Histology Lecture Notes PDF

BIOL131 Histology Microscopic study of tissues and organs through sectioning, staining, and examining those sections under a microscope. Imaging methods – from organs to cells Histology: the study of the structure of animal and plant tissues as visualised Human brain slice Cytology: study of th...

BIOL131 Histology Microscopic study of tissues and organs through sectioning, staining, and examining those sections under a microscope. Imaging methods – from organs to cells Histology: the study of the structure of animal and plant tissues as visualised Human brain slice Cytology: study of the microscopic appearance of cells under the microscope Neurons x40 Most cells are not visible to the naked eye 1 cm 1 mm 100 µm 10 µm 1 µm 100 nm 10 nm 1 nm 0.1 nm Frog egg Human egg Most plant and animal cells Nucleus Most bacteria Mitochondrion Smallest bacteria Superresolution Viruses microscopy Ribosomes Proteins Lipids Small molecules Atoms EM 0.1 m Human height Length of some nerve and muscle cells Chicken egg LM 1m Unaided eye 10 m http://www.cellsalive.com/howbig.htm Microscopy Three important variables of microscopy: 1. Magnification 2. Optical resolution 3. Contrast Light microscope Electron microscope x1000 magnification x 1 x 106 magnification Resolution down to 200nm Resolution down to 0.2nm Brightfield Scanning EM Phase-contrast Transmission EM Fluorescent Campbell and Reece: 11th Ed p 164 – 166 10th Ed. 168 – 170/ 9th Ed. p140-142 Resolution The separation distance at which 2 objects in an image can be apart and still distinguished as separate For example, if a microscope has resolution of 200nm, 2 items located 200nm apart will appear as 2 items, but 2 items located 100nm apart would appear to be 1 item. Single object Resolved Unresolved Electron microscopy Transmission EM • electron beam • 2D images Scanning EM • electrons scattered from surface of sample • lower resolution than TEM • 3D images Photoreceptors in retina https://webvision.med.utah.edu/2011/05/rod-photoreceptor-tem/#more-689 Science photo library F001/0041 The light microscope Eye piece Objective lens Condenser lens Light source What can you see down a light microscope? The light (fluorescent) microscope What can you see down a light (fluorescent) microscope? 3D reconstruction of artery in mouse brain using fluorescent immunohistochemistry Histology Visualise tissue structures → function & health Haematoxylin and Eosin stained intestinal tissue Hela cells (TEM) – Campbell and Reece et al. Brightfield microscopy Problem: Solution: Stain with dyes • Fix cells/tissue • Embed • Cut into thin sections New problems: - processing alters cell structure/molecules = may produce artefacts – only gives snapshot of dead cells – 2 dimensional image of a 3D structure New methods to monitor living tissue cultures Histology – start to finish Specimen dissected Weinberg: The Biology of Cancer Fixation Dehydration Embedding Sectioning Staining From here: pathologist review and report Fixation • Common fixatives • • formaldehyde (10% formalin) and glutaraldehyde alcohol Oestrogen receptor staining of breast carcinoma 3 hr formalin 8 hours formalin • Arrests biological activity • Prevents tissue degradation • autolytic degradation • bacteria (i.e. preserve) • Render the cells more amenable to staining https://www.leicabiosystems.com/knowledge-pathway/effects-of-fixation-and-tissueprocessing-on-immunocytochemistry/ Embedding of tissues • Fixed tissues require dehydration prior to embedding – remove water to prevent tissue damage • Support the tissue by embedding in a hard medium such as paraffin wax (LM) or freezing (LM) or plastic resin (EM) • Steps in tissue processing all cause artefacts (distortions in cell/tissue architecture e.g. shrinkage) Fine lines / Micro-chatter 15 seconds drying Freeze-thaw holes in tissue A review of artifacts in histopathology: J Oral Maxillofac Pathol. 