Intro To Ultrastructure & Histology 2024 PDF

AGRC1041 Introduction to Cell Ultrastructure & Histology Assoc Prof Dee Whitworth [email protected]  Introduction Vertebrate animals are a collection of organ systems Organs are composed of tissues Tissues consist of cells and their products Cells are indiv...

AGRC1041 Introduction to Cell Ultrastructure & Histology Assoc Prof Dee Whitworth [email protected]  Introduction Vertebrate animals are a collection of organ systems Organs are composed of tissues Tissues consist of cells and their products Cells are individually functional units, which vary in form, size and function Histology is the study of these cells and the tissues that they form  Histology  In order to examine these cells and tissues, we need to prepare them in such a way that they can be examined under the light microscope  This involves several steps: Fixation Processing Embedding Sectioning Staining  Fixation  Fixatives are chemicals used to preserve cellular structure halting the processes of autolysis & decay stabilising proteins  Must be buffered to match the pH and osmolarity of the tissue  Tissue is cut into small pieces  Use 10x volume of tissue  10% neutral buffered formalin (NBF)  Fixation Good fixation in 10% NBF Poor fixation in 10% NBF  Processing  In order to keep the tissue rigid so that it can be sectioned, it is infiltrated with a paraffin wax formulation (includes plasticising polymers)  As wax is not miscible with water, tissue must first be dehydrated through a series of increasing concentrations of alcohol  Tissue is then cleared of alcohol using xylene (or like) miscible with wax  Infiltrated with paraffin wax under vacuum  Embedding  Paraffin-infiltrated tissue is then embedded into a wax block  Sectioning Using a microtome, 3-10µm thick sections are cut from the block and mounted onto glass slides  Staining  Wax is removed by reversing the prior steps: xylene  decreasing alcohol series  water  To enable the identification of different cell types & their constituents, such as the nucleus & cytoplasm, the sections are stained  Staining  Many different types of stains specificities for different substrates  Haematoxylin & eosin (H & E) haematoxylin is alkaline: binds to acids, blueish eosin is acidic: binds to alkali, pinkish Masson’s trichrome Wright Giemsa H&E - connective tissues - - mast cells - - nuclei & cytoplasm -  Ultrastructure  To examine the ultrastructure of a cell we use a transmission electron microscope (TEM) can magnify up to 1.5 million times resolution of 0.14 nm (5x diameter of H atom)  TEM uses a beam of electrons instead of a beam of light  Electron beam produced by heating a tungsten filament  Ultrastructure  The optical system of TEMs must be evacuated of air to prevent distortion of the electron beam  A TEM contains many lenses which act to magnify the image produced by the electrons interacting with the tissue section  Ultrastructure  Tissue is fixed formaldehyde + glutaraldehyde osmium tetroxide  Then dehydrated  Embedded in resin & sectioned at 60-500nm on an ultramicrotome glass or diamond knife with water trough  Ultrastructure  Sections are mounted onto copper grids  And then stained with electron-absorbing heavy metal salts provide the contrast necessary to reveal the cells’ ultrastructure uranyl acetate & lead citrate  Four Main Tissue Types  Epithelial Tissue  Connective Tissue  Muscle  Nervous Tissues All organs & structures within the body consist of one or more of these tissue types These tissue types develop from one of three embryonic regions called the embryonic germ layers  The Embryonic Germ Layers  After fertilisation and multiple cell divisions, a series of cell re-arrangements occur, known as gastrulation  The Embryonic Germ Layers  As a result of gastrulation the embryo develops 3 cell regions known as the germ layers: Ectoderm – outer layer Mesoderm – middle layer Endoderm – inner layer The Embryonic Germ Layers give rise to:  ec me ec me en me me en ec Brown J L et al. (2008) PNAS ,105:12337-12342 Green: ectoderm Light red: mesoderm Dark red: endoderm  Epithelial Tissue  Sheets of cells that form a lining: externally: skin internally: lining the digestive, urogenital & respiratory tracts, blood vessels  Forms glands – glandular epithelium  Connective Tissue  Provides support: physically - eg cartilage & bone physiologically - eg blood  Packing between tissues: adipose tissue fascia, ligaments & tendons  Three main types: Connective Tissue Proper ligaments, tendons & adipose tissue Cartilage & Bone Blood  Muscle Tissue  3 main types: Skeletal (striated) – allows voluntary movement Smooth – not under voluntary control; found in the viscera Cardiac – the heart  Nervous Tissue  Brain, spinal cord, nerves & sensory receptors  Enables the body to process & respond to information from external & internal environments  Consists of neurons, which are responsible for transmitting electrical impulses, & support cells  Why study histology?!?  Veterinary Practice determine whether the tissue is healthy or diseased: fine needle aspirates & biopsies of suspicious lumps; specialise in pathology  Research study of development, gene function, regenerative medicine...