Cytology Flashcards
Tissue preparation
first cut into thinner pieces
- Fixation - Small tissue pieces placed in solutions of chemical to prevent
enzyme digestion > preserve the cell and tissue structure
Formulin used
preserve by cross-linking proteins and inactivating degradative
enzymes.
- Dehydration
The tissue is transferred through a series of increasingly
concentrated alcohol solutions, ending in 100%, which removes all
water.
3.Clearing
Alcohol is removed in toluene or other agents in which both
alcohol and paraffin are miscible.
4.Infiltration
The tissue is then placed in melted paraffin until it becomes
completely infiltrated with this substance.
5.Embedding
The paraffin-infiltrated tissue is placed in a small mold
with melted paraffin and allowed to harden
sectioning/trimming - to view on slide
Staining
-only necessary for light microscopy
-Paraffin sections are colourless
-tissues stained with dyes
Dyes can have basic or acidic properties due to ability to form electrostatic linkages with charged parts of the cell.
Basophilic tissue components:
-negative charge
-stain with basic dyes e.g toluidine blue,
Hematoxylin (blue and used for Nucleic acids), Sudan III lipids glycoproteins etc
Acidophilic tissue:
-positive charge
-stain with acidic dyes e.g Eosin (pink), fuschin, these stain mitochondria, collagen, cytoplasm and secretory granules.
H&E most common stain- covers most cell components. H gives dark blue colour to nucleic acids.
Masson and mallory stains are better for extracellular components.
H&E stain- nucleic acid would be purple due to dark blue from h and pink from e.
Periodic acid schiff- more effective on glycoproteins and polysaccharide/oligosaccharid regions.
Historical development
1590- First microseope with magnificaton 9× , created by two Dutch (Hans &Zacharias Jansen)
1609 - Improving and perfecting of the microscope (with focusing device) by Galileo Galilei
1665 - Robert Hooke used compound microscope and discovered first cell.
1674 - Antoine Van Leevwenhoek first to discover Protozoa, bacteria, blood cells and spermatozoa
1831-Robert Brown (scottish botanist) m ade the first description of the cell nucleus in detail
1838- schleidden and schwann postulated cell theory
histology-study of microscopic anatomy of cells/tissues of animals and plants
17th century- marphigi invented microscope for biological use - capillaries found in alveoli
19th century - regular study
Embryology- development of embryo from fertiliszation to foetus
aristotle proposed epigenesis - animla comes from egg with no form
18th century - thought semen contained a preformed embryo/homunculus
19th century - microscopy proved epigenesis.
Bright-field/ light Microscope
Brightfield microscope- most commonly used. Uses ordinary light passing through stained tissues .
3 lenses:
-Condenser- collects and focuses light onto object being observed on stage
- objective lens - enlarges and projects image toward eye piece.
- eyepiece: further magnified image to the eye or camera.
Resolving power is 0.2 micrometres
Phase contrast microscope
Doesn’t require staining
Produces visible images from transparent objects
Light changes when passing through structures with different refractive indexes. These changes cause structures to appear lighter or darker in comparison to each other.
Allows us to view living cells and cell cycle
Variation of this is interference microscopy which produces 3D image of a cell.
Confocal microscopy
Allows visualisation of specimen in 3D
Light from a. Laser source hits specimen and is reflected.
Beam splitter directs the reflected light to a pinhole and a detector.
Light from components of the specimen that are above or below the focused plane are blocked by the blind.
Laser scans the specimen so a larger area of specimen can be observed.
Mirror system moves laser beam across the specimen.
High resolution and sharp focus by using laser as focused light and a plate with a pinhole. Increases precision.
Can construct 3d image.
Polarising microscopy
Allows recognition of stained or unstained structures made of organised subunits. Repetitive macro molecular structure.
Observe specimens that are visible due to their optically anisotropic character.
E.g microfilaments, cellulose, collagen, microtubules.
Birefringence is to rotate direction of vibration of polarised light.
Fluorescence microscopy
UV light used on specimen and it emits visible light.
Can cause bleaching of image which is bad.
Fluorescein can be used bound to compounds whihc bind to specific cell components helping identify them.
Filter used to select specific wavelengths of light emitted by a substance.
Aridine orange used on Nucleic acids. They emit slightly different colours so can be identified.
Electron microscopy
Wavelengths are shorter than light so electron beams allow very high resolution images at high magnification.
TEM:
-Tissues prepared by embedding with resin or heavy metal ions which associate more with certain areas of tissue. These are called electron dense regions
-Cut into ultra thin sections using a diamond knife so electrons can pass through.
Electron gun/cathode beam goes through anode to specimen on the port.
Electron lose more energy passing through electron dense regions. through objective lens to fluorescent screen where image is formed
SEM:
Sample coated with gold/ palladium
ELectron beam shot at sample. causes coating to emit secondary electrons which are detected by a detector.
Creates 3D image.
HIstochemistry - lipids carbs proteins
carbs:
Periodic acid schiff - deep red colour
on glycongen, collagen, brushed borders
bleached fushin reacts with aldehyde group
lipids:
dyes soluble in lipids used
sudan iii - used on adipose tissue
sudan iv, sudan black and somium stain dark brown/ black e,g on myelin
protein:
PAS can be used but mistaken for carbs
alcian blue preferred
can be used with pas to separate from carbs
HIstochemistry - nucleic acids
Haematoxylin used on nucleic acids turns blue
Ethium bromide distinguishes between DNA and RNA
Feulgen reactions turn red in DNA
RNA stain requires higher conc of nucleic acid for detection
Basic stain used in DNA and RNA
Enzyme histochemistry
Method of localising cell structures using specific enzyme activity present in those structures
unfixed or mildly fixed tissue used to prserve enzymes sectioned on cryostat to reduce heat and organic solvent effect on enzymes.
tissue sections immersed in solution containing substate to be localised.
enzyme acts on substrate
this section then put into contact with a marker compound which reacts with product of enzyme substrate action.
FInal product from the marker is insoluble and visible by light or electron microscopy ( has colour or electron denstiy) precipitates over the enzyme site allowing region to be localised microscopically.
immunohistochemistry
Used for diagnostic and research purposees to detect proteins of interest.
Tissue which presumably contains protein of interest is added to solution containing a labelled antibody
Location of protein seen by microscopy depending on how the protein was labelled.
Based on specific reactions between antigen and antibodies labelled with visible markers e.g fluorescent compounds or peroxidase for light microscopy and gold particles for Transmission Electron Microscopy (TEM).
Direct immunohistochemistry is when antigen of interest is bound to a primary antibody specific for that antigen
Indirect uses an unlabelled primary antibody which is only detected when bound to a secondary antibody of that antigen
Indirect method is used more as a higher amount of antibody binding amplifies the signal detected.
cell culture
cells can be grown in vitro from a primary culture. and examined in their living state using phase contrast microscope.
- cells/tissues grown in solutions which serum and growth components added. cells dispersed into a single layer mechanically or enzymatically.
once isolated from tissue, cells grown in vitro fro long periods of time because they become immortal and constitute permanent cell line.
cells usually have finite lifespan but can transform to have longer ( similar to cancer)
cell fractionation
Breaking the cell:
-Detergent used to break cell membrane and allows components to be mixed into a homogenate.
-combined with mechanical method of machines to break it down.
Separating homogenates:
- Centrifugation separates cell components by density and mass.
- Most dense at bottom so collected.
