2 - Histology Flashcards
Histology
the study of the structure of animal and plant tissues as visualised
Cytology
study of the microscopic appearance of cells under the microscope
3 important variables of microscopy
- Magnification
- Optical resolution
- Contrast
Light vs electron microscope
x1000 magnification vs x 1 x 106 magnification
Resolution down to 200nm vs Resolution down to 0.2nm
Brightfield vs Scanning EM
Phase-contrast +Fluorescent vs Transmission EM
Resolution
The separation distance at which 2 objects in an image can be apart and still distinguished as separate
Electron microscopy
Transmission EM • electron beam • 2D images Scanning EM • electrons scattered from surface of sample • lower resolution than TEM • 3D images
Fluorescent
Shows locations of specific molecules in the cell.
Fluorescent substances absorb short- wavelength UV radiation, and emit longer-wavelength visible light.
Confocal microscope
Uses lasers and special optics for optical sectioning.
Only regions with a narrow depth of focus are imaged, regions above and below the plane appear black
Brightfield microscopy
Stain with dyes to help distinguish cells
• Fix cells/tissue
• Embed
• Cut into thin sections
New problems:
- processing alters cell structure/molecules = may produce artefacts
– only gives snapshot of dead cells
– 2D image of a 3D structure
New methods to monitor living tissue cultures
Fixation
• Common fixatives
-formaldehyde (10% formalin) and glutaraldehyde
- alcohol
• Arrests biological activity
• Prevents tissue degradation (autolytic + bacteria)
• Render the cells more amenable to staining
Fixation and 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)
Tissue sectioning maachines
Cryostat
Microtome
Vibratome
Immobilised tissues are held in place and sectioned thinly using a sharp blade
Cryostat
- tissues are frozen (-10-20C)
- section thickness ~10-40 mm
- tissue collected onto slides or “free-floating”
Microtome
- tissues are embedded in wax (room temp)
- section thickness ~5-40 mm
- tissue collected onto slides
Vibratome
- tissues are glued to holder (room temp or chilled)
- section thickness ~40-400 mm
- tissue collected onto slides or “free-floating”
Histology – start to finish
Specimen dissected Fixation Dehydration Embedding Sectioning Staining Pathologist review and report
Haematoxylin and Eosin (H&E)
Haematoxylin (basic dye)
• stains acidic structures a purplish blue
• nuclei, ribosomes and RER i.e. high DNA/RNA content
Eosin (acidic dye)
• stains basic structures red/pink
• e.g. cytoplasmic proteins
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
- Use antibodies or other methods to label a specific protein or cell status
- Positive stain = brown or black
Simple columnar epithelium
absorptive / secretory surfaces
Stratified epithelia
- protective function, e.g. skin
Brain tissue
thin axon for rapid cell-cell communication
Smooth muscle tissue
elongated cells to maximise contractile properties
Breast tissue
- Breast cancer usually forms in the ducts and lobules of the breast
- Alterations in lobule cells and invasion of adjacent adipose tissue = Malignant tumour
Carcinoma
neoplasm derived from epithelial cells of the skin or lining of internal organs
Normal vs. dysplasic colon
- villiform change of the epithelium
- crypt budding, branching, and crowding
- irregularity of crypt contour
- increased angiogenesis
Prostate cancer
- 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
• arise with high frequency but pose little risk because they are localised and small
Tumour has clear boundaries and has not grown into surrounding tissue
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
