Baraa Ayed AL-Odat. Israa Ayed. Heba kalbouneh

Transcription

1 1 Baraa Ayed AL-Odat Israa Ayed Heba kalbouneh

2 Introduction: "lecture #1" The name " histology " is derived from the Greek words: "histo" means a tissue and "logos" means the study of. So, Histology mean : the study of tissue. 1 -Histology is the microscopic Anatomy ( study the same structures: muscles and bones... but under microscope ) - remember that : Histology is looking at a two dimensional image of a three dimensional structure. Cells: are the smallest functional living units in our body. And the cells with similar structure,similar shape and similar function are grouped together to form a tissue. There are 4 types of tissues : 1)epithelial tissue 2)connective tissue 3)muscular tissue 4)nervous tissue - A group of tissue forms an organ. For example: in the stomach: the lining of stomach is composed of epithelial tissue and the wall of stomach is composed of muscles (muscular tissue ), the contraction of these muscles is important for digesting food and this contraction is controlled by nerves (nervous tissue ), all of these tissues are connected together by connective tissue. - A group of organs forms a system. ( respiratory system, gastrointestinal systemdigestion system -,urinary system and so on. For example : stomach, liver, pancreas, intestines. Form together the gastrointestinal system. - cell tissue organ system organism. The History of Histology : Bichat was the first anatomist (he was surgeon too) who identified tissue as the tissue without using microscope. for example when he studied a disease in the stomach, he knew that the disease in a certain part (certain tissue) not in the whole of them. Units that you must know in histology : * 1mille meter = 1000 micro meter (µm) * 1µm = 1000 nano meter (nm) * 1mm= nm

3 Microtechniques: " how to prepare a tissue in order to view it under a microscope". The principle of microtechneiques is to harden a tissue in order to cut it in to sections ( different methods but the same principle). We will talk about two microtechniques : 2 1) paraffin wax technique 2) freezing technique 1) paraffin wax technique : the principle is to replace the water inside a tissue with a wax material, ( we have transferred a tissue from a soft piece into a solid piece ). a)fixation :exposing the tissue to fixative material ( paraformalehyde - or simply formalin -) to preserve the structural and molecular composition of a tissue by preventing bacterial decomposition and enzymatic digestion. Parafomaldehyde interacts with proteins in the sample and cross links them together and then preserve the sample. the time depends on the size of the sample. b)dehydration: "de" means removal and " hydration " means water so, dehydration means the removal of water from a tissue in order to replace it by wax. We put a sample in ascending concentration of alcohol 70%,80%,90%,100% so, all parts will be filled with alcohol. We don t put it in 100% alcohol directly, because the tissue will shrink. c)clearing : is the intermediate step because the wax is not soluble in alcohol we need this step to remove alcohol and replace it with another material (xylene an organic solvent) that can be replaced with wax. d) embedding : we put a tissue in a small box containing the molten wax to create a support medium for a sample ( the tissue will be within a block of wax). e) cutting (sectioning): to cut a block of tissue in micrometer thickness sections, we use microtome ( "micro" means micrometer thickness and "tome" means cutting). we put a sample block in a block holder then we turn the handle up and down towards a knife, so we get a series - ribbon - of sections. f)mounting: we put a sections in water path in order to make them flat and then we put them into slides. g) staining : we cannot show and view the sample without staining. " stain" is a chemical substance which reacts with a tissue producing a color and then we put a cover slip on the slide and view it. 2) freezing technique : we put a sample in a liquid nitrogen,then we expose the tissue to a very low temperatures so the tissue will freeze and become solid ( by using a, so

4 liquid nitrogen the tissue will quickly be frozen). we cannot use ordinary microtome here because the block will melt, that s why we use special machine called cryostat (it's simply microtome but placed in a freezer), we control the temperature to less than zero. and then by turning a handle we get a series of sections. The quality of sections The speed of the method Paraffin wax Is good with less artifacts Slow. 12 hours. Freezing is poor with more artifacts. Fast. 20 minutes. االثار التي تظهر على العينة عند رؤيتها بالميكروسكوب ويكون سببها عمليات انتاج العينة )صناعي( : artifacts * Note: During the surgery, the surgeon need quick method ( freezing technique) that gives quick results.also, when we study lipids we cannot use paraffin wax because in this method we used xylene which dissolve lipids so, we should use freezing technique Staining: we will study 3 types of staining: 1) ordinary stains 2) immunohistochemistry and immunocytochemistry( "cyto" means the cell and "histo" mean the tissue ). 3) Hybridization techniques *immunohistochemistry: this method depends on the specific interaction between the antigen and the antibody. For example, if you have bacterial infection the bacteria is the antigen and your body will consider it as a foreign body and produce antibodies against it.the interaction between the antibody and the antigen will destroy the antigen to produce an infection. There are 2 types of immunohistochemistry(ihc): a) Direct IHC: if you want to determine the location of certain protein (for example actin protein ) in the cell ( Is it in the nucleus, cytoplasm or cell wall? ) we inject the actin protein in an animal so, the immune system will induce and produce antibodies against the antigen (actin protein ), then we take a sample of blood from the animal which contains the antibody. Then, we label the antibody with fluorescence material and inject it to the tissue, so it will interact with one antigen because it has one binding site and emits the signal. then, we view the tissue under the fluorescence microscope. b) Indirect( IHC) : the same concepts of direct IHC but we inject unlabeled antibody which called primary antibody on the tissue and then we inject other labeled 3