2018 May-Aug; 22(2): 279 Tissue sectioning Cryostat Microtome Vibratome • tissues are frozen (-1020C) • section thickness ~10-40 mm • tissue collected onto slides or “free-floating” • tissues are embedded in wax (room temp) • section thickness ~5-40 mm • tissue collected onto slides • tissues are glued to holder (room temp or chilled) • section thickness ~40-400 mm • tissue collected onto slides or “free-floating” Immobilised tissues are held in place and sectioned thinly using a sharp blade Basic stains: Haematoxylin and Eosin (H&E) Haematoxylin (basic dye) • stains acidic structures a purplish blue • nuclei, ribosomes and rough endoplasmic reticulum i.e. high DNA/RNA content Eosin (acidic dye) • stains basic structures red/pink • e.g. cytoplasmic proteins Animal cells: • Nucleus stains blue • Cytoplasm stains pink KIDNEY COLON Some other tissue stains Giemsa • standard method for staining blood cells • Nuclei stain dark blue to violet and cytoplasm pale blue Toluidine blue • basic stain that stains acidic components various shades of blue/purple Masson’s trichrome •stains connective tissue •nuclei/basophilic structures stain blue •collagen stains green or blue •cytoplasm bright red Periodic acid-Schiff rxn • stains complex carbohydrates purple/magenta • e.g. Mucin in goblet cells in the intestine Immunohistochemistry • Uses antibodies to label a specific protein or cell TUNEL stains apoptotic cells CD3 allows identification of T-cells Cell structure and morphology can indicate its function Simple columnar epithelium – absorptive / secretory surfaces Stratified epithelia - protective function, e.g. skin x200 x100 Brain tissue - thin axon for rapid cell-cell communication x40 Smooth muscle tissue - elongated cells to maximise contractile properties x480 x500 Young et al. Wheater’s Functional Histology © Elsevier Ltd Breast tissue – low magnification • Breast cancer usually forms in the ducts and lobules of the breast Normal • Alterations in lobule cells and invasion of adjacent adipose tissue https://www.protei natlas.org/learn/di ctionary/normal/br east Malignant tumour Cancer https://www.proteinatl as.org/learn/dictionary /pathology/breast+can cer x10 Breast tissue – high magnification Normal Cancer x40 x40 Cellular and nuclear pleomorphisms, nuclear hyperchromatism, nucleus:cytoplasm size Immunohistochemistry for oestrogen receptor alpha Cancer Normal x40 Nuclear expression of ORa is seen in a subset of glandular cells in breast lobuli and small ducts. x40 Ductal carcinoma showing nuclear expression of ORa in tumour cells. Colon Histology Muscularis mucosa Epithelium 4x Crypts of Lieberkuhn Lamina propria x40 Normal vs. dysplasic colon Normal Dysplasic • • • • villiform change of the epithelium crypt budding, branching, and crowding irregularity of crypt contour increased angiogenesis Carcinoma - neoplasm derived from epithelial cells of the skin or lining of internal organs Normal prostate histology x10 https://www.proteinatlas.org/learn/dictionary/normal/prostate+1 Normal prostate structure • Normal prostate contains ~60% luminal cells & 40% basal cells Basal cells Packer et al. 2016 BBA 1863 (6 Pt A): 1238-1260 luminal cells anti-keratin  50 x100 https://www.proteinatlas.org/learn/dictionary/normal/prostate/detail+1/magnification+1 Prostate cancer Basal cells luminal cells x100 https://www.proteinatlas.org/learn/dictionary/pathology/prostate+cancer/detail+ 1/magnification+1 • Cancer is characterised by luminal hyperproliferation, loss of the basal layer, breakdown of basement membrane, immune cell infiltration and stromal reactivity. • Luminal cells make up > 99% of tumours, basal cells constitute < 0.1% of tumour epithelial cells. Benign tumours • Benign tumors arise with high frequency but pose little risk because they are localised and small Colon Thyroid Tumour has clear boundaries and has not grown into surrounding tissues Human Tissue Act (2004) • Regulates activities - removal, storage, use and disposal of human tissue. • Consent - fundamental principle of the legislation and underpins the lawful removal, storage and use of body parts, organs and tissue. • DNA - unlawful to have human tissue with the intention of its DNA being analysed, without the consent of the person from whom the tissue came. • Tissue removed and stored for diagnosis does not fall under the Act