Functional Organization of the Body

Transcription

1 TR056/PG1006: Human Tissue Types Follows on from PG1003. Integrates knowledge of cell biology into an appreciaeon of the organisaeon of the human body at the Essue level. Understanding the structure and basic funceon of various Essue types is a foundaeon for the study of the formaeon of the Essues and organs (PG1007) and anatomy and physiology year 2 Lectures Week 1 Visualising Cell & Tissue structure (AK) Nervous Tissue 1 (ND) Nervous Tissue 2 (ND) Week 2 Skeletal Muscle (ND ) Cardiac & Smooth Muscle (ND) Lining Epithelia (AK) Week 3 Epithelial Specialisations (AK) Glandular Epithelia (AK) Connective Tissue Introduction (ND) Practicals Week 4 Haematoxylin & Eosin staining of rat kidney (continuous assessment 10%) Week 6 Human tissue types microscopy tutorial Theory session on TEM Centre for Microscopy & Analysis site visit TEM exam (continuous assessment 10%) Lectures Week 4 Blood Composition (SG) Blood Typing (SG) The Extracellular Matrix (ND) Week 5 Adipose Tissue (AG) Cartilage (AG) Bone (AG) Week 6 Body Cavities & Organ Systems (AK) Assessment Continuous (20%) TEM exam 10% Practical class report 10% Examination (annual) (80%) Short answer questions 60% Fill-in-the-blank/table 20% Annotation questions 20% Functional Organization of the Body Organism Body system Organ Tissue Cell: basic unit of life Subcellular Molecular Today s Lecture: Visualising Cell & Tissue structure Learning objectives: Revise briefly the major types of microscopy used for examining tissue Tissue processing Routine staining, enzyme histochemistry and immunohistochemistry 1

2 Basic Tissue Types Epithelial tissue: covers body & organ surfaces, lines body cavities, forms glands. Involved in protection, absorption, excretion, secretion, diffusion and filtration Connective tissue: binds, supports and protects body structures, stores energy and minerals *Nervous tissue: initiates and transmits nerve impulses to coordinate physiological function *Muscle tissue: contracts and relaxes to produce movement *Excitable tissues The Basic Tissues 2

3 The Upright (Visible) Light Microscope Compound lenses The Upright (Visible) Light Microscope Basic principle: Sample is illuminated and image is magnified by a series of lenses (compound lenses). A magnifying glass is a single lens microscope. Brightness, contrast, resolution (objective lens) and focus can be altered. Contrast can be improved by treating specimens with pigments or dyes that bind to specific structures within the specimen. Dyes absorb some wavelengths of the visible spectrum, thus we see colours as the mixture of transmi8ed light Typically used for viewing slides of prepared (fixed) tissues or cells stained for contrast, mounted on glass slides in a liquid phase and capped with a coverslip Phase-contrast microscopy can be used to view living cells eg in culture. SOURCE OF RADIATION Light bulb/halogen lamp White light MAGNIFICATION >1500X RESOLUTION 500nm 400X=10x40 Glial cell in culture 3

4 Fluorescence Microscopy Some chemicals are fluorescent, i.e., can be excited by individual wavelengths of light resulting in emission of light of a particular wavelength in the visible spectrum which we see as a defined wavelength of colour. Light enters the microscope and hits a dichroic mirror (reflects one range of wavelengths and allows another range to pass through). The dichroic mirror reflects UV light to the specimen, which excites fluorescence within molecules in the specimen. The objective lens collects the fluorescent-wavelength light produced; this passes through the dichroic mirror and a barrier filter (eliminates wavelengths other than fluorescent), making it to the eyepiece to form the image. High energy state Excite with defined wavelength Emission of energy FLUOROPHORE as longer wavelength light SOURCE OF RADIATION MAGNIFICATION RESOLUTION Mercury or Xenon Lamp UV light >1500X 10-30nm Biomolecules in sample are tagged by a fluorophore HeLa Cells Phalloidin stained actin DAPI stained nucleic acid Immunohistochemical detection of COX IV Transmission Electron Microscopy A beam of electrons is passed through an ultrathin section. (Scanning EM visualises sample surface) Same basic principle as light microscopy, but using electrons. Smaller wavelength, better resolution. High-resolution TEM, pm-range resolution Fine structure of cellular compartments, organelles and cytoskeleton SOURCE OF RADIATION MAGNIFICATION RESOLUTION Electron beam λ<1nm >1,000,000 <1nm graphics/microsco.gif 4

