1.-ders-histology.pdf

OVERVİEW OF HISTOLOGY Ankara Medipol University Department of Histology & Embryology Asst. Prof. Ender Deniz Asmaz 1 What is histology  Definition  Cell:  Tissue  Organ:  System Several organs with related functions Motor system Nervous system Circulatory system...

OVERVİEW OF HISTOLOGY Ankara Medipol University Department of Histology & Embryology Asst. Prof. Ender Deniz Asmaz 1 What is histology  Definition  Cell:  Tissue  Organ:  System Several organs with related functions Motor system Nervous system Circulatory system Respiratory system Digestive system Urinary system Reproductive system Endocrine system 2 Why to study histology  Anatomy: macrostructure  Biochemistry: chemical compounds and processes  Pathology: the relation between disease and the structures and functions of the body Although most medical students are not going to become histologists, a thorough knowledge of histology is fundamental for you as future doctors. 3 How to research on histology  Preparation of tissue for microscopic examination Paraffin section Frozen section  Microscopy  Problems in the interpretation of tissue sections 3 How to research on histology Knife Section Block Glass slide Light MICROTOME - a fancy meat- beam slicer - holds the wax block, & cuts off thin slices, as the block is slowly advanced mechanically Light microscope Paraffin section  Obtaining the specimen  Fixation  Dehydration  Clearing  Embedding  Sectioning  Staining  Fixation is used to: ✔ terminate cell metabolism, ✔ prevent enzymatic degradation of cells and tissues by ✔ autolysis (self-digestion), ✔ kill pathogenic microorganisms such as bacteria, fungi, ✔ and viruses, and Dehydration: Washing...> Removal of fixative Removal of water serial alcohol with increasing grade (70%...> 100% ethanol) Obtaining the specimen fresh as possible and small pieces  Remove the water & replace with wax-solvent Imbed the oriented specimen in molten wax 70 % ethanol Clearing 80 % ethanol Tap water 95 % ethanol Embedding 100 % label Fresh ethanol tissue xylene 4% formaldehyde fixative paraffin wax Tissue processor Automatic tissues processor moves the tissues around through the various agents on a preset time scale.  Tissue embedding Tissues are infiltrated in molten wax to replace the xyline. The molten wax drop into a plastic box; then Put the tissues into the box. The molten wax solidify into a block with the tissue inside.  After it is solid, hold the wax block & cut slices Knife 1-10μm Section Block Glass slide MICROTOME - a fancy meat- slicer - holds the wax block, & cuts off thin slices, as the block is slowly advanced Water-bath mechanically Mount the thin slices (sections) on slides Lift out floating section on the slide  Sectioning with microtome Rotation of the drive wheel moves the tissue-block holder up and down. Each turn of the drive wheel advances the specimen holder a controlled distance. After each forward move, the tissue block passes over the knife edge, which cuts the sections.  Sectioning with microtome Rotation of the drive wheel moves the tissue-block holder up and down. Each turn of the drive wheel advances the specimen holder a controlled distance. After each forward move, the tissue block passes over the knife edge, which cuts the sections. Picking sections up from water bath sections are floated on a warm-hot water bath that helps remove wrinkles. Paraffin section Unstained section on glass slide Tray of unstained slides in drying oven Sections are picked up on a glass slide and placed in a warm oven to help the section adhere to the slide. Staining QUESTION HERE ORDER OF STAINING  Deparaffinized： running through xylene to alcohol to water  Dye: acidic or basic compounds; electrostatic linkages with tissues  Hematoxylin & Eosin (H & E) staining Hematoxylin: stains cell nucleus and other acidic structure blue Eosin: stains the cytoplasm and collagen pink Basophilia: affinity for basic dyes Acidophilia: affinity for acid dyes Neutrophilia  Basic dyes react with anionic components of cells and tissue (components that carry a net negative charge) – basophilia (DNA-Phosphate in RNA ect) Acidic dyes react with cationic groups in cells and tissues, particularly with the ionized amino groups of proteins – acidophilia. Basic dyes Acidic dyes Methyl green Acid fuchsin Metihylene Blue Anilin Blue Pironin G Eosine Toluidin Blue Orange G It is a cross-section of kidney medullar which is made up of lots of tubules. The wall of them is epithelial cells. The cell nucleus is basophilic (blue) and the cytoplasm is acidophilic (pink). HE staining  Light Microscope  Gold staining Silver staining Frozen section  Snap frozen in a cold liquid or cold environment Frozen sections are performed with a cryostat. cryostat Cutting a frozen section Frozen section  It is necessary to get a rapid diagnosis of a pathologic process.  