2. Histology Flashcards
(39 cards)
Define a tissue
a collection of integrated cells which work together to carry out a specific function(s)
Define histology
the microscopic study of body tissues
How can you obtain a tissue sample?
Surgery e.g. tumour resection
Scraping methods e.g. scalpel, scrapes
Aspiration with needle e.g. synovial fluid, bone marrow
Venepuncture e.g. blood smears
Describe the preparation for a slide for light microscopy
Preserve the tissue within formalin
Harden the tissue with paraffin wax
Cut the sample into very thin slices with a microtome
Stain - commonly with haematoxylin and eosin
What is H&E and what does it stain?
Haematoxylin and Eosin
Haematoxylin stains DNA/RNA blue - acidic
Eosin stains cytoplasm and ECM pink - proteins
What is immunofluorescence?
Antibody labelled with a fluorescent marker
Fluorophore emits visible fluorescent light
What is immunohistochemistry?
Antibody tagged with an enzyme
Colourless substrate added - enzyme converts substrate into a coloured product e.g. peroxidases
What is phase contrast?
Converts phase shifts in light into brighntess changes which are visible, and no staining is required
How does confocal microscopy work?
A laser excites a fluorescent molecules and electrons in the dye are excited to a higher level.
As they relax a light with higher wavelength is emitted.
Emitted light sent through mirrors and a pinhole screen to a detector.
Define resolution
The smallest distance by which two objects can be separated and still be distinguishable as two separate objects.
Mathematically expressed as d = gamma/2 NA
- d = limit of resolution
- gamma = wavelength
- NA = numerical aperture
What are the resolving powers of microscopes?
- Compound light microscope up to 200nm - cells
- SEM up to 0.4nm
- TEM up to 0.078 nm
Outline light microscopy vs electron
Light: Can view images in natural colours
Cheap easy preparation
Can view living and moving objects
Electron: Only monochrome
Difficult, expensive preparation
Dead material
Transmission vs Scanning
Both fixed with glutaraldehyde, fix with epoxy resin
Transmission allows for thin cuts, scanning - surface
Explain the process of freeze fracture electron microscopy
The tissue is frozen at -160 and fractured by hitting with a knife edge. The fracture line passes through the plasma membrane exposing interior.
What are some requirement to image tissues using light microscopy?
Need to preserve to prevent from rotting (formalin)
Need to embed to be sliced thinly (paraffin wax)
Need to stain so we can see cell compartments (H&E)
Give examples of tissue procurement using an endometrial sample.
Endometrial Biopsy - suction small part of wall.
Curettage - scrape endometrium.
Pipelle - place into endometrium, twist and pull.
Hysterectomy - full removal to take a look at.
Why must fixation solutions be buffered?
Otherwise water causes swelling within the sample and artefacts.
Name some routine staining methods
Haematoxylin and eosin, masson’s trichrome (different colours) periodic acid-schiff stain
Describe the process of obtaining a frozen section
Specimen placed on metal disc frozen rapidly to -30. Cryosection - microtome within cold environment cuts the specimen then stained with H&E.
Paraffin wax formalin fixed vs frozen section
Paraffin:
fixed tissues, takes 24 hours, permanent, used in pathological diagnosis, long time but more permanent.
Frozen section:
fresh tissues, 10-20 mins, lasts only a few months but can be used during operations.
Describe the process to culture cells
Harvest cells
Isolate cells using appropriate enzymes, use of collagenase and DNAase as otherwise DNA from broken nucleus infects sample.
Centrifugation, place into appropriate growth medium.
What are some advantages of having access to cells within culture?
Allows manipulation of the cells and experiments to determine cells and thus tissue function.
Homogeneity of sample, less need for animal models.
What are some disadvantages of cell culture?
Hard to maintain, only grow small amount of tissue at high cost, cells can change phenotype, influence of other cells and tissues not maintained.
What is dark field?
A very specialised technique using living cells, illuminates the sample with light not collected by the objective lens. Produces appearance of dark background.