5 antibodies which called secondary antibodies. So, because the primary antibody can bind to more than one secondary antibody, the signal will intensify and more fluorescence will be emitted. *Hybridization technique : the same principle of IHC but instead of localizing protein, we will localize the presence or absence of certain sequence of gene in DNA OR RNA. We know that DNA is composed of douple helical structure of two strands " each strand is complementary to one another". If we have a sample of saliva and we want to know if it is infected by a certain virus. First, we look for (search for) the DNA or RNA sequence of the virus so, we make a complementary sequence of DNA or RNA of the virus and label it with fluorescence material then we inject it to the saliva and expose it to heat, after denaturation occurrence we remove any excess materials. by using a microscope we can detect if there is a signal, if yes, then the saliva is infected. Microscopes: there are 2 main types of microscopes : Light microscope ( the light is used ) and Electron microscope ( electron beam is used ). 1) LIGHT MICROSCOPE : a) Bright-field microscope : *The basic functional unit consists of a tube; having an objective lens at one end and an ocular lens at the other. *The objective lens enlarges the image of the object in the direction of the ocular lens. *The ocular lens further magnifies this image toward the observer s eye. *The total magnification is equal to = Magnification of ocular lens * Magnification of the objective lens. *Maximum magnification is 1500x. * tissues are dead because of stain using. b) phase contrast microscope : *It uses a lens system that produces visible images from transparent objects. *The structures appear lighter or darker relative to each other. *The light changes its speed and direction when passing in different media so, it will produce a 3D image. *The tissue remains alive because no stains are used, we use cell culture to grow cell outside the body in a dishes by using growth material. 4

6 c) fluorescence microscope : *Uses ultraviolet light. *When certain fluorescent substances are irradiated with ultra violet light, it emits light. *They appear as shiny particles on a dark background. *Placed in dark room. d) confocal microscope : * It is the most advanced light microscope. *Uses laser beams. *The laser can be moved (scanned) across the specimen as well as down into the specimen, it can produce 3D images. *Can be used in living and cultured cells and tissue sections. *3D image with high contrast than fluorescence microscope. *It is connected to computer *The thickness of sections is 10 µm usually but in this microscope software can make multiple layers from this 10µm section. 2) ELECTRON MICROSCOPE : *Uses electron beams instead of light *Provides the highest resolution of subcellular structures. *Electromagnets to focus the electrons ( versus glass lenses to focus the light). *Detect by fluorescent screen or photographic emulsion. *Requires ultrathin - very thin - sections ( µm). *Uses hard epoxy resin for embedding. *Ultrathin sections are produced by ultramicrotome ( Diamond or Glass knives). *No wax is used because sections are ultrathin so electrons will not able to penetrate the section. *the image on the microscope appears black and white because of the staining by heavy metals. So, there will be stained and unstained areas.electrons will pass through 5

7 unstained areas so it appears white(electron lucent ) and they cannot pass through stained areas so it appears black (electron dense). Types of electron microscopes : a- Transmission electron microscope (TEM) *It used to study ultra structures of the cells. *The image is 2D. *Used mainly in histology. b- Scanning electron microscope (SEM) *It used to study the surface of the specimen *The image is 3D because the electrons are reflected off the surface and captured by the detector. *The specimen is coated with gold. *We use SEM to study for example the lining of the stomach. *Used mainly in biology. Image Image produced by Magnification Resolution Time Section thickness Specimen placed on Light microscope colored Visible light Up to 1500X Resolving power to 0.25µm Frozen sections : within 20 minutes Paraffin wax :within 12hours 1-30 µm Glass slide Electron microscope Black and white Electron beam Up to 2,000,000X Resolving power to 0.1nm One day at least µm Copper mesh Don't forget to refer to slides in order to see images خ ل ق ت ب لس م ا ف ال ت ش ت ك ي... بالتوفيق. 6