It is also effective in the histochemical study of very sensitive enzymes or small molecules. Microscopy  Light microscopy Conventional light microscopy Phase-contrast microscopy Polarizing microscopy Fluorescence microscopy Confocal microscopy  Electron microscopy Transmission electron microscopy (TEM) Scanning electron microscopy (SEM) Conventional light microscopy  Mechanical parts  Optical parts Condenser collects and focuses light to illuminate the object Objective enlarges and projects the image of the object in the direction of the eyepieces. Eyepieces magnify this image and project it onto the viewer’s retina  Eyepiece LIGHT MICROSCOPE /Ocular Objective lenses Stage Max MAGNIFICATION Slide Body Eyepiece (10X) Objective (40X) Condenser = 400X Base Light source Schematic diagram of light microscope  Phase-contrast microscopy & differential interference microscopy  Phase-contrast microscopy light changes speed when passing through cellular and extracellular structures with different refractive indices.  Differential interference microscopy produces an three-dimesional image A B Cultured neural crest cells seen with different optical techniques. A: Conventional light microscopy. B: Phase contrast microscopy. C: Nomarski differential interference microscopy. C Under polarized light microscopy, collagen fibers appear brilliant or yellow. Fluorescence microscopy  Fluorescence microscopy tissue sections are irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum. The fluorescent substances appear brilliant or colored on a dark background.  Cytoplasm DNA Photomicrograph of kidney cells stained with acridine orange. DNA (within the nuclei) emits yellow light, and the RNA-rich cytoplasm appears reddish or orange. Confocal microscopy  A laser source  Different layers of the specimen are seen in different focus simultaneously.  Merged image of a three-dimension  Clearer image Different layers of the specimen are seen in different focus simultaneously. A merged image of a three- dimensional object could be got. a 3-D image of cultured cells The image of specimen is clearer than in common fluorescence microscope. Transmission electron microscope high resolution (0.1nm)  electron lucent electron dense TEM micrograph of hepatocyte Scanning electron microscopy  pseudo-three-dimensional views of the surfaces  A very thin metal coating  The electron beam interacts with this metal coating and produces reflected or emitted electrons. Schematic view of a transmission and scanning electron microscope SEM micrograph of the epithelium of stomach Problems in the interpretation of tissue sections  Distortions & artifacts caused by tissue processing shrinkage artifact Artificial spaces Wrinkles of the section precipitate of stain  Totality of the tissue  Two dimensions & three dimensions Shrinkage caused by tissue processing artificial spaces between the colloid and the follicular wall in the section of thyroid gland. Shrinkage of cells in hyaline cartilage Lipid droplets infat cells are lost during tissue preparation. Artifacts caused by tissue processing Mucous granules goblet cell goblet cell Mucous granules containing glycoprotein in the cytoplasm of goblet cells are lost during tissue preparation. Totality of the tissue Nissl bodies nucleus Sliver staining H&E staining Neurofibrils  A B How different 3-dimensional C structures may appear when thin-sectioned. A: Different sections through a hollow ball and a hollow tube. B: A section through a single coiled tube may appear as sections of many separate tubes. C: Sections through a solid ball (above) and sections through a solid cylinder (below).