5 Tissue Processing Tissue must be thin enough to allow dyes to permeate and stain and then allow light or electrons to traverse the tissue to allow for microscopic detection of contrast and hence biological detail Fixation: Chemicals preserve specimens without distorting them. Dehydration: Water removed from the specimen using ethanol. Particularly important for electron microscopy because water molecules deflect the electron beam, blurring the image. Embedding: Supports the tissue in wax or resin so that it can be cut into thin sections. Sectioning: Produces very thin slices for mounting. Sections are cut with a microtome or ultramicrotome to a few micrometres (light microscopy) or nanometres (electron microscopy) thickness. Mounting: Mounting on a slide protects the material so that it is suitable for viewing over a long period. Staining: Most biological material is transparent and needs staining to increase the contrast between different structures. Different stains are used for different types of tissues. Fixa@on Avoids decomposieon by autolysis and/or putrefaceon 1) DehydraEon reagent (ethanol, methanol) 2) Freezing then dehydraeon 3) Use of cross- linking chemical (formaldehyde, glutaraldehyde) Formaldehyde FixaEon *N.B. The aldehydes do not correctly preserve lipids and hence for fine structural analysis in TEM, glutaraldehyde and osmium tetroxide are added sequeneally 5

6 Clearing Embedding and DEHYDRATION: Once fixed, water is removed from the Essue through increasing concentraeons of alcohol CLEARING: Once dehydrated, alcohol is removed and Essue prepared for infiltraeon of a solidifying agent by use of solvents e.g. aromaec hydrocarbons (xylene) EMBEDDING: Tissue seceons are then placed in embedding medium (e.g. paraffin), the solvents are evaporated and the embedding agent hardens The now hardened Essue block can be cut into micron/nanometre thick seceons on a microtome/ultramicrotome *N.B. When using frozen seceons, Essue is placed in a polymer, snap frozen, cut on a cryomicrotome and then fixed and stained (No embedding) Staining Techniques Acidic/basic dyes (eg Haematoxylin & Eosin) Substrate reactions 1 (direct enzyme histochemistry) Substrate reactions 2 (immunohistochemistry) Colorimetric visualisation or antibodies can be fluorescently tagged Heavy metal staining contrast (TEM). Lead, uranium, tungsten scatter imaging electrons and thus give contrast between different structures 6

7 Enzyme Histochemistry Allows visualisation of chemicals/enzymes in tissue Chemical reaction used should be specific Reaction product should be detectable (eg coloured/electron-dense) and insoluble Examples: detection of phosphatases, dehydrogenases, peroxidases Frozen sections normally used, since fixing & clearing deactivate enzymes. Sections incubated in sustrates of enzymes of interest, products then detected. Example: peroxidases detected by incubating tissue with3 3 diaminobenzidine (DAB) and hydrogen peroxide Substrate reactions: Fluorescence-microscopy techniques useful for seeing structures and measuring physiological and biochemical events in living cells. Various fluorescent indicators available to study many physiologically important chemicals such as DNA, calcium, magnesium, sodium, protons (ph) and enzymes. Immunocytochemistry Antibody-antigen interaction: Specific Visualised by colorimetric or fluorescent means Counterstaining often used eg. DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that binds strongly to A-T rich regions in DNA HeLa Cells Phalloidin stained actin DAPI stained nucleic acid Immunohistochemical detection of COX IV 7