Lec 2 Examining Tissues and Cells Flashcards
define histology
why is it useful?
The microscopic study of body tissues
allows us to see/ visualise cells and their contents
useful in distinguishing pathology
Define Tissue
A collection of specialised cells which work together to carry out a specific function
Define the limit of resolution
smallest distance that two objects are distinguishable as separate objects
what are the general types of microscopy and what can they see?
light microscopy - allows us to see cells, mitochondria, bacteria ect
TEM and SEM allow us to see much smaller, DNA, proteins, viruses ect…
Compare light microscopy to electron microscopy
LM - natural colours,
cheap and easy,
view living and moving objects
smaller limit of resolution/ magnification
EM - only see monochrome
difficult and expensive
only view dead objects
Far greater resolution and magnification as electrons have a shorter wavelength
uses a beam of electrons to produce image
how do we obtain tissue samples?
Surgery – Tumour resection
Scraping methods – Curettes, scalpel scrapes
Aspiration with a needle – Bone marrow, synovial fluid
Venepuncture – Blood smears
How do we prepare a tissue sample for light microscopy?
1) preserve the tissue in formalin - causes fixation
2) harden with paraffin wax
3) very thin slices (transparent sample) with a mictrotome
4) stain with haemotxylin and eosin
what and why do we use H and E?
most common staining agent
haemotoxylin - stains the DNA blue
eosin - stains the cytoplasm pink
alone you get limited info, together they give a clear and complete picture
how do we prep a sample for EM
1) fix with glutaraldehyde
2) embed in epoxy resin
3) stain with osmium tetroxide - heavy e- dense metal
4) only for TEM - slice with a mictotome
what is a frozen section ?
compare to normal embedding techniques
frozen section - rapidly freeze the tissue
shatter with a diamond knife and H & E stained
frozen sections can use fresh tissue
they are far quicker - 10 mins this allows for mid surgery examinations
however tissue is broken and distorted so a far worse quality
what are immunohistochemistry and immunofluroescence
both use antibodies to bind to a protein
Immunofluorescence – Antibody labelled with a fluorescent marker
Immunohistochemistry – Antibody tagged with an enzyme
we add Colourless substrate – Enzyme converts substrate into a coloured product - produces image
E.g. Peroxidases
What are the three non staining methods of light microscopy ?
phase contrast
Dark Field
confocal microscopy
what is confocal microscopy?
uses electron excitement to produce a 3D image via taking multiple 2D images
what is dark feild
Unattenuated light is not collected by the objective lens, giving a dark background (enhancing contrast)
No staining required
What is phase contrast?
Converts phase shifts in light (invisible) into brightness changes (visible)
No staining required
all three non staining techniques use live cells in culture
what must we do with them, and what are the pros and cons?
we must maintain a constant internal enviroment, o2, co2 concs
nutrients, pH, temp ect..
this allows us to mainuplate cells in expriments live and determine function
it is hard to maintain, costly, 3D architecture lost,
dedifferentation and aneuploidy
loose infulene of other cells/tissue
undergo senecsence
do not behave like normal cells in tissue
we have absolute control of sample
less need for animal testing
TEM vs SEM
TEM - gives a 2D slice of cell
SEM - scans the surface of the cell to give a 3D image
how do we isolate cells for culture?
can use collegenase to break ECM
microdisection - manual separation
