contrast or dark field microscopy are used. Sometimes vital staining can be utilized e.g. to demonstrate phagocytosis of cells or to evaluate viability of cells using trypan blue dye. Permanent slides allow detailed observation of cell morphology. The preparation of permanent slides consists from fixation and following staining of cells or tissue slices. 10/9/2023 DR. Ahmed S. Alazzouni  Fixation terminates any ongoing living and autolytic biochemical processes in cells. Chemical or physical fixation is possible. During a physical fixation, used mostly for smears and touch preparations, biological sample is heated and dried at laboratory temperature or above a burner flame. Chemical fixation requires a liquid chemical fixative (e.g. formol, methanol, ethanol etc.). Fixation is followed by staining in different types of solutions which is based on affinity interactions between cell structures and stain components. They are informative (e.g. Nile Blue) or specific stains (e.g. Giemsa stain for chromatin visualization). Some dyes have both fixative and staining effect (e.g. Lugol’s solution, orcein). 10/9/2023 DR. Ahmed S. Alazzouni The cell The cell (cellula – Latin, kytos – Greek) is the basic morphological, functional and reproductive unit of all unicellular and multicellular organisms. It is autonomous and dynamic system which is characterized by basic life manifestations (metabolism, growth, irritability, reproduction and development). Science which deals with the study of cells is called cytology. 10/9/2023 DR. Ahmed S. Alazzouni Cell theory In 1838 Schleiden and Schwann formulated the cell theory in which they appointed plant and animal cells as elementary constituents of all living organisms. In 1855 Virchow revised cell theory and summarized it into three general points: 1) The cell is basic unit of all organisms; 2) Every cell consists of nucleus and cytoplasm; 3) Every cell originated from existing cell („Omnis cellula e cellula“). 10/9/2023 DR. Ahmed S. Alazzouni Cell types 10/9/2023 DR. Ahmed S. Alazzouni Eukaryotic cell Eukaryotic cells (Fig. 10) form unicellular and multicellular organisms. According to way of nutrition we distinguish autotrophic (protophyta) and heterotrophic (protozoa) organisms. Eukaryotic multicellular organisms are fungi, plants and animals. They undergo process of differentiation and specialization. They are composed from tissues, which are organizes to organs and organ system 10/9/2023 DR. Ahmed S. Alazzouni Shape and size of cells The shape and size of cells is genetically determined and is related to their location and function in the body. The basic cell shape is spherical (e.g. human oocyte, leukocytes) and other shapes are derived from it (Fig. 11) – biconcave disc (e.g. human erythrocytes), squamous (e.g. epithelial cells of skin or oesophagus), cuboidal (e.g. germinal epithelium of ovary, epithelium of ducts of many glands), columnar (e.g. epithelial cells of small intestine), polygonal (hepatocytes), spindle-shaped (e.g. fibroblast, myocytes), multi-polar (e.g. neurons, astrocytes), pear-shaped (e.g. Purkinje cells), pyramidal cells (e.g. pyramidal neurons) etc. Cells may have different projections. For example cells with fibrous projections (e.g. motor neurons, astrocytes); with irregular cytoplasmic projections (e.g. leukocytes, pericytes); with flattened projections (e.g. cells of tendons); with cilia and microvilli (e.g. cells of the small intestine, respiratory tract and uterus); with flagellum (e.g. sperm). 10/9/2023 DR. Ahmed S. Alazzouni Cell morphology Morphology of cells: a. biconcave disc – human erythrocyte; b. spherical cell –oocyt; c. columnar – enterocytes; d. polygonal cells – hepatocytes; e. spindle-shaped cell – myocytes; f. multi-polar – neurons; g. pear-shaped cell – Purkinje cells 10/9/2023 DR. Ahmed S. Alazzouni  Cells according size are: small, which reach size to 10 μm (e.g. erythrocytes, lymphocytes). The smallest cells are occurred in the stratum granulosum of cerebellum; middle sized, their size varied from 10 to 30 μm (e.g. plasmatic cells, chondrocytes). Most of cells in human tissues are of this size; big, with size over 30 μm (human ova, megakaryocytes, motoric neurons). 10/9/2023 DR. Ahmed S. Alazzouni Molecular structure of cell membranes Cell membranes (biomembranes) are important part of all cells. Their discovery is closely related to the upgrading of microscopic techniques, especially with the design of transmission electron microscopy, by which was observed typical trilaminar structure. Further observations showed that biomembranes in the cell are similar in structure and slight differences in chemical composition are due to cell differentiation and specialization. 10/9/2023 DR. Ahmed S. Alazzouni  Function of cell membrane Every cell is surrounded by the cytoplasmic membrane, which separates intracellular from extracellular space. Its average thickness is 60 – 10 nm. Cell membrane is selectively permeable boundary, which ensures the maintenance of dynamic equilibrium between cell and environment. It contains enzymes, receptors, transport proteins, signalling systems and antigens. It performs different functions, e.g. intake of substances, interactions, recognition of signals, etc. The biological membrane is a part of many important cellular organelles. It makes their border and also it is involved in the execution of physiological processes (e.g. oxidative phosphorylation). 10/9/2023 DR. Ahmed S. Alazzouni  The main components of cell membranes The main components of cell membranes are phospholipids. The molecule of phospholipids is composed of polar (hydrophilic) head and two non-polar (hydrophobic) fatty acids chains. In the aqueous environment the hydrophilic parts are oriented towards the water around them and fatty acids chains to each other, creating so-called phospholipid bilayer.Given that phospholipids are not chemically bound to each other, their lateral movement is possible. 10/9/2023 DR. Ahmed S. Alazzouni Cell membrane protiens integral, which affect the hydrophobic parts of the phospholipid bilayer or transgress it. They are hardly separable from the biomembranes; peripheral, which lie outside the lipid bilayer. They are associated with electrostatic bonds and can be easily separated from biomembranes. Membrane proteins perform various functions. They are part of biomembrane structure (structural proteins). 1- Some of them are involved in the transport of ions across the membrane (pump and ion channels) or in the transfer of substances along the electrochemical gradient by facilitated diffusion. 2- Many of them are part of the receptors that are able to specifically bind hormones, neurotransmitters and other signal molecules. 3- Some have the role of enzymes. 10/9/2023 DR. Ahmed S. Alazzouni 10/9/2023 DR. Ahmed S. Alazzouni